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Sample records for rapid multiplex analysis

  1. Rapid analysis of Saccharomyces cerevisiae genome rearrangements by multiplex ligation-dependent probe amplification.

    Directory of Open Access Journals (Sweden)

    Jason E Chan

    Full Text Available Aneuploidy and gross chromosomal rearrangements (GCRs can lead to genetic diseases and the development of cancer. We previously demonstrated that introduction of the repetitive retrotransposon Ty912 onto a nonessential chromosome arm of Saccharomyces cerevisiae led to increased genome instability predominantly due to increased rates of formation of monocentric nonreciprocal translocations. In this study, we adapted Multiplex Ligation-dependent Probe Amplification (MLPA to analyze a large numbers of these GCRs. Using MLPA, we found that the distribution of translocations induced by the presence of Ty912 in a wild-type strain was nonrandom and that the majority of these translocations were mediated by only six translocation targets on four different chromosomes, even though there were 254 potential Ty-related translocation targets in the S. cerevisiae genome. While the majority of Ty912-mediated translocations resulted from RAD52-dependent recombination, we observed a number of nonreciprocal translocations mediated by RAD52-independent recombination between Ty1 elements. The formation of these RAD52-independent translocations did not require the Rad51 or Rad59 homologous pairing proteins or the Rad1-Rad10 endonuclease complex that processes branched DNAs during recombination. Finally, we found that defects in ASF1-RTT109-dependent acetylation of histone H3 lysine residue 56 (H3K56 resulted in increased accumulation of both GCRs and whole-chromosome duplications, and resulted in aneuploidy that tended to occur simultaneously with GCRs. Overall, we found that MLPA is a versatile technique for the rapid analysis of GCRs and can facilitate the genetic analysis of the pathways that prevent and promote GCRs and aneuploidy.

  2. Rapid Multiplex Microbial Detector Project

    Data.gov (United States)

    National Aeronautics and Space Administration — ORBITEC, in collaboration with Lucigen, proposes a rapid nucleic acid-based detector for spaceflight water systems to enable simultaneous quantification of multiple...

  3. A Rapid Screening Analysis of Antioxidant Compounds in Native Australian Food Plants Using Multiplexed Detection with Active Flow Technology Columns

    Directory of Open Access Journals (Sweden)

    Emmanuel Janaka Rochana Rupesinghe

    2016-01-01

    Full Text Available Conventional techniques for identifying antioxidant and phenolic compounds in native Australian food plants are laborious and time-consuming. Here, we present a multiplexed detection technique that reduces analysis time without compromising separation performance. This technique is achieved using Active Flow Technology-Parallel Segmented Flow (AFT-PSF columns. Extracts from cinnamon myrtle (Backhousia myrtifolia and lemon myrtle (Backhousia citriodora leaves were analysed via multiplexed detection using an AFT-PSF column with underivatised UV-VIS, mass spectroscopy (MS, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH• derivatisation for antioxidants as detection methods. A number of antioxidant compounds were detected in the extracts of each leaf extract.

  4. Economic analysis of rapid multiplex polymerase chain reaction testing for meningitis/encephalitis in pediatric patients.

    Science.gov (United States)

    Duff, Steve; Hasbun, Rodrigo; Ginocchio, Christine C; Balada-Llasat, Joan-Miquel; Zimmer, Louise; Bozzette, Samuel A

    2018-01-10

    We assessed the possible economic impact of a rapid test in pediatric patients with suspected community-acquired meningitis/encephalitis. Modeling simulated diagnosis, clinical decisions, resource use/costs of standard of care (SOC) and two cerebrospinal fluid testing strategies using FilmArray ® (FA), a US FDA-cleared system that provides results in approximately 1 h. Pathogens detected by FA caused approximately 75% of cases, 97% of which would be accurately diagnosed with FA.  Mean cost/case ranged from $17,599 to $22,025.  Syndromic testing is less expensive than SOC. Testing all suspected cases yielded greater savings ($3481/case) than testing only those with abnormal cerebrospinal fluid ($2157/case). Greater economic benefits are achievable with syndromic testing of all cases, rather than SOC or targeted syndromic testing.

  5. Rapid and not culture-dependent assay based on multiplex PCR-SSR analysis for monitoring inoculated yeast strains in industrial wine fermentations.

    Science.gov (United States)

    Cordero-Bueso, Gustavo; Rodríguez, María Esther; Garrido, Carlos; Cantoral, Jesús Manuel

    2017-01-01

    Wine industry needs a simple method for rapid diagnosis of the dominance of inoculated strains that could be performed routinely during the fermentation process. We present a suitable, high-throughput, and low-cost method to monitor rapidly the dominance of inoculated yeast strains in industrial fermentations of red and white wines using an activated carbon cleaning pretreatment, and a rapid DNA extraction method plus multiplex PCR-SSR analysis. We apply this technique directly to samples of fermenting wines without previously isolating yeast colonies. Results are obtained in a maximum time of 4.5 h.

  6. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

    Science.gov (United States)

    Hanson, Erin K; Ballantyne, Jack

    2013-01-01

    Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive

  7. Multiplex PCR system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis.

    Science.gov (United States)

    Huang, H-S; Tsai, C-L; Chang, J; Hsu, T-C; Lin, S; Lee, C-C

    2017-12-05

    To provide a summary of evidence for the diagnostic accuracies of three multiplex PCR systems (mPCRs)-BioFire FilmArray RP (FilmArray), Nanosphere Verigene RV+ test (Verigene RV+) and Hologic Gen-Probe Prodesse assays-on the detection of viral respiratory infections. A comprehensive search up to 1 July 2017 was conducted on Medline and Embase for studies that utilized FilmArray, Verigene RV+ and Prodesse for diagnosis of viral respiratory infections. A summary of diagnostic accuracies for the following five viruses were calculated: influenza A virus (FluA), influenza B virus, respiratory syncytial virus, human metapneumovirus and adenovirus. Hierarchical summary receiver operating curves were used for estimating the viral detection performance per assay. Twenty studies of 5510 patient samples were eligible for analysis. Multiplex PCRs demonstrated high diagnostic accuracy, with area under the receiver operating characteristic curve (AUROC) equal to or more than 0.98 for all the above viruses except for adenovirus (AUROC 0.89). FilmArray, Verigene RV+ and ProFlu+ (the only Prodesse assay with enough data) demonstrated a summary sensitivity for FluA of 0.911 (95% confidence interval, 0.848-0.949), 0.949 (95% confidence interval, 0.882-0.979) and 0.954 (95% confidence interval, 0.871-0.985), respectively. The three mPCRs were comparable in terms of detection of FluA. Point estimates calculated from eligible studies showed that the three mPCRs (FilmArray, Verigene RV+ and ProFlu+) are highly accurate and may provide important diagnostic information for early identification of respiratory virus infections. In patients with low pretest probability for FluA, these three mPCRs can predict a low possibility of infection and may justify withholding empirical antiviral treatments. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  8. Rapid Detection of Mycobacterium tuberculosis Strains Resistant to Isoniazid and/or Rifampicin: Standardization of Multiplex Polymerase Chain Reaction Analysis.

    Science.gov (United States)

    Collantes, Jimena; Solari, Francesca Barletta; Rigouts, Leen

    2016-12-07

    Drug susceptibility testing using molecular techniques can enhance the identification of drug-resistant Mycobacterium tuberculosis Two multiplex real-time polymerase chain reaction (qPCR) assays were developed to detect the most common resistance-associated mutations to isoniazid (katGS315T, inhA-15C → T), and rifampicin (rpoBH526Y and rpoBS531L). To assess the species specificity of the qPCR, we selected 31 nontuberculous mycobacteria (NTM) reference strains belonging to 17 species from the public collection of mycobacterial cultures (BCCM/ITM). Additionally, we tested 17 isoniazid and/or rifampicin-resistant strains with other mutations in the target genes to assess mutation specificity. The limit of detection for all the targeted mutations was 20 bacilli/reaction. Multiplex 1 showed 90%, 95%, and 100% efficiency for wild type (WT), Mut katGS315T, and Mut rpoBS531L, respectively; whereas Multiplex 2 showed 97%, 94%, and 90% efficiency for WT, Mut inhA-15, and Mut rpoBH526Y, respectively. Three of 17 strains that presented other mutations in the target genes were identified as rifampicin resistant and only 3/31 NTM showed a similar melting temperature to rpoBL531 and/or katGT315 mutants. Thus, our proposed cascade of specific tuberculosis detection followed by drug resistance testing showed sensitivities for katGS315T, rpoBS531L, rpoBH526Y, and inhA-15 detection of 100%, 100%, 100%, and 96%, respectively; and specificities of 98%, 95%, 100%, and 100, respectively. © The American Society of Tropical Medicine and Hygiene.

  9. A handheld flow genetic analysis system (FGAS): towards rapid, sensitive, quantitative and multiplex molecular diagnosis at the point-of-care level.

    Science.gov (United States)

    Shu, Bowen; Zhang, Chunsun; Xing, Da

    2015-06-21

    A handheld flow genetic analysis system (FGAS) is proposed for rapid, sensitive, multiplex and real-time quantification of nucleic acids at the point-of-care (POC) level. The FGAS includes a helical thermal-gradient microreactor and a microflow actuator, as well as control circuitry for temperature, fluid and power management, and smartphone fluorescence imaging. All of these features are integrated into a field-portable and easy-to-use molecular diagnostic platform powered by lithium batteries. Due to the unique design of the microreactor, not only steady temperatures for denaturation and annealing/extension but also a linear thermal gradient for spatial high-resolution melting can be achieved through simply maintaining a single heater at constant temperature. The smartphone fluorescence imaging system has a wide field of view that captures all PCR channels of the microreactor in a single snapshot without the need for any mechanical scanning. By these designs, the FGAS enables real-time monitoring of the temporal and spatial fluorescence signatures of amplicons during continuous-flow amplification. On the current FGAS, visual detection of as little as 10 copies per μL of genomic DNA of Salmonella enterica was achieved in 15 min, with real-time quantitative detection of the DNA over 6 orders of magnitude concentration from 10(6) to 10(1) copies per μL also completed in 7.5-15 min. In addition, multiple pathogenic DNA targets could be simultaneously discriminated with direct bar-chart readout or multiplex spatial melting in serial flow. We anticipate that the FGAS has great potential to become a next-generation gene analyzer for POC molecular diagnostics.

  10. Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing.

    Science.gov (United States)

    Varga, Aniko; James, Delano

    2005-02-01

    A real-time multiplex PCR procedure with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid and reliable identification of Plum pox virus (PPV) isolates of strains D and M. Members of the different strains were identified by their distinctive melting temperatures (T(m)s); 84.3-84.43 degrees C for D isolates, and 85.34-86.11 degrees C for M isolates. The associated amplicon sizes were 114 and 380 bp, respectively. The procedure was used for detection and identification of PPV in both herbaceous and woody hosts. The Tm for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Universal primers included in the study detected all isolates of PPV tested, amplifying a 74 bp fragment. The Tm of this fragment varied from 80.12 to 81.52 degrees C and may have supplementary value for PPV identification. SYBR Green-based detection was compared to detection using a hybridization LUX fluorogenic primer. Better resolution of the melting peaks was observed with SYBR Green I, than with the LUX primers, hence strain identification with SYBR Green I was more reliable. This is a simple approach to PPV strain identification with the relatively inexpensive dye SYBR Green I, and eliminates any need for electrophoretic analysis of amplicons or RFLP patterns using ethidium bromide.

  11. Multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis - a systemic review and meta-analysis.

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    Shy-Shin Chang

    Full Text Available BACKGROUND: Blood culture is viewed as the golden standard for the diagnosis of sepsis but suffers from low sensitivity and long turnaround time. LightCycler SeptiFast (LC-SF is a real-time multiplex polymerase chain reaction test able to detect 25 common pathogens responsible for bloodstream infections within hours. We aim to assess the accuracy of LC-SF by systematically reviewing the published studies. METHOD: Related literature on Medline, Embase, and Cochrane databases was searched up to October 2012 for studies utilizing LC-SF to diagnose suspected sepsis and that provided sufficient data to construct two-by-two tables. RESULTS: A total of 34 studies enrolling 6012 patients of suspected sepsis were included. The overall sensitivity and specificity for LC-SF to detect bacteremia or fungemia was 0·75 (95% CI: 0·65-0·83 and 0·92 (95%CI:0·90-0·95, respectively. LC-SF had a high positive likelihood ratio (10·10 and a moderate negative likelihood ratio (0·27. Specifically, LC-SF had a sensitivity of 0·80 (95%CI: 0·70-0·88 and a specificity of 0·95(95%CI: 0·93-0·97 for the bacteremia outcome, and a sensitivity of 0·61 (95%CI: 0·48-0·72 and a specificity of 0·99 (95%CI: 0·99-0·99 for the fungemia outcome. High heterogeneity was found in the bacteremia outcome subgroup but not in the fungemia outcome subgroup. CONCLUSION: LC-SF is of high rule-in value for early detection of septic patients. In a population with low pretest probability, LC-SF test can still provide valuable information for ruling out bacteremia or fungemia.

  12. Rapid detection of methicillin-resistant staphylococci by multiplex PCR

    African Journals Online (AJOL)

    Administrator

    2010-11-08

    Nov 8, 2010 ... Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16: 11141-11156. Fang H, Hedin G (2003). Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay.

  13. Use of Multiplex Terminal Restriction Fragment Length Polymorphism for Rapid and Simultaneous Analysis of Different Components of the Soil Microbial Community▿

    OpenAIRE

    Singh, Brajesh K.; Nazaries, Loic; Munro, Stacey; Ian C Anderson; Campbell, Colin D.

    2006-01-01

    A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples ...

  14. Rapid detection and grouping of porcine bocaviruses by an EvaGreen(®) based multiplex real-time PCR assay using melting curve analysis.

    Science.gov (United States)

    Zheng, Xiaowen; Liu, Gaopeng; Opriessnig, Tanja; Wang, Zining; Yang, Zongqi; Jiang, Yonghou

    2016-08-01

    Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen(®)-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.3 ± 0.34 °C for PBoV G1, 78.2 ± 0.37 °C for PBoV G2, and 85.0 ± 0.29 °C for PBoV G3. Non-specific reactions were not observed when other pig viruses were used to assess the EG-mPCR assay. The sensitivity of the EG-mPCR assay using purified plasmid constructs containing the specific viral target fragments was 100 copies for PBoV G1, 50 for PBoV G2 and 100 for PBoV G3. The assay is able to detect and distinguish three PBoV groups with intra-assay and inter-assay variations ranging from 0.13 to 1.59%. The newly established EG-mPCR assay was validated with 227 field samples from pigs. PBoV G1, G2 and G3 was detected in 15.0%, 25.1% and 41.9% of the investigated samples and coinfections of two or three PBoV groups were also detected in 25.1% of the cases, indicating that all PBoV groups are prevalent in Chinese pigs. The agreement of the EG-mPCR assay with an EvaGreen-based singleplex real-time PCR (EG-sPCR) assay was 99.1%. This EG-mPCR will serve as a rapid, sensitive, reliable and cost effective alternative for routine surveillance testing of multiple PBoVs in pigs and will enhance our understanding of the epidemiological features and possible also pathogenetic changes associated with these viruses in pigs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach

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    Sinclair Andrew H

    2009-09-01

    Full Text Available Abstract Background Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems. Results We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 × 104 cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method. Conclusion This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 105 cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

  16. SCAR makers and multiplex PCR-based rapid molecular typing of Lentinula edodes strains.

    Science.gov (United States)

    Wu, Xueqian; Li, Haibo; Zhao, Weiwei; Fu, Lizhong; Peng, Huazheng; He, Liang; Cheng, Junwen; Wei, Hailong; Wu, Qingqi

    2010-11-01

    Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.

  17. Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

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    Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce

    2018-01-01

    In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

  18. Development a rapid and accurate multiplex real time PCR method for the detection Chlamydia trachomatis and Mycoplasma hominis.

    Science.gov (United States)

    Safarkar, Roya; Mehrabadi, Jalil Fallah; Noormohammadi, Zahra; Mirnejad, Reza

    2017-11-01

    Sexually transmitted diseases easily spread among sexually active people and often have no symptoms. Rapid and accurate method for detecting these infections are necessary in early stages. The traditional detection methods of them are difficult and time-consuming. In this study, multiplex real time PCR was optimized for rapid identification of Chlamydia trachomatis and Mycoplasma hominis in a single tube and was performed with our designed primers. The sensitivity test was carried out to designed primers with diluted genomic DNA. To defined the specificity, non STD bacteria were used as DNA template. This study indicated that the developed multiplex real time PCR can be an effective alternative procedure to the conventional methods for rapid and accurate identification of C Chlamydia trachomatis and Mycoplasma hominis. Multiplex real-time PCR Results of them were checked with melting curves. The sensitivity of our designed primer by multiplex real time PCR for Chlamydia trachomatis and Mycoplasma hominis were 4.78×1010 and 8.35×1010 , respectively, Which the primers did not amplify any product from a non-STD species. Multiplex real time PCR by our new primers and analysis of melting curves were successfully usable for rapid and accurate detection of Chlamydia trachomatis and Mycoplasma hominis. This assay instead of traditional culture method, has considerable potential to be rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous and direct detection. © 2017 Wiley Periodicals, Inc.

  19. A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs

    NARCIS (Netherlands)

    R. Alghafri (Rashed); W. Goodwin (Will); A. Ralf (Arwin); M.H. Kayser (Manfred); S. Hadi (Sibte)

    2015-01-01

    textabstractAbstract A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other

  20. Multiplexed plasmon sensor for rapid label-free analyte detection.

    Science.gov (United States)

    Rosman, Christina; Prasad, Janak; Neiser, Andreas; Henkel, Andreas; Edgar, Jonathan; Sönnichsen, Carsten

    2013-07-10

    Efficient and cost-effective multiplexed detection schemes for proteins in small liquid samples would bring drastic advances to fields like disease detection or water quality monitoring. We present a novel multiplexed sensor with randomly deposited aptamer functionalized gold nanorods. The spectral position of plasmon resonances of individual nanorods, monitored by dark-field spectroscopy, respond specifically to different proteins. We demonstrate nanomolar sensitivity, sensor recycling, and the potential to upscale to hundreds or thousands of targets.

  1. New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

    Science.gov (United States)

    Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

    2015-01-01

    We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

  2. New multiplex PCR methods for rapid screening of genetically modified organisms in foods

    Directory of Open Access Journals (Sweden)

    Nelly eDatukishvili

    2015-07-01

    Full Text Available We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs. New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

  3. Rapid DNA haplotyping using a multiplex heteroduplex approach: application to Duchenne muscular dystrophy carrier testing.

    Science.gov (United States)

    Prior, T W; Wenger, G D; Papp, A C; Snyder, P J; Sedra, M S; Bartolo, C; Moore, J W; Highsmith, W E

    1995-01-01

    A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles.

  4. Spatial analysis of various multiplex cinema types

    Directory of Open Access Journals (Sweden)

    Young-Seo Park

    2016-03-01

    Full Text Available This study identifies the spatial characteristics and relationships of each used space according to the multiplex type. In this study, multiplexes are classified according to screen rooms and circulation systems, and each used space is quantitatively analyzed. The multiplex type based on screen rooms and moving line systems influences the relationship and characteristics of each used space in various ways. In particular, the structure of the used space of multiplexes has a significant effect on profit generation and audience convenience.

  5. Nanoscale Test Strips for Multiplexed Blood Analysis Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The goal of our nanoscale test strips, or nanostrips, is to provide rapid, low-cost, powerful multiplexed analyses in a diminutive form so that whole body health...

  6. Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

    Science.gov (United States)

    Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

    2018-02-01

    The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

  7. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM analysis [v1; ref status: indexed, http://f1000r.es/2hj

    Directory of Open Access Journals (Sweden)

    Erin K. Hanson

    2013-12-01

    Full Text Available Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE or quantitative RT-PCR (qRT-PCR platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions, IL1F7 (skin, ALAS2 (blood, MMP10 (menstrual blood, HTN3 (saliva and TGM4 (semen.  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green. Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively

  8. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM analysis [v2; ref status: indexed, http://f1000r.es/314

    Directory of Open Access Journals (Sweden)

    Erin K. Hanson

    2014-02-01

    Full Text Available Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE or quantitative RT-PCR (qRT-PCR platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions, IL1F7 (skin, ALAS2 (blood, MMP10 (menstrual blood, HTN3 (saliva and TGM4 (semen.  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green. Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively

  9. Development of multiplex-PCR assay for rapid detection of Candida spp.

    Directory of Open Access Journals (Sweden)

    Ni Made A. Tarini

    2010-05-01

    Full Text Available Aim Candida spp. infection commonly occur in immunocompromised patients. Biochemical assay for identification of Candida spp. is time-consuming and shows many undetermined results. Specific detection for antibody, antigen and metabolites of Candida spp. had low sensitivity and specificity. In this study, we developed a rapid diagnostic method, Multiplex-PCR, to identify Candida spp.Methods Five Candida spp. isolates were cultured, identifi ed with germ tube and API® 20 C AUX (BioMerieux® SA kit. Furthermore, DNA was purified by QIAamp DNA mini (Qiagen® kit for Multiplex-PCR assay.Results DNA detection limit by Multiplex-PCR assays for C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata were 4 pg, 0.98 pg, 0.98 pg, 0.5 pg and 16 pg respectively. This assay was also more sensitive than culture in that Multiplex-PCR could detect 2.6-2.9 x 100 CFU/ml, whereas culture 2.6-2.9 x 102 CFU/ml.Conclusion Multiplex-PCR is much more sensitive than culture and thus, can be recommended as a sensitive and specific assay for identification of Candida spp. (Med J Indones 2010; 19:83-7Keywords: Candida spp., multiplex-PCR

  10. Rapid DNA haplotyping using a multiplex heteroduplex approach: Application to Duchenne muscula dystrophy carrier detection

    Energy Technology Data Exchange (ETDEWEB)

    Prior, T.W.; Wenger, G.D.; Moore, J. [Ohio State Univ., Columbus, OH (United States)] [and others

    1994-09-01

    A new strategy has been developed for rapid haplotype analysis. It is based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. The method is simple, rapid, does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using 12 commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. As a result of expanding the number of detectable polymorphisms throughout the dystrophin gene, we show how the method can easily be combined with dinucleotide analysis to improve the accuracy of carrier detection in the nondeletion cases. The technique is also shown to be used as an effective screen for improving carrier detection in several families with deletions. The finding of heterozygosity within the deletion identifies the at-risk female as a noncarrier. Using this method, we have identified and incorporated 3 new dystrophin polymorphisms (one of which in exon 16 is unique to African Americans). The method may be used other genetic diseases when mutations are unknown, or there are few dinucleotide markers in the gene proximity, or for the identification of haplotype backgrounds of mutant alleles.

  11. Rapid detection of methicillin-resistant staphylococci by multiplex PCR

    African Journals Online (AJOL)

    A rapid and sensitive method for excluding the presence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples was developed. The combination of MRSA detection by mecA coaA PCR with prior enrichment in selective broth was tested for 300 swabs. PCR identified 26 MRSApositive samples, ...

  12. Nanoscale Test Strips for Multiplexed Blood Analysis

    Science.gov (United States)

    Chan, Eugene

    2015-01-01

    A critical component of the DNA Medicine Institute's Reusable Handheld Electrolyte and Lab Technology for Humans (rHEALTH) sensor are nanoscale test strips, or nanostrips, that enable multiplexed blood analysis. Nanostrips are conceptually similar to the standard urinalysis test strip, but the strips are shrunk down a billionfold to the microscale. Each nanostrip can have several sensor pads that fluoresce in response to different targets in a sample. The strips carry identification tags that permit differentiation of a specific panel from hundreds of other nanostrip panels during a single measurement session. In Phase I of the project, the company fabricated, tested, and demonstrated functional parathyroid hormone and vitamin D nanostrips for bone metabolism, and thrombin aptamer and immunoglobulin G antibody nanostrips. In Phase II, numerous nanostrips were developed to address key space flight-based medical needs: assessment of bone metabolism, immune response, cardiac status, liver metabolism, and lipid profiles. This unique approach holds genuine promise for space-based portable biodiagnostics and for point-of-care (POC) health monitoring and diagnostics here on Earth.

  13. New multiplex PCR methods for rapid screening of genetically modified organisms in foods

    OpenAIRE

    Nelly eDatukishvili; Tamara eKutateladze; Inga eGabriadze; Kakha eBitskinashvili; Boris eVishnepolsky

    2015-01-01

    We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of C...

  14. Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues.

    Science.gov (United States)

    Wang, Yu; Woehrstein, Johannes B; Donoghue, Noah; Dai, Mingjie; Avendaño, Maier S; Schackmann, Ron C J; Zoeller, Jason J; Wang, Shan Shan H; Tillberg, Paul W; Park, Demian; Lapan, Sylvain W; Boyden, Edward S; Brugge, Joan S; Kaeser, Pascal S; Church, George M; Agasti, Sarit S; Jungmann, Ralf; Yin, Peng

    2017-10-11

    To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ∼300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

  15. Development of a novel multiplex PCR assay for rapid detection of virulence associated genes of Pasteurella multocida from pigs

    National Research Council Canada - National Science Library

    Rajkhowa, S

    2015-01-01

    Significance and Impact of the Study: The study reports the development and evaluation of a novel multiplex PCR assay for the rapid detection of 11 important VAGs of Pasteurella multocida isolates from pigs...

  16. Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples

    DEFF Research Database (Denmark)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas

    2017-01-01

    , in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair...... of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using...... and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection....

  17. Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR.

    Science.gov (United States)

    Torriani, S; Zapparoli, G; Malacrinò, P; Suzzi, G; Dellaglio, F

    2004-01-01

    To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.

  18. Multiplex pyrosequencing of InDel markers for forensic DNA analysis.

    Science.gov (United States)

    Bus, Magdalena M; Karas, Ognjen; Allen, Marie

    2016-12-01

    The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator ® DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator ® DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof-of-principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost-effective alternative for some applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. multiplex

    DEFF Research Database (Denmark)

    2015-01-01

    Algebraic procedures for the analysis of multiple social networks are delivered with this package. Among other things, it is possible to create and manipulate multivariate network data with different formats, and there are effective ways available to treat multiple networks with routines that com......Algebraic procedures for the analysis of multiple social networks are delivered with this package. Among other things, it is possible to create and manipulate multivariate network data with different formats, and there are effective ways available to treat multiple networks with routines...... that combine algebraic systems like the partially ordered semigroup or the semiring structure together with the relational bundles occurring in different types of multivariate network data sets. As well an algebraic approach for two-mode networks is made through Galois derivations between families of the pair...

  20. Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

    Science.gov (United States)

    Benitez, Alvaro J; Winchell, Jonas M

    2016-04-01

    We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

  1. Rapid, Time-Division Multiplexed, Direct Absorption- and Wavelength Modulation-Spectroscopy

    Directory of Open Access Journals (Sweden)

    Alexander Klein

    2014-11-01

    Full Text Available We present a tunable diode laser spectrometer with a novel, rapid time multiplexed direct absorption- and wavelength modulation-spectroscopy operation mode. The new technique allows enhancing the precision and dynamic range of a tunable diode laser absorption spectrometer without sacrificing accuracy. The spectroscopic technique combines the benefits of absolute concentration measurements using calibration-free direct tunable diode laser absorption spectroscopy (dTDLAS with the enhanced noise rejection of wavelength modulation spectroscopy (WMS. In this work we demonstrate for the first time a 125 Hz time division multiplexed (TDM-dTDLAS-WMS spectroscopic scheme by alternating the modulation of a DFB-laser between a triangle-ramp (dTDLAS and an additional 20 kHz sinusoidal modulation (WMS. The absolute concentration measurement via the dTDLAS-technique allows one to simultaneously calibrate the normalized 2f/1f-signal of the WMS-technique. A dTDLAS/WMS-spectrometer at 1.37 µm for H2O detection was built for experimental validation of the multiplexing scheme over a concentration range from 50 to 3000 ppmV (0.1 MPa, 293 K. A precision of 190 ppbV was achieved with an absorption length of 12.7 cm and an averaging time of two seconds. Our results show a five-fold improvement in precision over the entire concentration range and a significantly decreased averaging time of the spectrometer.

  2. The power of multiplexed functional analysis of genetic variants.

    Science.gov (United States)

    Gasperini, Molly; Starita, Lea; Shendure, Jay

    2016-10-01

    New technologies have recently enabled saturation mutagenesis and functional analysis of nearly all possible variants of regulatory elements or proteins of interest in single experiments. Here we discuss the past, present, and future of such multiplexed (functional) assays for variant effects (MAVEs). MAVEs provide detailed insight into sequence-function relationships, and they may prove critical for the prospective clinical interpretation of genetic variants.

  3. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

    Science.gov (United States)

    García, L; Tercero, J C; Legido, B; Ramos, J A; Alemany, J; Sanz, M

    1998-01-01

    The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens.

  4. Rapid multiplex PCR assay to identify respiratory viral pathogens: moving forward diagnosing the common cold.

    Science.gov (United States)

    Layman, Clifton P; Gordon, Sarah M; Elegino-Steffens, Diane U; Agee, Willie; Barnhill, Jason; Hsue, Gunther

    2013-09-01

    Upper respiratory tract infections (URIs) can be a serious burden to the healthcare system. The majority of URIs are viral in etiology, but definitive diagnosis can prove difficult due to frequently overlapping clinical presentations of viral and bacterial infections, and the variable sensitivity, and lengthy turn-around time of viral culture. We tested new automated nested multiplex PCR technology, the FilmArray(®) system, in the TAMC department of clinical investigations, to determine the feasibility of replacing the standard viral culture with a rapid turn-around system. We conducted a feasibility study using a single-blinded comparison study, comparing PCR results with archived viral culture results from a convenience sample of cryopreserved archived nasopharyngeal swabs from acutely ill ED patients who presented with complaints of URI symptoms. A total of 61 archived samples were processed. Viral culture had previously identified 31 positive specimens from these samples. The automated nested multiplex PCR detected 38 positive samples. In total, PCR was 94.5% concordant with the previously positive viral culture results. However, PCR was only 63.4% concordant with the negative viral culture results, owing to PCR detection of 11 additional viral pathogens not recovered on viral culture. The average time to process a sample was 75 minutes. We determined that an automated nested multiplex PCR is a feasible alternative to viral culture in an acute clinical setting. We were able to detect at least 94.5% as many viral pathogens as viral culture is able to identify, with a faster turn-around time.

  5. Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

    Science.gov (United States)

    Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

    2012-11-01

    Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

  6. Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.

    Science.gov (United States)

    Wang, Hye-Young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok; Uh, Young; Lee, Hyeyoung

    2014-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10(3) CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

    Science.gov (United States)

    Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

  8. Performance analysis of a majority logic multiplex system

    Science.gov (United States)

    Maseng, T.

    1980-09-01

    The code division multiplexing (CDM) of digitally modulated baseband signals is normally referred to as majority logic multiplexing (MLM). The employment of MLM techniques in spread spectrum multiple access (SSMA) systems is especially attractive because it allows many different types of baseband channels to be easily combined. The traffic level can be gradually reduced to obtain a desired increase in processing gain, without affecting the active channels' share of the transmitter power. The code integrity and the processing gain of the SSMA equipment can be preserved by suitable design. This paper contains a general performance analysis of an MLM system that uses random codes. The basic MLM principle is extended to handle the combining of traffic channels with different bit rates. Calculated values of performance deterioration due to noise are compared with experimental results and found to be in close agreement.

  9. Capacity analysis of spectrum sharing spatial multiplexing MIMO systems

    KAUST Repository

    Yang, Liang

    2014-12-01

    This paper considers a spectrum sharing (SS) multiple-input multiple-output (MIMO) system operating in a Rayleigh fading environment. First the capacity of a single-user SS spatial multiplexing system is investigated in two scenarios that assume different receivers. To explicitly show the capacity scaling law of SS MIMO systems, some approximate capacity expressions for the two scenarios are derived. Next, we extend our analysis to a multiple user system with zero-forcing receivers (ZF) under spatially-independent scheduling and analyze the sum-rate. Furthermore, we provide an asymptotic sum-rate analysis to investigate the effects of different parameters on the multiuser diversity gain. Our results show that the secondary system with a smaller number of transmit antennas Nt and a larger number of receive antennas Nr can achieve higher capacity at lower interference temperature Q, but at high Q the capacity follows the scaling law of the conventional MIMO systems. However, for a ZF SS spatial multiplexing system, the secondary system with small Nt and large Nr can achieve the highest capacity throughout the entire region of Q. For a ZF SS spatial multiplexing system with scheduling, the asymptotic sum-rate scales like Ntlog2(Q(KNtNp-1)/Nt), where Np denotes the number of antennas of the primary receiver and K represents the number of secondary transmitters.

  10. Multiplex network analysis of employee performance and employee social relationships

    Science.gov (United States)

    Cai, Meng; Wang, Wei; Cui, Ying; Stanley, H. Eugene

    2018-01-01

    In human resource management, employee performance is strongly affected by both formal and informal employee networks. Most previous research on employee performance has focused on monolayer networks that can represent only single categories of employee social relationships. We study employee performance by taking into account the entire multiplex structure of underlying employee social networks. We collect three datasets consisting of five different employee relationship categories in three firms, and predict employee performance using degree centrality and eigenvector centrality in a superimposed multiplex network (SMN) and an unfolded multiplex network (UMN). We use a quadratic assignment procedure (QAP) analysis and a regression analysis to demonstrate that the different categories of relationship are mutually embedded and that the strength of their impact on employee performance differs. We also use weighted/unweighted SMN/UMN to measure the predictive accuracy of this approach and find that employees with high centrality in a weighted UMN are more likely to perform well. Our results shed new light on how social structures affect employee performance.

  11. Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex.

    Science.gov (United States)

    Koh, Seng Fook; Tay, Sun Tee; Sermswan, Rasana; Wongratanacheewin, Surasakdi; Chua, Kek Heng; Puthucheary, Savithri D

    2012-09-01

    We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Performance of the Real Fungus-ID kit based on multiplex RT-PCR assay for the rapid detection and identification of Trichophyton spp. and Microsporum spp. in clinical specimens with suspected dermatophyte infection.

    Science.gov (United States)

    Wang, H-Y; Kim, H; Choi, E H; Lee, H

    2016-01-01

    The aim of this study was to evaluate the performance of a commercially available multiplex RT-PCR assay for the rapid detection and identification of dermatophytes directly from clinical samples and cultures. The multiplex RT-PCR assay was used to evaluate 118 clinical isolates from various specimen types and a total of 140 known specimens were compared with both conventional methods, commercially available PCR-REBA, and ITS sequence analysis. In this study, multiplex RT-PCR assay yield significantly more positive results than culture (91·9 vs 39·5%) and conventional methods including KOH microscopy (91·9 vs 71·3%). Although the results among the multiplex RT-PCR, PCR-REBA and ITS sequence analysis were concordant (100%) in 118 clinical isolates, concordant results between multiplex RT-PCR assay and culture were at 66% (78/118). The overall positive rates for the PCR-REBA, multiplex RT-PCR assay and ITS sequence analysis were 98·8, 91·9, and 52·9% respectively. In addition, the concordance rate of multiplex RT-PCR assay and the PCR-REBA assay was 93% (95% confidence interval (CI), 89·9-96·1, P culture can take up to 2-3 weeks. The use of the multiplex RT-PCR molecular diagnostic assay was rapid and reliable for detecting pathogen infections. Even though the use of molecular diagnostic technology is more expensive than conventional methods, the clinical and economic benefit of saving time relative to expense remains to be elucidated. Therefore, the multiplex RT-PCR assay may provide the essential information to accelerate therapeutic decisions for earlier and adequate antibiotic treatment in the acute phase of fungal pathogen infections. © 2015 The Society for Applied Microbiology.

  13. A multiplex PCR for rapid identification of Brassica species in the triangle of U.

    Science.gov (United States)

    Koh, Joshua C O; Barbulescu, Denise M; Norton, Sally; Redden, Bob; Salisbury, Phil A; Kaur, Sukhjiwan; Cogan, Noel; Slater, Anthony T

    2017-01-01

    Within the Brassicaceae, six species from the genus Brassica are widely cultivated throughout the world as oilseed, condiment, fodder or vegetable crops. The genetic relationships among the six Brassica species are described by U's triangle model. Extensive shared traits and diverse morphotypes among Brassica species make identification and classification based on phenotypic data alone challenging and unreliable, especially when dealing with large germplasm collections. Consequently, a major issue for genebank collections is ensuring the correct identification of species. Molecular genotyping based on simple sequence repeat (SSR) marker sequencing or the Illumina Infinium Brassica napus 60K single nucleotide polymorphism (SNP) array has been used to identify species and assess genetic diversity of Brassica collections. However, these methods are technically challenging, expensive and time-consuming, making them unsuitable for routine or rapid screening of Brassica accessions for germplasm management. A cheaper, faster and simpler method for Brassica species identification is described here. A multiplex polymerase chain reaction (MPCR) consisting of new and existing primers specific to the Brassica A, B and C genomes was able to reliably distinguish all six Brassica species in the triangle of U with 16 control samples of known species identity. Further validation against 120 Brassica accessions previously genotyped showed that the MPCR is highly accurate and comparable to more advanced techniques such as SSR marker sequencing or the Illumina Infinium B. napus 60K SNP array. In addition, the MPCR was sensitive enough to detect seed contaminations in pooled seed samples of Brassica accessions. A cheap and fast multiplex PCR assay for identification of Brassica species in the triangle of U was developed and validated in this study. The MPCR assay can be readily implemented in any basic molecular laboratory and should prove useful for the management of Brassica

  14. Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

    Science.gov (United States)

    Peters, Sarah E.; Wang, Jinhong; Hernandez-Garcia, Juan; Weinert, Lucy A.; Luan, Shi-Lu; Chaudhuri, Roy R.; Angen, Øystein; Aragon, Virginia; Williamson, Susanna M.; Langford, Paul R.; Rycroft, Andrew N.; Wren, Brendan W.; Maskell, Duncan J.; Tucker, Alexander W.

    2015-01-01

    Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis. PMID:26424843

  15. Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

    Science.gov (United States)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Quyen, Than Linh; Engelsmann, Pia; Wolff, Anders; Bang, Dang Duong

    2017-04-01

    Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. A novel electromagnetic apparatus for rapid multiplex single molecule force spectroscopy.

    Science.gov (United States)

    Shen, Yi; Czajkowsky, Daniel M; Li, Xiaowei; Sun, Jielin; Hu, Jun; Shao, Zhifeng

    2013-02-01

    Single-molecule force spectroscopy has revolutionized our ability to probe the details of molecular structures and interactions, but the numbers of individual measurements required for achieving a statistically reliable result can sometimes prove daunting. To overcome this problem, a number of instruments have recently been developed that are capable of monitoring the behavior of tens of individual biomolecules simultaneously. In this work, we have constructed a novel electromagnetic apparatus for multiplex single molecule force measurements utilizing magnetic microspheres. In this system, the magnetic field is generated with an electron-lens of an electron microscope mated with a high voltage flash light circuit to rapidly attain a stable magnetic field. We show that this instrument can generate a uniform magnetic force of up to -20 pN within 5 ms, over a region spanning 1 mm. The successful application of this apparatus to the force-dependent extension of dsDNA fully validates this approach. Furthermore, the lens-like design of the pole piece is fully compatible with optical imaging, thus allowing for the integration of single molecule fluorescence capabilities that should make this system a particularly powerful apparatus for multi-dimensional characterization of fast processes within interacting single molecules.

  17. Clinical usefulness of multiplex PCR lateral flow in MRSA detection: a novel, rapid genetic testing method.

    Science.gov (United States)

    Nihonyanagi, Shin; Kanoh, Yuhsaku; Okada, Kiyomi; Uozumi, Toshiki; Kazuyama, Yukumasa; Yamaguchi, Tokiko; Nakazaki, Nobuhiko; Sakurai, Keizou; Hirata, Yasuyoshi; Munekata, Shinichi; Ohtani, Shinichi; Takemoto, Tsuyoshi; Bandoh, Yuki; Akahoshi, Tohru

    2012-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) with exogenous cassette DNA containing the methicillin-resistant gene mecA (SCCmec) poses a problem as a drug-resistant bacterium responsible for hospital- and community-acquired infections. The frequency of MRSA detection has recently been increasing rapidly in Japan, and SCCmec has also been classified more diversely into types I-V. A rapid test is essential for early diagnosis and treatment of MRSA infections, but detection by conventional methods requires at least two days. The newly developed multiplex PCR lateral flow method allows specific amplification of femA to detect S. aureus, mecA to detect SCCmec, and kdpC to detect SCCmec type II; moreover, PCR products can be evaluated visually in about 3 h. In the present study, we developed a PCR lateral flow method for MRSA using this method and investigated its clinical usefulness in the detection of MRSA. The results showed a diagnostic concordance rate of 91.7% for MRSA and methicillin-susceptible S. aureus between bacteriological examination and PCR lateral flow, and a high level of specificity in PCR lateral flow. In addition, a higher detection rate for S. aureus using the same sample was observed for PCR lateral flow (70.2%) than for bacteriological tests (48.6%). The above results show that PCR lateral flow for MRSA detection has high sensitivity, specificity, and speed, and its clinical application as a method for early diagnosis of MRSA infections appears to be feasible.

  18. A multiplex RT-PCR for rapid and simultaneous detection of viruses and viroids in chrysanthemum.

    Science.gov (United States)

    Song, A; You, Y; Chen, F; Li, P; Jiang, J; Chen, S

    2013-01-01

    Chrysanthemum plants are subject to serious virus diseases, so detection and identification of virus pathogens is important to prevent the virus spread. A reliable one-step multiplex RT-PCR was developed to simultaneously detect two viruses and two viriods: chrysanthemum virus B, tomato Aspermy virus, chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid. In addition, we investigated the detection limit and the efficiency of single and multiplex RT-PCR assays. The results showed that the multiplex RT-PCR assay proved to be as sensitive as the single one. In conclusion, this technique is potentially useful in routine diagnosis of chrysanthemum viruses and viroids. The multiplex RT-PCR assay described in this study is the first report of simultaneous detection of virus and viroid in chrysanthemum, which provides a fast, convenient, cost-saving way to detect the virus and viroid mixed infections in plants. © 2012 The Society for Applied Microbiology.

  19. Sensitive Flow-through Immunoassay for Rapid Multiplex Determination of Cereal-borne Mycotoxins in Feed and Feed Ingredients.

    Science.gov (United States)

    Beloglazova, Natalia V; Graniczkowska, Kinga; Njumbe Ediage, Emmanuel; Averkieva, Olga; De Saeger, Sarah

    2017-08-23

    An easy-to-operate membrane-based flow-through test for multiplex screening of four mycotoxins (zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A) in a variety of cereal-based feed ingredients and compound feeds, such as wheat, barley, soybean, wheat bran, rice, rice bran, maize, rapeseed meal, and sunflower meal, and various types of complete feed (duckling feed, swine feed, broiler feed, piglet feed) was developed and validated. First, the antibodies were evaluated by enzyme-linked immunosorbent assay and then employed in the membrane rapid test. The cutoff levels for zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A were 50, 200, 1, and 10 μg/kg, respectively, based on European regulations and consumers' requirements. As sample pretreatment, consecutive steps of extraction, dilution, solid-phase extraction by addition of C18 sorbent, and final filtration of supernatant were followed. Both the sample preparation and the analysis procedure were simple, cost-effective, and easy to perform on-site in a nonlaboratory environment. The impact of sample processing on the result of the experiment was investigated supported by experimental design. The validation procedure was performed on the basis of Commission Regulation 2006/401/EC. The numbers of false-positive and false-negative outcomes were <5%, going along with the Commission Decision 2002/657/EC. Liquid chromatography-tandem mass spectrometry was performed as a confirmatory technique.

  20. Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

    Science.gov (United States)

    Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

    2017-08-01

    Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of 4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

  1. Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions.

    Science.gov (United States)

    Stadhouders, Ralph; Kolovos, Petros; Brouwer, Rutger; Zuin, Jessica; van den Heuvel, Anita; Kockx, Christel; Palstra, Robert-Jan; Wendt, Kerstin S; Grosveld, Frank; van Ijcken, Wilfred; Soler, Eric

    2013-03-01

    Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1-8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.

  2. Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets

    DEFF Research Database (Denmark)

    Wittig, Rainer; Salowsky, Rüdiger; Blaich, Stephanie

    2005-01-01

    Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality...... of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array...... analyses. The screening of additional melanoma cell lines resulted in distinct expression patterns for ten candidate genes. Our approach reveals a rapid and easy-to-handle alternative for candidate gene set evaluation from limited amounts of RNA....

  3. Rapid detection of carbapenemase genes by multiplex real-time PCR.

    Science.gov (United States)

    Monteiro, Jussimara; Widen, Raymond H; Pignatari, Antonio C C; Kubasek, Carly; Silbert, Suzane

    2012-04-01

    To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48). A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA). Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

  4. Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

    Directory of Open Access Journals (Sweden)

    Andrea Santos Lima

    2013-07-01

    Full Text Available Introduction The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM Results Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23 were characterized as the Mycobacterium tuberculosis complex (MTBC and 20% (n=8 as nontuberculous mycobacteria (NTM, with 22.5% (n=9 of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13 of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Conclusions Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

  5. Rapid detection of chromosomal aneuploidies in uncultured amniocytes by multiplex ligation-dependent probe amplification (MLPA)

    NARCIS (Netherlands)

    Hochstenbach, R; Meijer, J; van de Brug, J; Vossebeld-Hoff, I; Jansen, R; van der Luijt, R B; Sinke, R J; Page-Christiaens, G C M L; Ploos van Amstel, J-K; de Pater, J M

    2005-01-01

    OBJECTIVE: To test whether multiplex ligation-dependent probe amplification (MLPA) can be used for the detection of aneuploidy of chromosomes 13, 18, 21, X, and Y in uncultured amniocytes. METHODS: We performed a prospective study based on 527 amniotic fluid samples. Chromosome copy numbers were

  6. Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay.

    Science.gov (United States)

    Kim, Suk Ran; Ki, Chang-Seok; Lee, Nam Yong

    2009-03-01

    Acute viral respiratory infections are among the most common causes of human disease. Rapid and accurate diagnosis of viral respiratory infections is important for providing timely therapeutic interventions. This study evaluated a new multiplex PCR assay (Seegene Inc., Seoul, Korea) for simultaneous detection and identification of 12 respiratory viruses using two primer mixes. The viruses included parainfluenza viruses 1, 2, and 3, human metapneumovirus, human coronavirus 229E/NL63 and OC43, adenovirus, influenza viruses A and B, human respiratory syncytial viruses A and B, and human rhinovirus A. The analytical sensitivity of the assay was 10-100 copies per reaction for each type of virus. There was no cross-reactivity with common bacterial or viral pathogens. A comparison with conventional viral culture and immunofluorescence was carried out using 101 respiratory specimens from 92 patients. Using viral culture, 57 specimens (56.4%) were positive without co-infection. The same viruses were identified in all 57 specimens using the multiplex PCR. Seven of the 57 specimens (12.3%) were found to be co-infected with other respiratory viruses, and 19 of 44 (43.2%) specimens which were negative by culture were positive by the multiplex PCR. The Seeplex Respiratory Virus Detection assay represents a significant improvement over the conventional methods for the detection of a broad spectrum of respiratory viruses.

  7. Comparative multiplex dosage analysis in spinocerebellar ataxia type 2 patients.

    Science.gov (United States)

    Calì, F; Chiavetta, V; Ragalmuto, A; Vinci, M; Ruggeri, G; Schinocca, P; Romano, V

    2013-04-12

    We developed a new application of comparative multiplex dosage analysis (CMDA) for evaluation of the ataxin 2 gene. Expansions of the triplet CAG can cause spinocerebellar ataxia type 2 (SCA2), a neurodegenerative disease with an autosomal-dominant mode of inheritance. Molecular diagnosis of SCA2 is routinely based on the use of conventional PCR to detect the CAG expansion. However, PCR does not amplify an allele with an expansion of many triplets (>80), which is typically found in infantile and juvenile forms of SCA2, thus leading to false negatives. We propose the analysis of the ATXN2 gene by CMDA to complement existing methods currently used for the detection of large expansions of the CAG repeat. Using CMDA, the presence of any longer mutated allele in a heterozygous patient or fetus would be inferred due to dosage variation of the very frequent normal allele #22. CMDA can be completed in 1 day, at very low cost, and would be a useful tool for prenatal diagnosis and for diagnosis of presymptomatic forms of early-onset SCA2.

  8. Performance Analysis of Statistical Time Division Multiplexing Systems

    Directory of Open Access Journals (Sweden)

    Johnson A. AJIBOYE

    2010-12-01

    Full Text Available Multiplexing is a way of accommodating many input sources of a low capacity over a high capacity outgoing channel. Statistical Time Division Multiplexing (STDM is a technique that allows the number of users to be multiplexed over the channel more than the channel can afford. The STDM normally exploits unused time slots by the non-active users and allocates those slots for the active users. Therefore, STDM is appropriate for bursty sources. In this way STDM normally utilizes channel bandwidth better than traditional Time Division Multiplexing (TDM. In this work, the statistical multiplexer is viewed as M/M/1queuing system and the performance is measured by comparing analytical results to simulation results using Matlab. The index used to determine the performance of the statistical multiplexer is the number of packets both in the system and the queue. Comparison of analytical results was also done between M/M/1 and M/M/2 and also between M/M/1 and M/D/1 queue systems. At high utilizations, M/M/2 performs better than M/M/1. M/D/1 also outperforms M/M1.

  9. Development of a novel multiplex PCR assay for rapid detection of virulence associated genes of Pasteurella multocida from pigs.

    Science.gov (United States)

    Rajkhowa, S

    2015-09-01

    As the pathogenicity of Pasteurella multocida is associated with various virulence factors (VFs), the aim of the study was to develop a novel multiplex PCR (m-PCR) assay for the rapid detection of important virulence associated genes (VAGs) of P. multocida isolates from pigs. The target recognized VFs used in the study were diverse adhesins (ptfA and pfhA), toxins (toxA), siderophores (tonB and hgbA), sialidases (nanB, nanH) and outer membrane proteins (ompA, ompH, oma87 and plpB). The primers for the genes encoding these VFs were designed by primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) using gene sequences available in Genbank. The detection limit of the developed assay was 10(2)  CFU ml(-1) . The m-PCR did not produce any nonspecific amplification products when tested against Bordetella bronchiseptica which also commonly infects pigs. We applied m-PCR to the field samples, and the results obtained were the same as the single PCR results. The developed assay would be very useful for veterinary diagnostic laboratories and for others interested in the rapid virulence profiling of porcine P. multocida isolates circulating in the piggeries. The study reports the development and evaluation of a novel multiplex PCR assay for the rapid detection of 11 important VAGs of Pasteurella multocida isolates from pigs. Rapid and simultaneous detection of recognized VFs of the organism are essential to know the virulo-types of P. multocida isolates circulating in the piggeries. The developed novel assay will be very useful for the rapid detection of VAGs of P. multocida isolates from pigs. © 2015 The Society for Applied Microbiology.

  10. A multiplex panel of short-amplicon insertion-deletion DNA polymorphisms for forensic analysis

    National Research Council Canada - National Science Library

    Santos, V R D; Pena, H B; Pena, S D J

    2015-01-01

    .... To optimize the panel for application with degraded DNA samples, which are commonly encountered in fo-rensic analysis, we have significantly reduced the amplicon size of the INDELs and developed a new multiplex panel...

  11. Multiplexed DNA Methylation Analysis of Target Regions Using Microfluidics (Fluidigm).

    Science.gov (United States)

    Adamowicz, Martyna; Maratou, Klio; Aitman, Timothy J

    2018-01-01

    Whole genome shotgun bisulfite sequencing is a method used to generate genome-wide methylation profiles. There are many available protocols to validate the results of this genome-wide method, but they mostly share the limitation of measuring methylation at a small number of CpG positions in small numbers of samples. We developed a multiplexed DNA methylation analysis protocol, which allows for the simultaneous quantitative measurement of cytosine methylation at single nucleotide resolution in 48 PCR amplicons and 48 samples utilizing the microfluidic system established by Fluidigm. Following bisulfite conversion of 500 ng of the target DNA, a PCR reaction is performed using a 48.48 Access Array, which allows parallel amplification of 48 samples by 48 primer pairs. The products of each reaction are labeled with individual, sample specific tags, pooled in a single library and sequenced using the Illumina MiSeq sequencer. The advantages of this system are: speed, small amount of input material, single nucleotide resolution, high coverage of each locus, low cost of simultaneously assaying multiple CpG loci in multiple DNA samples and high reproducibility.

  12. [Application of multiplex ligation-dependent probe amplification for rapid detection of aneuploidies and structural chromosomal abnormalities in prenatal diagnosis].

    Science.gov (United States)

    Zhang, Jingjing; Hu, Ping; Luo, Chunyu; Ji, Qiuqing; Zhou, Jing; Liu, An; Ma, Dingyuan; Xu, Zhengfeng

    2014-02-01

    To explore the value of multiplex ligation-dependent probe amplification (MLPA) for rapid detection of aneuploidies and structural chromosomal abnormalities during prenatal diagnosis. Two hundred and eight six amniotic fluid samples were analyzed with both MLPA and conventional karyotyping. Structural abnormalities were verified with array comparative genomic hybridization. Ten cases of trisomy 21, 2 cases of trisomy 18, 1 case of trisomy 13, 1 case of mosaic trisomy 21, 1 case of 45,X, 1 case of large deletion of Xp, 1 case of trisomy 18p and 1 case of large deletion of 18p and 18q were identified. The same results were derived by both MLPA and conventional karyotyping. Structural abnormalities were verified by array comparative genomic hybridization (aCGH) with 100% accuracy. In addition to aneuploidies, MLPA can rapidly identify large deletions and duplications of chromosomes 21, 18, 13, X and Y. MLPA is supplementary to conventional karyotyping for identification of such chromosomal abnormalities prenatal diagnosis.

  13. Rapid spectro-polarimetry to probe molecular symmetry in multiplex coherent anti-Stokes Raman scattering.

    Science.gov (United States)

    Würthwein, Thomas; Brinkmann, Maximilian; Hellwig, Tim; Fallnich, Carsten

    2017-11-21

    We present the simultaneous detection of the spectrum and the complete polarization state of a multiplex coherent anti-Stokes Raman scattering signal with a fast division-of-amplitude spectro-polarimeter. The spectro-polarimeter is based on a commercial imaging spectrograph, a birefringent wedge prism, and a segmented polarizer. Compared to the standard rotating-retarder fixed-analyzer spectro-polarimeter, only a single measurement is required and an up to 21-fold reduced acquisition time is shown. The measured Stokes parameters allow us to differentiate between vibrational symmetries and to determine the depolarization ratio ρ by data post-processing.

  14. Rapid screening of pyogenic Staphylococcus aureus for confirmation of genus and species, methicillin resistance and virulence factors by using two novel multiplex PCR.

    Science.gov (United States)

    Haque, Abdul; Haque, Asma; Saeed, Muhammad; Azhar, Aysha; Rasool, Samreen; Shan, Sidra; Ehsan, Beenish; Nisar, Zohaib

    2017-01-01

    Emergence of methicillin resistant Staphylococcus aureus (MRSA) is a major medical problem of current era. These bacteria are resistant to most drugs and rapid diagnosis can provide a clear guideline to clinicians. They possess specific virulence factors and relevant information can be very useful. We designed this study to develop multiplex PCRs to provide rapid information. We studied 60 Staphylococcus aureus isolates and detected methicillin resistance by cefoxitin sensitivity and targeting of mecA gene. After initial studies with uniplex PCRs we optimized two multiplex PCRs with highly reproducible results. The first multiplex PCR was developed to confirm genus, species and methicillin resistance simultaneously, and the second multiplex PCR was for screening of virulence factors. We found 38.33% isolates as methicillin resistant. α -toxin, the major cytotoxic factor, was detected in 40% whereas β-hemolysin was found in 25% cases. Panton Valentine leucocidin was detected in 8.33% and toxic shock syndrome toxin in5% cases. The results of uniplex and multiplex PCRs were highly compatible. These two multiplex PCRs when run simultaneously can provide vital information about methicillin resistance and virulence status of the isolate within a few hours as compared to several days needed by routine procedures.

  15. Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

    Energy Technology Data Exchange (ETDEWEB)

    Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

    2007-06-26

    A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

  16. Rapid genetically modified organism (GMO screening of various food products and animal feeds using multiplex polymerase chain reaction (PCR

    Directory of Open Access Journals (Sweden)

    Lisha, V.

    2017-01-01

    Full Text Available modified crops which brought up a controversy on the safety usage of genetically modified organisms (GMOs. It has been implemented globally that all GMO products and its derived ingredients should have regulations on the usage and labelling. Thus, it is necessary to develop methods that allow rapid screening of GMO products to comply with the regulations. This study employed a reliable and flexible multiplex polymerase chain reaction (PCR method for the rapid detection of transgenic elements in genetically modified soy and maize along with the soybean LECTIN gene and maize ZEIN gene respectively. The selected four common transgenic elements were 35S promoter (35S; Agrobacterium tumefaciens nopaline synthase terminator (NOS; 5-enolypyruvylshikimate-3-phosphate synthase (epsps gene; and Cry1Ab delta-endotoxin (cry1Ab gene. Optimization of the multiplex PCR methods were carried out by using 1% Roundup ReadyTM Soybean (RRS as the certified reference material for soybean that produced fourplex PCR method detecting 35S promoter, NOS terminator, epsps gene and soybean LECTIN gene and by using 1% MON810 as the certified reference material for maize that produced triplex PCR method detecting 35S promoter, cry1Ab gene and maize ZEIN gene prior to screening of the GMO traits in various food products and animal feeds. 1/9 (11.1% of the animal feed contained maize and 1/15 (6.7% of the soybean food products showed positive results for the detection of GMO transgenic gene. None of the maize food products showed positive results for GMO transgenic gene. In total, approximately 4% of the food products and animal feed were positive as GMO. This indicated GMOs have not widely entered the food chain. However, it is necessary to have an appropriate screening method due to GMOs’ unknown potential risk to humans and to animals. This rapid screening method will provide leverage in terms of being economically wise, time saving and reliable.

  17. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  18. Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

    Science.gov (United States)

    Hasanpour, Mojtaba; Najafi, Akram

    2017-06-01

    Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (T m ) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Qualitative analysis of meat and meat products by multiplex ...

    African Journals Online (AJOL)

    Specific-species primers, designed according to the conservative regions of 16S rRNA, were used after alignment of the available sequences in the GenBank database. The primers generated ... it was not indicated on the label. Key words: Multiplex polymerase chain reaction (M-PCR), meat products, food, salami, sausage.

  20. Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

    Science.gov (United States)

    Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

    2014-06-01

    Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

  1. [Evaluation of the efficacies of rapid antigen test, multiplex PCR, and real-time PCR for the detection of a novel influenza A (H1N1) virus].

    Science.gov (United States)

    Hwang, Yusun; Kim, Kyounghee; Lee, Miae

    2010-04-01

    In April 2009, a novel influenza A (H1N1) virus was detected in the US, and at the time of conducting this study, H1N1 infection had reached pandemic proportions. In Korea, rapid antigen tests and PCR assays have been developed to detect the H1N1 virus. We evaluated the efficacies of rapid antigen test, multiplex PCR, and real-time PCR for detecting the H1N1 virus. From August to September 2009, we tested 734 samples obtained from nasopharyngeal swab or nasal swab using rapid antigen test (SD Influenza Antigen, Standard Diagnostics, Inc., Korea) and multiplex PCR (Seeplex FluA ACE Subtyping, Seegene, Korea). We also tested 224 samples using the AdvanSure real-time PCR (LG Life Sciences, Korea) to compare the results obtained using real-time PCR with those obtained using multiplex PCR. Furthermore, 99 samples were tested using the AdvanSure real-time PCR and the AccuPower real-time PCR (Bioneer, Korea). In comparison with the results of multiplex PCR, the sensitivity and specificity of the rapid antigen test were 48.0% and 99.8%, respectively. The concordance rate for multiplex PCR and the AdvanSure real-time PCR was 99.6% (kappa=0.991, P=0.000), and that for the AdvanSure real-time PCR and the AccuPower real-time PCR was 97.0% (kappa=0.936, P=0.000). The rapid antigen test is significantly less sensitive than PCR assay; therefore, it is not useful for H1N1 detection; however multiplex PCR, the AdvanSure real-time PCR, and the Accu-Power real-time PCR can be useful for H1N1 detection.

  2. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array‐based multiplex assay

    DEFF Research Database (Denmark)

    Lievens, B.; Frans, I.; Heusdens, C.

    2011-01-01

    for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV‐1, CyHV‐2 and CyHV‐3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were......Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been...... one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad‐range PCR amplification combined with DNA array hybridization...

  3. Bridging online and offline social networks: Multiplex analysis

    Science.gov (United States)

    Filiposka, Sonja; Gajduk, Andrej; Dimitrova, Tamara; Kocarev, Ljupco

    2017-04-01

    We show that three basic actor characteristics, namely normalized reciprocity, three cycles, and triplets, can be expressed using an unified framework that is based on computing the similarity index between two sets associated with the actor: the set of her/his friends and the set of those considering her/him as a friend. These metrics are extended to multiplex networks and then computed for two friendship networks generated by collecting data from two groups of undergraduate students. We found that in offline communication strong and weak ties are (almost) equally presented, while in online communication weak ties are dominant. Moreover, weak ties are much less reciprocal than strong ties. However, across different layers of the multiplex network reciprocities are preserved, while triads (measured with normalized three cycles and triplets) are not significant.

  4. Analysis of subsystems in wavelength-division-multiplexing networks

    OpenAIRE

    Liu, Fenghai; Jeppesen, Palle; Pedersen, Rune S.J.

    2001-01-01

    Wavelength division multiplexing (WDM) technology together with optical amplification has created a new era for optical communication. Transmission capacity is greatly increased by adding more and more wavelength channels into a single fiber, as well as by increasing the line rate of each channel. WDM not only can be used to increase transmission capacity, but also to introduce a new dimension to design and implement flexible, reliable, cost effective optical networks. Optical signals may pas...

  5. A sensitive and a rapid multiplex polymerase chain reaction for the identification of Candida species in concentrated oral rinse specimens in patients with diabetes.

    Science.gov (United States)

    Sampath, Asanga; Weerasekera, Manjula; Gunasekara, Chinthika; Dilhari, Ayomi; Bulugahapitiya, Uditha; Fernando, Neluka

    2017-03-01

    Oral candidiasis is being frequently recognized in patients with diabetes, and is associated with multiple pathogens including Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis. The aim of this study was to evaluate a usefulness of a Multiplex Polymerase Chain Reaction as a rapid diagnostic tool for identification of four oral Candida pathogens in patients with diabetes. A multiplex PCR was optimized to identify four Candida species in concentrated oral rinse samples. Common reverse primer, ITS4 and four species-specific forward primers targeting ITS1 and ITS2 regions of yeast genome were used. Species-specific single amplicon were detected by agarose gel electrophoresis. Performance efficacy of multiplex PCR was compared with phenotypic identification. Out of 100 oral rinse samples, 72 were culture positive and of these 43 were at risk of oral Candida infection (>600cfu/ml). Multiple Candida species including C. albicans, C. parapsilosis and C. tropicalis were identified in 22 samples which had risk of oral Candida infection. In total, 85 patients were positive for Candida by multiplex PCR and of them 49 had multiple Candida species. All 43 colonized specimens were also positive by multiplex PCR. C. albicans was the most predominant organism (75/85) followed by C. parapsilosis (47/85), C. tropicalis (17/85) and C. glabrata (6/85). In specimens with multiple species, the two most common organisms were C. albicans and C. parapsilosis. Multiplex PCR yielded a sensitivity of 10 Candida cells/ml of oral rinse sample. Multiplex PCR is found to be rapid, sensitive and specific than phenotypic identification methods in discriminating multiple Candida species in oral rinse specimens.

  6. Rapid aneuploidy diagnosis by multiplex ligation-dependent probe amplification and array comparative genomic hybridization in pregnancy with major congenital malformations

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chen

    2011-03-01

    Conclusions: Prenatal diagnosis of major congenital malformations should alert one to the possibility of chromosomal abnormalities. Multiplex ligation-dependent probe amplification and aCGH have the advantage of rapid aneuploidy diagnosis of common aneuploidies in cases with major congenital malformations.

  7. Clinical impact of a commercially available multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis

    Directory of Open Access Journals (Sweden)

    Linde Hans-Jörg

    2009-08-01

    Full Text Available Abstract Background Timely identification of pathogens is crucial to minimize mortality in patients with severe infections. Detection of bacterial and fungal pathogens in blood by nucleic acid amplification promises to yield results faster than blood cultures (BC. We analyzed the clinical impact of a commercially available multiplex PCR system in patients with suspected sepsis. Methods Blood samples from patients with presumed sepsis were cultured with the Bactec 9240™ system (Becton Dickinson, Heidelberg, Germany and aliquots subjected to analysis with the LightCycler® SeptiFast® (SF Test (Roche Diagnostics, Mannheim, Germany at a tertiary care centre. For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the SF result, based on a fictive assumption that it was done either on a once or twice daily basis, was made. Results Of 101 blood samples from 77 patients, 63 (62% yielded concordant negative results, 14 (13% concordant positive and 9 (9% were BC positive only. In 14 (13% samples pathogens were detected by SF only, resulting in adjustment of antibiotic therapy in 5 patients (7,7% of patients. In 3 samples a treatment adjustment would have been made earlier resulting in a total of 8 adjustments in all 101 samples (8%. Conclusion The addition of multiplex PCR to conventional blood cultures had a relevant impact on clinical management for a subset of patients with presumed sepsis.

  8. Power analysis dataset for QCA based multiplexer circuits

    Directory of Open Access Journals (Sweden)

    Md. Abdullah-Al-Shafi

    2017-04-01

    Full Text Available Power consumption in irreversible QCA logic circuits is a vital and a major issue; however in the practical cases, this focus is mostly omitted.The complete power depletion dataset of different QCA multiplexers have been worked out in this paper. At −271.15 °C temperature, the depletion is evaluated under three separate tunneling energy levels. All the circuits are designed with QCADesigner, a broadly used simulation engine and QCAPro tool has been applied for estimating the power dissipation.

  9. Analysis of tolerances in polytopic-multiplexing holographic data storage.

    Science.gov (United States)

    Ide, Tatsuro

    2016-04-10

    A fast Fourier transformation (FFT)-based simulator was developed to analyze a recovered image of a hologram formed by polytopic multiplexing holographic data storage and to determine the positional tolerances of optical devices. By focusing the positional shift of 4f configuration devices (phase mask, SLM, polytopic filter, and camera), the number of FFT iterations was reduced, and the calculation speed was improved. Using the simulator, the positional tolerances of these devices were determined. Also, to validate the simulator, simulated readout images under defocuses of the phase mask and camera were compared with experimentally obtained images and found to agree well.

  10. Rapid sex identification of papaya (Carica papaya) using multiplex loop-mediated isothermal amplification (mLAMP).

    Science.gov (United States)

    Hsu, Te-Hua; Gwo, Jin-Chywan; Lin, Kuan-Hung

    2012-10-01

    Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (papaya production.

  11. Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks.

    Science.gov (United States)

    Wei, B; Cha, S-Y; Kang, M; Park, I-J; Moon, O-K; Park, C-K; Jang, H-K

    2013-05-01

    Infections with Pasteurella multocida, Salmonella enterica, Riemerella anatipestifer, and Escherichia coli result in high morbidity and mortality, which cause significant economic loss in the poultry industry. It can be difficult to distinguish these pathogens based on clinical signs because these pathogens can cause similar clinical signs and coinfections can occur. Thus, rapid and sensitive detection of these 4 major bacterial pathogens are important in ducks. The aim of this study was to develop a multiplex PCR (mPCR) assay for simultaneously detecting and identifying these 4 pathogenic bacteria in a single tube reaction. The target genes used were KMT1 of P. multocida, the invasion protein gene of S. enterica, 16S rDNA of R. anatipestifer, and the alkaline phosphatase gene of E. coli. The detection limit of the assay for all bacterial DNA was 10 pg. The mPCR did not produce any nonspecific amplification products when tested against other related pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, Mycoplasma gallinarum, Mycoplasma synoviae, and Mycoplasma gallisepticum, which can also infect ducks. We applied mPCR to field samples, and the results were the same as the single PCR results. These results suggest that mPCR for the 4 bacteria is a useful and rapid technique to apply to field samples.

  12. Reagent deposition for rapid multiplex pathogen identification in human blood culture samples

    DEFF Research Database (Denmark)

    Mogensen, Klaus Bo; Machado, Ana Manuel; Dufva, Martin

    2014-01-01

    viewed in a dual-color microscope configuration. The test takes 20-30 min to perform. In order to lower the cost of the test, rapid automated reagent deposition is needed. Here, ultrasonic spray coating of polyvinyl alcohol/PNA-probes on microscope glass slides is presented. Different wetting regimes...

  13. Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.

    Science.gov (United States)

    Tian, Huan; Cao, Liching; Tan, Yuping; Williams, Stephen; Chen, Lili; Matray, Tracy; Chenna, Ahmed; Moore, Sean; Hernandez, Vincent; Xiao, Vivian; Tang, Mengxiang; Singh, Sharat

    2004-09-08

    We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1beta. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise, as evaluated by measuring qualified Z' factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection and the isothermal linear amplification characteristics of the Invader assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.

  14. A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

    Science.gov (United States)

    Forghani, Fereidoun; Wei, Shuai; Oh, Deog-Hwan

    2016-05-01

    Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .

  15. A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA

    Directory of Open Access Journals (Sweden)

    Gonzalo J. Domingo

    2013-04-01

    Full Text Available This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays.

  16. Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis.

    Science.gov (United States)

    Webster, B L; Rollinson, D; Stothard, J R; Huyse, T

    2010-03-01

    Schistosoma haematobium and S. bovis are widespread schistosome species causing human and cattle schistosomiasis, respectively, in Africa. The sympatric occurrence of these two species and their ability to infect the same Bulinus intermediate snail hosts necessitates precise methods of identification of the larval stages. A rapid diagnostic 'mulitplex' one-step polymerase chain reaction protocol (RD-PCR) was developed using cytochrome oxidase subunit 1 (COX1) mitochondrial DNA (mtDNA) to discriminate between S. haematobium and S. bovis. A single forward primer and two species-specific reverse primers were used to produce a polymerase chain reaction (PCR) fragment of 306 bp and 543 bp for S. bovis and S. haematobium, respectively. Serial dilutions were carried out on various lifecycle stages and species combinations to test the sensitivity and specificity of the primers. This RD-PCR proved highly sensitive, detecting a single larval stage and as little as 0.78 ng of genomic DNA (gDNA) from an adult schistosome, providing a cost-effective, rapid and robust molecular tool for high-throughput screening of S. haematobium and S. bovis populations. In areas where human and cattle schistosomiasis overlap and are transmitted in close proximity, this mitochondrial assay will be a valuable identification tool for epidemiological studies, especially when used in conjunction with other nuclear diagnostic markers.

  17. Development of a multiplex PCR for the genetic analysis of paddlefish (Polyodon spathula Walbaum,1792 populations

    Directory of Open Access Journals (Sweden)

    K. Kurta

    2017-12-01

    Full Text Available Purpose. Paddlefish is commercially important species owing to its biological features and consumer characteristics, namely it produces valuable and delicious fish products, such as high quality meat and black caviar. Consequently, its cultivation under Ukrainian fish farm conditions and further realization in domestic and foreign markets are economically efficient. However, the paddlefish broodstock in Ukraine requires the efficient solution of increasing its productivity, identification and assessment of its genetic variation. Thus, the aim of our study was to develop and implement a multiplex PCR-analysis of paddlefish (Polyodon spathula for population-genetic monitoring of its artificial broodstocks in Ukraine. Methodology. A multiplex PCR was used for the study. The multiplex PCR development was performed for four microsatellite DNA markers: Psp12, Psp21, Psp26 and Psp28. Each investigated DNA loci, for which the multiplex PCR was optimized, was selected in such a way that the colored PCR products labeled with fluorescent dye did not overlap the length of the amplified fragments. Evaluation of the multiplex PCR effectiveness and processing of the data were performed by fragment analysis of DNA on the genetic analyzer ABI Prism 3130 (Applied Biosystem, USA. The size of the identified alleles was determined using the "Gene Mapper 3.7" program (Applied Biosystems, USA and LIZ-500 size standard (Applied Biosystems, USA. Results. Based on the results of capillary electrophoresis of multiplex PCR products, it was found that the amplified fragments for each of the four studied loci: Psp12, Psp21, Psp26 and Psp28 in one PCR reaction were within the expected size range. Data analysis on the electrophoregram demonstrated that Psp21 had the highest peak intensity at 611 fluorescent units (FU and the lowest peak intensity at 105 FU was observed for Psp26 locus. In the multiplex PCR after proper interpretation of the data we identified heterozygous

  18. Multiplex analysis of tumor multidrug-resistance genes expression with photonic suspension array.

    Science.gov (United States)

    Yang, Zixue; Chen, Baoan; Pei, Xiaoping; Shangguan, Fengqi

    2012-07-21

    An easy-operated suspension array based on silica colloidal crystal beads is developed for multiplex analysis of tumor multidrug-resistance genes expression, such as multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1), and potentially single nucleotide polymorphism. In order to obtain high fluorescence intensity, controlled PCR was used to amplify targets at the samples pretreatment stage. By optimizing the conditions a hybridization procedure, which is similar to nucleic acids analysis with binary probes, was established. Small amounts of analytes 10(-19) M could be detected by the method. The K562 cell, human myeloma cell, and its multidrug-resistance string, adriamycin-selected P-glycoprotein-overexpressed K562/A02, were analyzed by using an established procedure to validate feasibility. Clinical blood samples were detected by our method and real-time PCR simultaneously to validate accuracy. Moreover, when combined with multiplex controlled PCR, the method successfully meets the requirements of multiplex analysis. Hence, the method presented is a good method for multiplex analysis of tumor multidrug-resistance genes expression.

  19. Improved Multiplex Polymerase Chain Reaction for Rapid Staphylococcus Aureus Detection in Meat and Milk Matrices

    Directory of Open Access Journals (Sweden)

    Šramková Zuzana

    2016-06-01

    Full Text Available Staphylococcal food poisoning represents one of the most frequently occurring intoxications, caused by staphylococcal enterotoxins (SE-s and staphylococcal enterotoxin-like proteins (SEl-s. Therefore, there is a need for rapid, sensitive and specific detection method for this human pathogen and its toxin genes in food matrices. The present work is focused on Staphylococcus aureus detection by a nonaplex polymerase chain reaction, which targets the 23S rRNA gene for identification of S. aureus at the species level, genes for classical SE-s (SEA, SEC, SED, new SE-s (SEH, SEI, SEl-s (SEK, SEL and tsst-1 gene (toxic shock syndrome toxin. Primers were properly designed to avoid undesirable interactions and to create a reliably identifiable profile of amplicons when visualized in agarose gel. According to obtained results, this approach is able to reach the detection sensitivity of 12 colony forming units from milk and meat matrices without prior culturing and DNA extraction.

  20. Analysis of complex contagions in random multiplex networks

    CERN Document Server

    Yagan, Osman

    2012-01-01

    We study the diffusion of influence in random multiplex networks where links can be of $r$ different types, and for a given content (e.g., rumor, product, political view), each link type is associated with a content dependent parameter $c_i$ in $[0,\\infty]$ that measures the relative bias type-$i$ links have in spreading this content. In this setting, we propose a linear threshold model of contagion where nodes switch state if their "perceived" proportion of active neighbors exceeds a threshold \\tau. Namely, a node connected to $m_i$ active neighbors and $k_i-m_i$ inactive neighbors via type-$i$ links will turn active if $\\sum{c_i m_i}/\\sum{c_i k_i}$ exceeds its threshold \\tau. Under this model, we obtain the condition, probability and expected size of global spreading events. Our results extend the existing work on complex contagions in several directions by i) providing solutions for coupled random networks whose vertices are neither identical nor disjoint, (ii) highlighting the effect of content on the dyn...

  1. Oncogenic human papillomavirus genotyping by multiplex PCR and fragment analysis.

    Science.gov (United States)

    Souho, Tiatou; Bennani, Bahia

    2014-02-01

    Human papillomavirus (HPV) detection and genotyping are determinant in cervical cancer prevention. They help to identify women at risk and allow the establishment of epidemiologic profiles. Many PCR-based genotyping methods have been developed. They require many steps or specialized equipment increasing the assay duration and cost. This affects their routine use, especially in developing countries. Therefore, the aim of this study was to develop a new HPV genotyping method that can be routinely used also in low incomes countries. For this purpose, fifteen high risk HPV type specific reverse primers were designed on L1 gene and fluorescently labeled. These primers were used on two multiplex PCR with one common forward primer (MY11). The lengths of products were revealed by capillary electrophoresis. This technique identifies sixteen high risk HPVs (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 68, 73 and 82). It was optimized on HPV genome plasmids and evaluated on artificial and cervical samples. All the sixteen targeted genotypes were identified specifically and repeatedly in simple and multiple infections in both artificial and clinical samples. The developed technique is sensitive, specific, easy to perform and appropriate for routine laboratory use and high throughput screening programs. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis

    Directory of Open Access Journals (Sweden)

    Tu Jing

    2012-01-01

    Full Text Available Abstract Background The multiplexing becomes the major limitation of the next-generation sequencing (NGS in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Results Here we introduce pair-barcode sequencing (PBS, an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc., 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. Conclusions By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.

  3. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

    OpenAIRE

    fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

    2014-01-01

    Objective: To analyse molecular detection of coliforms and shorten the time of PCR. Methods: Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated...

  4. Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR.

    Science.gov (United States)

    Guion, Chase E; Ochoa, Theresa J; Walker, Christopher M; Barletta, Francesca; Cleary, Thomas G

    2008-05-01

    Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.

  5. Detection of Diarrheagenic Escherichia coli by Use of Melting-Curve Analysis and Real-Time Multiplex PCR ▿

    Science.gov (United States)

    Guion, Chase E.; Ochoa, Theresa J.; Walker, Christopher M.; Barletta, Francesca; Cleary, Thomas G.

    2008-01-01

    Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx1 and stx2 for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli. PMID:18322059

  6. Rapid multiplex high resolution melting method to analyze inflammatory related SNPs in preterm birth

    Directory of Open Access Journals (Sweden)

    Pereyra Silvana

    2012-01-01

    Full Text Available Abstract Background Complex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM analysis is a new method in which polymerase chain reaction (PCR and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB. Methods Here, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism. Results We were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB. Conclusions Several gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing

  7. A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis.

    Directory of Open Access Journals (Sweden)

    Wentao Xu

    Full Text Available In this study, a novel universal primer-multiplex-PCR (UP-M-PCR method adding a universal primer (UP in the multiplex PCR reaction system was described. A universal adapter was designed in the 5'-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP. PCR products were analyzed on sequencing gel electrophoresis (SGE which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on.

  8. A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

    Science.gov (United States)

    Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

    2012-01-01

    In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on. PMID:22272223

  9. The Microwave SQUID Multiplexer

    Science.gov (United States)

    Mates, John Arthur Benson

    2011-12-01

    This thesis describes a multiplexer of Superconducting Quantum Interference Devices (SQUIDs) with low-noise, ultra-low power dissipation, and great scalability. The multiplexer circuit measures the magnetic flux in a large number of unshunted rf SQUIDs by coupling each SQUID to a superconducting microwave resonator tuned to a unique resonance frequency and driving the resonators from a common feedline. A superposition of microwave tones measures each SQUID simultaneously using only two coaxial cables between the cryogenic device and room temperature. This multiplexer will enable the instrumentation of arrays with hundreds of thousands of low-temperature detectors for new applications in cosmology, materials analysis, and nuclear non-proliferation. The driving application of the Microwave SQUID Multiplexer is the readout of large arrays of superconducting transition-edge sensors, by some figures of merit the most sensitive detectors of electromagnetic signals over a span of more than nine orders of magnitude in energy, from 40 GHz microwaves to 200 keV gamma rays. Modern transition-edge sensors have noise-equivalent power as low as 10-20 W / Hz1/2 and energy resolution as good as 2 eV at 6 keV. These per-pixel sensitivities approach theoretical limits set by the underlying signals, motivating a rapid increase in pixel count to access new science. Compelling applications, like the non-destructive assay of nuclear material for treaty verification or the search for primordial gravity waves from inflation use arrays of these detectors to increase collection area or tile a focal plane. We developed three generations of SQUID multiplexers, optimizing the first for flux noise 0.17 muPhi0 / Hz1/2, the second for input current noise 19 pA / Hz1/2, and the last for practical multiplexing of large arrays of cosmic microwave background polarimeters based on transition-edge sensors. Using the last design we demonstrated multiplexed readout of prototype polarimeters with the

  10. A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

    Science.gov (United States)

    Xu, Benjin; Liu, Ling; Liu, Li; Li, Xinping; Li, Xiaofang; Wang, Xin

    2012-11-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA. © 2012 Institute of Food Technologists®

  11. Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

    Science.gov (United States)

    Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

    2015-02-01

    Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

  12. Phenylketonuria mutation analysis in Northern Ireland: A rapid stepwise approach

    Energy Technology Data Exchange (ETDEWEB)

    Zschocke, J.; Graham, C.A.; Nevin, N.C. [Queen`s Univ., Belfast (Australia)] [and others

    1995-12-01

    We present a multistep approach for the rapid analysis of phenylketonuria (PKU) mutations. In the first step, three common mutations and a polymorphic short tandem repeat (STR) system are rapidly analyzed with a fluorescent multiplex assay. In the second step, minihaplotypes combining STR and VNTR data are used to determine rare mutations likely to be present in an investigated patient, which are then confirmed by restriction enzyme analysis. The remaining mutations are analyzed with denaturant gradient-gel electrophoresis and sequencing. The first two steps together identify both mutations in 90%-95% of PKU patients, and results can be obtained within 2 d. We have investigated 121 Northern Irish families with hyperphenylalaninemia, including virtually all patients born since 1972, and have found 34 different mutations on 241 of the 242 mutant alleles. Three mutations (R408W, 165T, and F39L) account for 57.5% of mutations, while 14 mutations occur with a frequency of 1%-6%. The present analysis system is efficient and inexpensive and is particularly well suited to routine mutation analysis in a diagnostic setting. 19 refs., 5 tabs.

  13. Validation of microsatellite multiplexes for parentage analysis in a coral reef fish (Lutjanus carponotatus, Lutjanidae)

    KAUST Repository

    Harrison, Hugo B.

    2014-05-25

    Parentage analysis is an important tool for identifying connectivity patterns in coral reef fishes, but often requires numerous highly polymorphic markers. We isolated 21 polymorphic microsatellite markers from the stripey snapper, Lutjanus carponotatus and describe their integration into three multiplex PCRs. All markers were highly polymorphic with a mean of 24.9 ± 1.8 SE alleles per locus and an average observed heterozygosity of 0.797 ± 0.038 SE across 285 genotyped individuals. Using a simulated dataset, we conclude that the complete marker set provides sufficient resolution to resolve parent–offspring relationships in natural populations with 99.6 ± 0.1 % accuracy in parentage assignments. This multiplex assay provides an effective means of investigating larval dispersal and population connectivity in this fishery-targeted coral reef fish species and informing the design of marine protected area networks for biodiversity conservation and fisheries management.

  14. Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

    Science.gov (United States)

    Gopaul, Krishna K; Sells, Jessica; Lee, Robin; Beckstrom-Sternberg, Stephen M; Foster, Jeffrey T; Whatmore, Adrian M

    2014-12-11

    The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

  15. Cytokines profiling by multiplex analysis in experimental arthritis: which pathophysiological relevance for articular versus systemic mediators?

    Science.gov (United States)

    Paquet, Joseph; Goebel, Jean-Christophe; Delaunay, Camille; Pinzano, Astrid; Grossin, Laurent; Cournil-Henrionnet, Christel; Gillet, Pierre; Netter, Patrick; Jouzeau, Jean-Yves; Moulin, David

    2012-03-13

    We have taken advantage of the large screening capacity of a multiplex immunoassay to better define the respective contribution of articular versus systemic cytokines in experimental arthritis. We performed a follow up (from 7 hours to 14 days) multiplex analysis of 24 cytokines in synovial fluid and sera of rats developing Antigen-Induced Arthritis (AIA) and confronted their protein level changes with molecular, biochemical, histological and clinical events occurring in the course of the disease. The time-scheduled findings in arthritic joints correlated with time-dependent changes of cytokine amounts in joint effusions but not with their blood levels. From seven hours after sensitization, high levels of chemokines (MCP-1, MIP1α, GRO/KC, RANTES, eotaxin) were found in synovial fluid of arthritic knees whereas perivascular infiltration occurred in the synovium; local release of inflammatory cytokines (IFNγ, IL-1β, IL-6) preceded the spreading of inflammation and resulted in progressive degradation of cartilage and bone. Finally a local overexpression of several cytokines/adipocytokines poorly described in arthritis (IL-13, IL-18, leptin) was observed. Distinct panels of cytokines were found in arthritic fluid during AIA, and the expected effect of mediators correlated well with changes occurring in joint tissues. Moreover, multiplex analysis could be helpful to identify new pathogenic mediators and to elucidate the mechanisms supporting the efficacy of putative targeted therapies.

  16. DNA from buccal swabs suitable for high-throughput SNP multiplex analysis.

    Science.gov (United States)

    McMichael, Gai L; Gibson, Catherine S; O'Callaghan, Michael E; Goldwater, Paul N; Dekker, Gustaaf A; Haan, Eric A; MacLennan, Alastair H

    2009-12-01

    We sought a convenient and reliable method for collection of genetic material that is inexpensive and noninvasive and suitable for self-collection and mailing and a compatible, commercial DNA extraction protocol to meet quantitative and qualitative requirements for high-throughput single nucleotide polymorphism (SNP) multiplex analysis on an automated platform. Buccal swabs were collected from 34 individuals as part of a pilot study to test commercially available buccal swabs and DNA extraction kits. DNA was quantified on a spectrofluorometer with Picogreen dsDNA prior to testing the DNA integrity with predesigned SNP multiplex assays. Based on the pilot study results, the Catch-All swabs and Isohelix buccal DNA isolation kit were selected for our high-throughput application and extended to a further 1140 samples as part of a large cohort study. The average DNA yield in the pilot study (n=34) was 1.94 microg +/- 0.54 with a 94% genotyping pass rate. For the high-throughput application (n=1140), the average DNA yield was 2.44 microg +/- 1.74 with a >or=93% genotyping pass rate. The Catch-All buccal swabs are a convenient and cost-effective alternative to blood sampling. Combined with the Isohelix buccal DNA isolation kit, they provided DNA of sufficient quantity and quality for high-throughput SNP multiplex analysis.

  17. Rapid detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in food, using multiplex real-time PCR.

    Science.gov (United States)

    Mayr, A M; Lick, S; Bauer, J; Thärigen, D; Busch, U; Huber, I

    2010-02-01

    A multiplex real-time PCR assay based on four differently labeled TaqMan probes for detection and differentiation of the thermophilic Campylobacter species C. jejuni, C. coli, and C. lari was established and validated in food products. This assay combines two previously published PCR assays for C. jejuni and C. coli with a newly developed detection assay for C. lari and an internal amplification control system. The selectivity of the method was determined by analyzing 70 Campylobacter strains and 43 strains of other bacteria. The sensitivity was 50 fg of C. jejuni and C. lari DNA and 500 fg of C. coli DNA per PCR. It was possible to detect 1 to 10 CFU/25 g of food before preenrichment of all three species. More than 400 samples of various foods (poultry, seafood, and meat) were analyzed after 48 h of preenrichment parallel to the conventional diagnostic method of culture and biochemical identification. Using the established real-time PCR assay, 55.4% of the samples were recognized as positive for thermophilic Campylobacter species, whereas with the conventional method only 40.3% of the samples were positive. The real-time PCR assay also detected contaminations with two different Campylobacter species in 32.6% of the analyzed poultry samples, a finding of epidemiological interest. Compared with the original PCR method, which was established for the differentiation of bacterial isolates of C. jejuni and C. coli, this new method also detects and distinguishes C. lari, was validated as an analytical tool for food analysis, and provides reliable and extensive results within 2 days.

  18. Development of a multiplex real-time PCR assay for the rapid diagnosis of neonatal late onset sepsis.

    Science.gov (United States)

    van den Brand, Marre; Peters, Remco P H; Catsburg, Arnold; Rubenjan, Anna; Broeke, Ferdi J; van den Dungen, Frank A M; van Weissenbruch, Mirjam M; van Furth, A Marceline; Kõressaar, Triinu; Remm, Maido; Savelkoul, Paul H M; Bos, Martine P

    2014-11-01

    The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

    Science.gov (United States)

    Fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

    2014-01-01

    Objective To analyse molecular detection of coliforms and shorten the time of PCR. Methods Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Conclusions Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN. PMID:25182727

  20. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number).

    Science.gov (United States)

    Fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

    2014-05-01

    To analyse molecular detection of coliforms and shorten the time of PCR. Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN.

  1. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

    Directory of Open Access Journals (Sweden)

    Qiuying Huang

    2011-04-01

    Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

  2. Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, L.S.; Hoffman, E.P. (Univ. of Pittsburgh Schoool of Medicine, Pittsburgh, PA (United States)); Tarleton, J. (Self Memorial Hospital, Greenwood, SC (United States)); Popovich, B. (Children' s Hosptial and Health Center, San Diego, CA (United States)); Seltzer, W.K. (Univ. of Colorado Health Sciences Center, Denver, CO (United States))

    1992-10-01

    The authors have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)[sub n] repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. The authors present the successful application of this protocol in families who proved refractory to more traditional analyses. 22 refs., 3 figs.

  3. Comparison of molecular and extract-based allergy diagnostics with multiplex and singleplex analysis.

    Science.gov (United States)

    Huss-Marp, Johannes; Gutermuth, Jan; Schäffner, Ina; Darsow, Ulf; Pfab, Florian; Brockow, Knut; Ring, Johannes; Behrendt, Heidrun; Jakob, Thilo; Ahlgrim, Christoph

    ImmunoCAP ISAC 112, is a commercially available molecular allergy IgE multiplex test. Data on the comparison of this rather novel test with extract-based as well as molecular ImmunoCAP singleplex IgE tests is missing. To perform a comparison between the ISAC multiplex IgE assay and the ImmunoCAP singleplex test results. Serum samples of 101 adults with grass pollen allergy were analysed for sIgE to 112 allergenic molecules represented on the ISAC test as well as to common atopy-related extract-based allergy tests with the ImmunoCAP System (house dust mite [d1], cat [e1], dog [e5], cow's milk [f2], hen's egg [f1], hazelnut [f17], celery [f85], Alternaria alternate [m6], as well as pollen from birch [t3], hazel [t4], mugwort [w6], and ragweed [w1]). Subsequently statistical analysis was performed with the Spearman rank correlation test and the Clopper-Pearson method in order to compare the ISAC multiplex results with the sIgE singleplex results. The positive percent agreements (PPA) and negative percent agreement (NPA) of corresponding allergens between the ISAC sIgE test and the extract-based singleplex ImmunoCAP results at cutoff 0.1 kUA/l varied between 60-100 % for PPA and 78-97 % for NPA. When taking into account corresponding allergens molecular testing with the ISAC multiplex test correlates well with ImmunoCAP singleplex results.

  4. An experimental study for rapid detection and quantification of endodontic microbiota following photo-activated disinfection via new multiplex real-time PCR assay.

    Science.gov (United States)

    Pourhajibagher, Maryam; Raoofian, Reza; Ghorbanzadeh, Roghayeh; Bahador, Abbas

    2018-01-11

    The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas. gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively. Copyright © 2018. Published by Elsevier B.V.

  5. Millstone: software for multiplex microbial genome analysis and engineering.

    Science.gov (United States)

    Goodman, Daniel B; Kuznetsov, Gleb; Lajoie, Marc J; Ahern, Brian W; Napolitano, Michael G; Chen, Kevin Y; Chen, Changping; Church, George M

    2017-05-25

    Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

  6. Analysis of multiplex gene expression maps obtained by voxelation

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    Smith Desmond J

    2009-04-01

    Full Text Available Abstract Background Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. Results To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in

  7. Simultaneous Screening of 24 Target Genes of Foodborne Pathogens in 35 Foodborne Outbreaks Using Multiplex Real-Time SYBR Green PCR Analysis

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    Hiroshi Fukushima

    2010-01-01

    Full Text Available A set of 8 multiplex real-time SYBR Green PCR (SG-PCR assays including 3 target primers and an internal amplification control (IAC primer was simultaneously evaluated in 3 h or less with regard to detection of 24 target genes of 23 foodborne pathogens in 7 stool specimens of foodborne outbreak using a 96-well reaction plate. This assay, combined with DNA extraction (QIAamp DNA Stool Mini kit, offered detection of greater than 103-104 foodborne pathogens per g in stool specimens. The products formed were identified using melting point temperature (Tm curve analysis. This assay was evaluated for the detection of foodborne pathogens in 33 out of 35 cases of foodborne outbreak, using 4 different PCR instruments in 5 different laboratories. No interference from the multiplex real-time SG-PCR assay, including IAC, was observed in stool specimens in any analysis. We found multiplex real-time SG-PCR assay for simultaneous detection of 24 target genes of foodborne pathogens to be comprehensive, rapid, inexpensive, accurate, of high selectivity, and good for screening probability.

  8. Development of a simultaneous identification method for 13 animal species using two multiplex real-time PCR assays and melting curve analysis.

    Science.gov (United States)

    Ishida, Natsumi; Sakurada, Makoto; Kusunoki, Hiroshi; Ueno, Yasuhiro

    2017-11-26

    We developed a simple and rapid method for animal species identification in the forensic science field based on mitochondrial DNA using two multiplex real-time PCRs and analysis of the resultant SYBR Green I melting curves. This method was designed to identify nine domestic animals simultaneously (dog, cat, rabbit, cattle, pig, chicken, goat, sheep and horse) and four wild animals (deer, raccoon-dog, monkey and bear) by comparing the different melting temperatures of the amplicons produced from samples originating from each species. For this analysis, we targeted various mitochondrial genes, including those encoding cytochrome b (cytb), NADH dehydrogenase 5 (ND5), cytochrome c oxidase 3 (COX3), tRNA-ND5, and tRNA-ATP synthase 8 (ATP8). For practical applications, this study presents a validation of this assay including its specificity, sensitivity and robustness. The limits of detection in the multiplex reactions were 10 pg for eight of the nine animals, excluding horse (1 pg for horse). The method was able to correctly identify the animal species from artificial forensic samples including blood stains, saliva, hair and bone, and samples digested in artificial gastric fluid, and for 17 forensic casework samples. The data from the multiplex real-time PCR assays are obtainable only 30 min after DNA extraction of the samples, making the assays useful for screening samples containing DNA from unknown animal origin in the forensic field. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Rapid Detection of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in Fresh Chicken Meat and By-Products in Bangkok, Thailand, Using Modified Multiplex PCR.

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    Saiyudthong, S; Phusri, K; Buates, S

    2015-07-01

    A multiplex PCR assay for simultaneous detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari was developed and validated to assess the occurrence of these bacteria in fresh chicken meat and by-products in Bangkok, Thailand, by using a new combination of four previously published PCR primers for C. jejuni, C. coli, C. lari, and a universal 16S rDNA gene as an internal control. The specificity was determined by using 13 strains of other bacteria. With pure culture DNA, the detection limit was 0.017 ng/PCR for C. jejuni and C. coli and was 0.016 ng/PCR for C. lari. It can detect 10 CFU of C. jejuni, C. coli, and C. lari in 2 g of chicken meat within a 16-h enrichment time. Our multiplex PCR assay was applied for identification of Campylobacter spp. in 122 supermarket samples and 108 fresh market samples. Of the 230 samples evaluated by multiplex PCR, 54.0, 3.3, and 10.7% of supermarket samples were positive for C. jejuni, C. coli, and mixed C. jejuni and C. coli, respectively, and 56.5 and 33.3% of fresh market samples were positive for C. jejuni and mixed C. jejuni and C. coli, respectively. No sample was positive for C. lari. Fresh market samples had significantly higher C. jejuni and C. coli contamination than those from supermarkets (relative risk: 1.3; P = 0.0001). Compared with the culture method (a gold standard), the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of multiplex PCR were 97.7, 86.8, 96.1, 92.0, and 95.2%, respectively. No significant difference was observed between results from two methods (P = 0.55). Therefore, the established multiplex PCR was not only rapid and easy to perform but had a high sensitivity and specificity to distinguish between C. jejuni, C. coli, and C. lari, even in samples containing mixed contamination. Our study indicated that fresh chicken meat and by-products from fresh markets were significantly less hygienic than those

  10. MULTIPLEX ANALYSIS OF BIOMARKERS OF NEOANGIOGENESIS AND INFLAMMATION IN HEART TRANSPLANT RECIPIENTS

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    O. P. Shevchenko

    2015-01-01

    Full Text Available Aim of study: multiplex analysis of the levels of biomarkers of neoangiogenesis and inflammation in cardiac transplant recipients. Materials and methods. 59 pts. with heart failure III–IV according to NYHA FC, waiting for a heart transplant, aged 22 to 73 years, 48 males and 11 females. 41 recipient (30 men and 11 women had dilated cardiomyopathy, 18 – coronary heart disease (CHD. The concentration of VEGF-A, VEGF-D, PlGF, PDGF-BB, FGF, sCD40L, MCP-1 was measured using xMAP technology, the sets of reagents Simplex ProcartaPlexTM (Affymetrix, USA. Results. There are four levels of seven biomarkers of neoangiogenesis and inflammation method for multiplex analysis in patients with heart failure. A year after transplantation, the mean levels of biomarkers VEGF-A (p = 0.001, PDGF-BB (p = 0.018, MCP-1 (p = 0.003 was significantly decreased, and the others had a tendency to decrease relative to the level before transplantation. It was shown individual differences of levels of VEGF-A, VEGF-D and PlGF before and after transplantation. There were found different dynamics of the concentrations of biomarkers and growth factors before and after heart transplantation in patients with cardiovascular complications and without them. Conclusion. Multiplex analysis allows to measure the concentration range of analyte biomarkers of neoangiogenesis, inflammation in one sample of blood serum of patients with severe heart failure and after transplantation. There are marked individual differences in the concentration of biomarkers in different clinical situations that may have clinical significance in the conduct and supervision of recipients after transplantation.

  11. Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

    Science.gov (United States)

    Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A

    2015-01-01

    Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

  12. Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

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    Banya Banowary

    Full Text Available Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR and high resolution melt (HRM curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO of C. jejuni and putative aspartokinase (asp gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours technique for differentiation between C. jejuni and C. coli isolates.

  13. Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

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    Ullal, Adeeti V; Weissleder, Ralph

    2015-01-01

    We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

  14. Systems pathology by multiplexed immunohistochemistry and whole-slide digital image analysis.

    Science.gov (United States)

    Blom, Sami; Paavolainen, Lassi; Bychkov, Dmitrii; Turkki, Riku; Mäki-Teeri, Petra; Hemmes, Annabrita; Välimäki, Katja; Lundin, Johan; Kallioniemi, Olli; Pellinen, Teijo

    2017-11-14

    The paradigm of molecular histopathology is shifting from a single-marker immunohistochemistry towards multiplexed detection of markers to better understand the complex pathological processes. However, there are no systems allowing multiplexed IHC (mIHC) with high-resolution whole-slide tissue imaging and analysis, yet providing feasible throughput for routine use. We present an mIHC platform combining fluorescent and chromogenic staining with automated whole-slide imaging and integrated whole-slide image analysis, enabling simultaneous detection of six protein markers and nuclei, and automatic quantification and classification of hundreds of thousands of cells in situ in formalin-fixed paraffin-embedded tissues. In the first proof-of-concept, we detected immune cells at cell-level resolution (n = 128,894 cells) in human prostate cancer, and analysed T cell subpopulations in different tumour compartments (epithelium vs. stroma). In the second proof-of-concept, we demonstrated an automatic classification of epithelial cell populations (n = 83,558) and glands (benign vs. cancer) in prostate cancer with simultaneous analysis of androgen receptor (AR) and alpha-methylacyl-CoA (AMACR) expression at cell-level resolution. We conclude that the open-source combination of 8-plex mIHC detection, whole-slide image acquisition and analysis provides a robust tool allowing quantitative, spatially resolved whole-slide tissue cytometry directly in formalin-fixed human tumour tissues for improved characterization of histology and the tumour microenvironment.

  15. Multiplex PCR assay for direct identification of group B streptococcal alpha-protein-like protein genes.

    Science.gov (United States)

    Creti, Roberta; Fabretti, Francesca; Orefici, Graziella; von Hunolstein, Christina

    2004-03-01

    We developed a group B streptococcus multiplex PCR assay which allows, by direct analysis of the amplicon size, determination of the surface protein antigen genes of alpha-C protein, epsilon protein, Rib, Alp2, Alp3, and Alp4. The multiplex PCR assay offers a rapid and simple method of subtyping Streptococcus agalactiae based on surface protein genes.

  16. C6 Peptide-Based Multiplex Phosphorescence Analysis (PHOSPHAN) for Serologic Confirmation of Lyme Borreliosis.

    Science.gov (United States)

    Pomelova, Vera G; Korenberg, Edward I; Kuznetsova, Tatiana I; Bychenkova, Tatiana A; Bekman, Natalya I; Osin, Nikolay S

    2015-01-01

    A single-tier immunoassay using the C6 peptide of VlsE (C6) from Borrelia burgdorferi sensu stricto (Bb) has been proposed as a potential alternative to conventional two-tier testing for the serologic diagnosis of Lyme disease in the United States and Europe. To evaluate the performance of C6 peptide based multiplex Phosphorescence Analysis (PHOSPHAN) for the serologic confirmation of Lyme borreliosis (LB) in Russian patients. Serum samples (n = 351) were collected from 146 patients with erythema migrans (EM); samples from 131 of these patients were taken several times prior to treatment and at different stages of recovery. The control group consisted of 197 healthy blood donors and 31 patients with other diseases, all from the same highly endemic region of Russia. All samples were analyzed by PHOSPHAN for IgM and IgG to Bb C6, recombinant OspC and VlsE proteins, and C6 peptides from B. garinii and B. afzelii. IgM and IgG to Bb C6 were identified in 43 and 95 out of 131 patients (32.8 and 72.5%, respectively); seroconversion of IgM antibodies was observed in about half of the patients (51.2%), and of IgG antibodies, in almost all of them (88.4%). Additional detection of OspC-IgM and VlsE-IgM or IgG to C6 from B. garinii or B. afzelii did not contribute significantly to the overall sensitivity of the multiplex immunoassay. The multiplex phosphorescence immunoassay is a promising method for simultaneously revealing the spectrum of antibodies to several Borrelia antigens. Detection of IgM and IgG to Bb C6 in the sera of EM patients provides effective serologic confirmation of LB and, with high probability, indicates an active infection process.

  17. Metal Affinity-Enabled Capture and Release Antibody Reagents Generate a Multiplex Biomarker Enrichment System that Improves Detection Limits of Rapid Diagnostic Tests.

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    Bauer, Westley S; Gulka, Christopher P; Silva-Baucage, Lidalee; Adams, Nicholas M; Haselton, Frederick R; Wright, David W

    2017-10-03

    Multi-antigen rapid diagnostic tests (RDTs) are highly informative, simple, mobile, and inexpensive, making them valuable point-of-care (POC) diagnostic tools. However, these RDTs suffer from several technical limitations-the most significant being the failure to detect low levels of infection. To overcome this, we have developed a magnetic bead-based multiplex biomarker enrichment strategy that combines metal affinity and immunospecific capture to purify and enrich multiple target biomarkers. Modifying antibodies to contain histidine-rich peptides enables reversible loading onto immobilized metal affinity magnetic beads, generating a novel class of antibodies coined "Capture and Release" (CaR) antibody reagents. This approach extends the specificity of immunocapture to metal affinity magnetic beads while also maintaining a common trigger for releasing multiple biomarkers. Multiplex biomarker enrichment is accomplished by adding magnetic beads equipped with CaR antibody reagents to a large sample volume to capture biomarkers of interest. Once captured, these biomarkers are magnetically purified, concentrated, and released into a RDT-compatible volume. This system was tailored to enhance a popular dual-antigen lateral flow malaria RDT that targets Plasmodium falciparum histidine-rich protein-II (HRPII) and Plasmodium lactate dehydrogenase (pLDH). A suite of pLDH CaR antibody reagents were synthesized, characterized, and the optimal CaR antibody reagent was loaded onto magnetic beads to make a multiplex magnetic capture bead that simultaneously enriches pLDH and HRPII from Plasmodium falciparum parasitized blood samples. This system achieves a 17.5-fold improvement in the dual positive HRPII/pan-pLDH detection limits enabling visual detection of both antigens at levels correlating to 5 p/μL. This front-end sample processing system serves as an efficient strategy to improve the sensitivity of RDTs without the need for modifications or remanufacturing.

  18. Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses.

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    Zhujun Zhang

    Full Text Available Avian influenza virus (AIV can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.

  19. Using multiplex PCR amplification and typing kits for the analysis of DNA evidence in a serial killer case.

    Science.gov (United States)

    Hochmeister, M N; Budowle, B; Eisenberg, A; Borer, U V; Dirnhofer, R

    1996-01-01

    Analysis of DNA evidence in a serial killer case was performed using the AmpliType HLA-DQ alpha-, AmpliType PM-, and the GenePrint STR Multiplex System PCR Amplification Kits. In addition, a sex typing procedure using the X-Y homologous gene amelogenin was carried out. DNA profiles from a single hair with attached sheath material, recovered from underneath the seat cover of the suspect's car seat were compared with DNA profiles derived from reference head hairs from a homicide victim. From the evidentiary sample only 9 ng of human DNA could be recovered. In a sample, where the quantity of DNA becomes a critical issue a powerful route is the simultaneous amplification of several loci (multiplex PCR). This is the first report where commercially available multiplex PCR amplification and typing kits have been introduced for the analysis of DNA evidence in a serial killer case and the analysis has been admitted in court.

  20. Evaluation of a rapid single multiplex microsatellite-based assay for use in forensic genetic investigations in dogs.

    Science.gov (United States)

    Clark, Leigh Anne; Famula, Thomas R; Murphy, Keith E

    2004-10-01

    To develop a set of microsatellite markers, composed of a minimal number of these markers, suitable for use in forensic genetic investigations in dogs. Blood, tissue, or buccal epithelial cells from 364 dogs of 85 breeds and mixed breeds and 19 animals from related species in the family Canidae. 61 tetranucleotide microsatellite markers were characterized on the basis of number and size of alleles, ease of genotyping, chromosomal location, and ability to be coamplified. The range in allele size, number of alleles, total heterozygosity, and fixation index for each marker were determined by use of genotype data from 383 dogs and related species. Polymorphism information content was calculated for several breeds of dogs. 7 microsatellite markers could be coamplified. These markers were labeled with fluorescent dyes, multiplexed into a single reaction, and optimized for resolution in a commercial genetic analyzer. The multiplex set was used to identify sires for 2 mixed litters. The test was not species specific; genotype information collected for wolves, coyotes, jackals, New Guinea singing dogs, and an African wild dog could not distinguish between these species. This set of 7 microsatellite markers is useful in forensic applications (ie, identification of dogs and determination of parentage) in closely related animals and is applicable to a wide range of species belonging to the family Canidae.

  1. Development of a rapid multiplex SSR genotyping method to study populations of the fungal plant pathogen Zymoseptoria tritici.

    Science.gov (United States)

    Gautier, Angélique; Marcel, Thierry C; Confais, Johann; Crane, Charles; Kema, Gert; Suffert, Frédéric; Walker, Anne-Sophie

    2014-06-18

    Zymoseptoria tritici is a hemibiotrophic ascomycete fungus causing leaf blotch of wheat that often decreases yield severely. Populations of the fungus are known to be highly diverse and poorly differentiated from each other. However, a genotyping tool is needed to address further questions in large collections of isolates, regarding regional population structure, adaptation to anthropogenic selective pressures, and dynamics of the recently discovered accessory chromosomes. This procedure is limited by costly and time-consuming simplex PCR genotyping. Recent development of genomic approaches and of larger sets of SSRs enabled the optimization of microsatellite multiplexing. We report here a reliable protocol to amplify 24 SSRs organized in three multiplex panels, and covering all Z. tritici chromosomes. We also propose an automatic allele assignment procedure, which allows scoring alleles in a repeatable manner across studies and laboratories. All together, these tools enabled us to characterize local and worldwide populations and to calculate diversity indexes consistent with results reported in the literature. This easy-to-use, accurate, repeatable, economical, and faster technical strategy can provide useful genetic information for evolutionary inferences concerning Z. tritici populations. Moreover, it will facilitate the comparison of studies from different scientific groups.

  2. Rapid Authentication of the Herbal Medicine Plant Species Aralia continentalis Kitag. and Angelica biserrata C.Q. Yuan and R.H. Shan Using ITS2 Sequences and Multiplex-SCAR Markers

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    Wook Jin Kim

    2016-02-01

    Full Text Available Accurate identification of the plant species that are present in herbal medicines is important for quality control. Although the dried roots of Aralia continentalis (Araliae Continentalis Radix and Angelica biserrata (Angelicae Pubescentis Radix are used in the same traditional medicine, namely Dok-Hwal in Korean and Du-Huo in Chinese, the medicines are described differently in the national pharmacopeia. Further confusion arises from the distribution of dried Levisticum officinale and Heracleum moellendorffii roots as the same medicine. Medicinal ingredients from all four plants are morphologically similar, and discrimination is difficult using conventional methods. Molecular identification methods offer rapidity and accuracy. The internal transcribed spacer 2 (ITS2 region of the nuclear ribosomal RNA gene (rDNA was sequenced in all four plant species, and the sequences were used to design species-specific primers. Primers for each species were then combined to allow sample analysis in a single PCR reaction. Commercial herbal medicine samples were obtained from Korea and China and analyzed using the multiplex assay. The assay successfully identified authentic medicines and also identified inauthentic or adulterated samples. The multiplex assay will be a useful tool for identification of authentic Araliae Continentalis Radix and/or Angelicae Pubescentis Radix preparations in Korea and China.

  3. Rapid Authentication of the Herbal Medicine Plant Species Aralia continentalis Kitag. and Angelica biserrata C.Q. Yuan and R.H. Shan Using ITS2 Sequences and Multiplex-SCAR Markers.

    Science.gov (United States)

    Kim, Wook Jin; Moon, Byeong Cheol; Yang, Sungyu; Han, Kyeong Suk; Choi, Goya; Lee, A Yeong

    2016-02-29

    Accurate identification of the plant species that are present in herbal medicines is important for quality control. Although the dried roots of Aralia continentalis (Araliae Continentalis Radix) and Angelica biserrata (Angelicae Pubescentis Radix) are used in the same traditional medicine, namely Dok-Hwal in Korean and Du-Huo in Chinese, the medicines are described differently in the national pharmacopeia. Further confusion arises from the distribution of dried Levisticum officinale and Heracleum moellendorffii roots as the same medicine. Medicinal ingredients from all four plants are morphologically similar, and discrimination is difficult using conventional methods. Molecular identification methods offer rapidity and accuracy. The internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal RNA gene (rDNA) was sequenced in all four plant species, and the sequences were used to design species-specific primers. Primers for each species were then combined to allow sample analysis in a single PCR reaction. Commercial herbal medicine samples were obtained from Korea and China and analyzed using the multiplex assay. The assay successfully identified authentic medicines and also identified inauthentic or adulterated samples. The multiplex assay will be a useful tool for identification of authentic Araliae Continentalis Radix and/or Angelicae Pubescentis Radix preparations in Korea and China.

  4. Multiplexed extraction and quantitative analysis of pharmaceuticals from DBS samples using digital microfluidics.

    Science.gov (United States)

    Lafrenière, Nelson M; Shih, Steve C C; Abu-Rabie, Paul; Jebrail, Mais J; Spooner, Neil; Wheeler, Aaron R

    2014-02-01

    Dried blood spot (DBS) sampling is emerging as a valuable technique in a variety of fields, including clinical and preclinical testing of pharmaceuticals. Despite this popularity, current DBS sampling and analysis processes remain laborious and time consuming. Digital microfluidics, a microscale liquid-handling technique, characterized by the manipulation of discrete droplets on open electrode arrays, offers a potential solution to these problems. We report a new digital microfluidic method for multiplexed extraction and analysis of pharmaceuticals in DBS samples. In the new method, four DBS samples are extracted in microliter-sized droplets containing internal standard, and the extract is delivered to dedicated nanoelectrospray ionization emitters for direct analysis by tandem mass spectometry and selected reaction monitoring. The new method allows for an order of magnitude reduction in processing time and approximately three-times reduction in extraction solvent relative to conventional techniques, while maintaining acceptable analytical performance for most drugs tested.

  5. A multiplex bead-based assay for immune gene expression analysis in shrimp.

    Science.gov (United States)

    Arayamethakorn, Sopacha; Karoonuthaisiri, Nitsara; Rungrassamee, Wanilada

    2017-10-20

    Here, we developed a 9-plex bead-based array as a tool to evaluate molecular effects on transcription levels of immune-related genes in the black tiger shrimp (Penaeus monodon). The bead array technology allows simultaneous detection of multiple target genes in a single sample, reducing time, labor and cost. The oligonucleotide probes were designed to target eight immune-related genes that involve in antimicrobial activity, melanization, pathogen pattern recognition proteins, lysozyme and one housekeeping gene as an internal control. The nine probes were coupled to carboxylated-magnetic bead sets. The 9-plex PCR primers were designed and optimized for conditions to allow multiplex detection. The specificity of the assay was validated and the sensitivity was determined to be 10(3) copies/μL for all target genes. The 9-plex immune gene expression assay was applied to determine transcript levels in gills of P. monodon under exposure to a shrimp pathogen, Vibrio harveyi, and gene expression patterns were consistent to patterns observed under a traditional realtime PCR method. While realtime PCR method gave a better sensitivity but limited multiplexity, our 9-plex immune gene expression assay was able to simultaneously measure expression of multiple target genes, providing useful alternative assay in the need of higher-throughput gene expression analysis such as evaluation of immune stimulatory effects in different feed additives under various dosages and time points in shrimp. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Multiplex Y-STRs analysis using the ion torrent personal genome machine (PGM).

    Science.gov (United States)

    Zhao, Xueying; Ma, Ke; Li, Hui; Cao, Yu; Liu, Wenbin; Zhou, Huaigu; Ping, Yuan

    2015-11-01

    Massively parallel sequencing (MPS) technologies allow parallel sequencing analyses of many targeted regions of multiple samples at desirable depth of coverage. Routine use of MPS for forensic genetics is on the horizon. In this study, we explore the application of MPS technology in forensic Y-STR analysis. We designed a multiplex assay with 13 Y-STR loci (DYS19, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS437, DYS438, DYS439, DYS448, DYS456, DYS635, GATA-H4) for the purpose of MPS. The multiplex Y-STR assay was amplified in 42 unrelated male individuals and amplicons were sequenced simultaneously using the ion torrent personal genome machine (PGM) system. All loci were detected successfully, except for DYS389 II that exhibited a failure rate of 1.8% due to the relatively long amplicon sizes. We observed 7, 3, 2, 6 and 5 new alleles, respectively in DYS389 II, DYS390, DYS437, DYS448 and DYS635 due to the presence of sub-repeat composition differences, and a new allele in DYS438 because of nucleotide substitution. One allele of DYS390 was inconsistent with allele call from conventional capillary electrophoresis (CE) because of 4 bp deletions upstream of the core repeat unit. This study demonstrates that Y-STR typing by MPS can provide more genetic information, holding the promise for high discriminatory power. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. Fiber Attachment Module Experiment (FAME): Using a Multiplexed Miniature Hollow Fiber Membrane Bioreactor Solution for Rapid Process Testing

    Science.gov (United States)

    Lunn, Griffin; Wheeler, Raymond; Hummerick, Mary; Birmele, Michele; Richards, Jeffrey; Coutts, Janelle; Koss, Lawrence; Spencer, Lashelle.; Johnsey, Marissa; Ellis, Ronald

    Bioreactor research, even today, is mostly limited to continuous stirred-tank reactors (CSTRs). These are not an option for microgravity applications due to the lack of a gravity gradient to drive aeration as described by the Archimedes principle. This has led to testing of Hollow Fiber Membrane Bioreactors (HFMBs) for microgravity applications, including possible use for wastewater treatment systems for the International Space Station (ISS). Bioreactors and filtration systems for treating wastewater could avoid the need for harsh pretreatment chemicals and improve overall water recovery. However, the construction of these reactors is difficult and commercial off-the-shelf (COTS) versions do not exist in small sizes. We have used 1-L modular HFMBs in the past, but the need to perform rapid testing has led us to consider even smaller systems. To address this, we designed and built 125-mL, rectangular reactors, which we have called the Fiber Attachment Module Experiment (FAME) system. A polycarbonate rack of four square modules was developed with each module containing removable hollow fibers. Each FAME reactor is self-contained and can be easily plumbed with peristaltic and syringe pumps for continuous recycling of fluids and feeding, as well as fitted with sensors for monitoring pH, dissolved oxygen, and gas measurements similar to their larger counterparts. The first application tested in the FAME racks allowed analysis of over a dozen fiber surface treatments and three inoculation sources to achieve rapid reactor startup and biofilm attachment (based on carbon oxidation and nitrification of wastewater). With these miniature FAME reactors, data for this multi-factorial test were collected in duplicate over a six-month period; this greatly compressed time period required for gathering data needed to study and improve bioreactor performance.

  8. Rapid Generation of Multiplexed Cell Cocultures Using Acoustic Droplet Ejection Followed by Aqueous Two-Phase Exclusion Patterning

    Science.gov (United States)

    Fang, Yu; Frampton, John P.; Raghavan, Shreya; Sabahi-Kaviani, Rahman; Luker, Gary

    2012-01-01

    The development of tools for patterning cocultures of cells is a fundamental interest among cell biologists and tissue engineers. Although a variety of systems exist for micropatterning cells, the methods used to generate cell micropatterns are often cumbersome and difficult to adapt for tissue engineering purposes. This study combines acoustic droplet ejection and aqueous two-phase system exclusion patterning to introduce a method for patterning cocultures of cells in multiplexed arrays. This new method uses focused acoustic radiation pressure to eject discrete droplets of uniform size from the surface of a dextran solution containing cells. The size of droplets is controlled by adjusting ultrasound parameters, such as pulse, duration, and amplitude. The ejected dextran droplets are captured on a cell culture substrate that is manipulated by a computer-controlled 3D positioning system according to predesigned patterns. Polyethylene glycol solution containing an additional cell type is then added to the culture dish to produce a two-phase system capable of depositing different types of cells around the initial pattern of cells. We demonstrate that our method can produce patterns of islands or lines with two or more cell types. Further, we demonstrate that patterns can be multiplexed for studies involving combinations of multiple cell types. This method offers a tool to transfer cell-containing samples in a contact-free, nozzle-less manner, avoiding sample cross-contamination. It can be used to pattern cell cocultures without complicated fabrication of culture substrates. These capabilities were used to examine the response of cancer cells to the presence of a ligand (CXCL12) secreted from surrounding cocultured cells. PMID:22356298

  9. Multiplex SNP analysis on whole genome amplified DNA from archived dried bloodspots, a validation study

    DEFF Research Database (Denmark)

    Tvedegaard, Kristine C.; Parner, Erik; Hooper, Craig W.

    on whole genome amplified (WGA) DNA from archived dried bloodspots. METHODS AND MATERIAL: The chemically synthesized new base pair (isoC and isoG) allow for the creation of new DNA strands that provide superior specificity and allow development of assays with greater sensitivity than conventional methods...... is a further development of allele specific primer extension (ASPE) for multiplex SNP analysis based on the Luminex 100 IS platform. It uses isobases (isoC and isoG) and the software MultiCode-PLx platform for data analysis and data handling. We validate the EraGen multicode system in two 6-plex assays used....... To validate the method 900 WGA DNA samples were genotyped in duplets. 10-20 % of all samples were sequenced to be used as reference. Accuracy and repeatability was estimated for each SNP. Robustness was estimated as rate of conclusive outcomes for each SNP. RESULTS: The accuracy ranged from 98...

  10. Rapid identification of ST131 Escherichia coli by a novel multiplex real-time allelic discrimination assay.

    Science.gov (United States)

    François, Patrice; Bonetti, Eve-Julie; Fankhauser, Carolina; Baud, Damien; Cherkaoui, Abdessalam; Schrenzel, Jacques; Harbarth, Stephan

    2017-09-01

    Escherichia coli sequence type 131 is increasingly described in severe hospital infections. We developed a rapid real-time allelic discrimination assay for the rapid identification of E. coli ST131 isolates. This rapid assay represents an affordable alternative to sequence-based strategies before completing characterization of potentially highly virulent isolates of E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. How Rapid is Rapid Prototyping? Analysis of ESPADON Programme Results

    Directory of Open Access Journals (Sweden)

    Ian D. Alston

    2003-05-01

    Full Text Available New methodologies, engineering processes, and support environments are beginning to emerge for embedded signal processing systems. The main objectives are to enable defence industry to field state-of-the-art products in less time and with lower costs, including retrofits and upgrades, based predominately on commercial off the shelf (COTS components and the model-year concept. One of the cornerstones of the new methodologies is the concept of rapid prototyping. This is the ability to rapidly and seamlessly move from functional design to the architectural design to the implementation, through automatic code generation tools, onto real-time COTS test beds. In this paper, we try to quantify the term “rapid” and provide results, the metrics, from two independent benchmarks, a radar and sonar beamforming application subset. The metrics show that the rapid prototyping process may be sixteen times faster than a conventional process.

  12. Multiplex Reverse Transcription-Polymerase Chain Reaction untuk Deteksi Cepat Virus Flu Burung H5N1 (MULTIPLEX REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR RAPID DETECTION OF H5N1 AVIAN INFLUENZA VIRUS

    Directory of Open Access Journals (Sweden)

    Raden Wasito

    2015-05-01

    Full Text Available Avian influenza virus subtype H5N1 (AIV H5N1 is highly pathogenic and fatal in poultry. The virusis still endemic with low virulence rate, although it may play a critical role in causing high morbidity andmortality rates in poultry in Indonesia. In general, diagnostic approach for AIV H5N1 is based onconventional serological and viral isolation methods that have the potential to produce consumings oftime and relatively expensive cost within the laboratory without compromising test utility. Thus, amolecular approach of multiplex reverse transcription-polymerase chain reaction (mRT-PCR was developedand applied for the detection of matrix gene type A influenza viruses, AIV subtype subtype H5hemagglutinin gene with simultaneous detection of N1 nucleoprotein gene. Thirty sera specimens fromthe diseased commercial chickens that were specifically amplified positive-RT-PCR for AIV H5N1 wereselected for mRT-PCR. The mRT-PCR products were visualized by agarose gel electrophoresis and consistedof DNA fragments of AIV of 245 bp, 545 bp and 343 bp for M, H5 and N1 genes, respectively. Thus, themRT-PCR that can rapidly differentiate simultaneously between these genes is very important for thecontrol and even eradication of AIV transmission in poultry in Indonesia.

  13. Simultaneous Confirmatory Analysis of Different Transgenic Maize (Zea mays) Lines Using Multiplex Polymerase Chain Reaction−Restriction Analysis and Capillary Gel Electrophoresis with Laser Induced Fluorescence Detection

    OpenAIRE

    García-Cañas, Virginia; Cifuentes, Alejandro

    2008-01-01

    A novel analytical procedure based on the combination of multiplex PCR, restriction analysis, and CGE-LIF to unambiguosly and simultaneously confirm the presence of multiple lines of genetically modified corn is proposed. This methodology is based on the amplification of event-specific DNA regions by multiplex PCR using 6-FAM-labeled primers. Subsequently, PCR products are digested by a mixture containing specific restriction endonucleases. Thus, restriction endonucleases selectively recogniz...

  14. Rapid and simple method by combining FTA™ card DNA extraction with two set multiplex PCR for simultaneous detection of non-O157 Shiga toxin-producing Escherichia coli strains and virulence genes in food samples.

    Science.gov (United States)

    Kim, S A; Park, S H; Lee, S I; Ricke, S C

    2017-12-01

    The aim of this research was to optimize two multiplex polymerase chain reaction (PCR) assays that could simultaneously detect six non-O157 Shiga toxin-producing Escherichia coli (STEC) as well as the three virulence genes. We also investigated the potential of combining the FTA™ card-based DNA extraction with the multiplex PCR assays. Two multiplex PCR assays were optimized using six primer pairs for each non-O157 STEC serogroup and three primer pairs for virulence genes respectively. Each STEC strain specific primer pair only amplified 155, 238, 321, 438, 587 and 750 bp product for O26, O45, O103, O111, O121 and O145 respectively. Three virulence genes were successfully multiplexed: 375 bp for eae, 655 bp for stx1 and 477 bp for stx2. When two multiplex PCR assays were validated with ground beef samples, distinctive bands were also successfully produced. Since the two multiplex PCR examined here can be conducted under the same PCR conditions, the six non-O157 STEC and their virulence genes could be concurrently detected with one run on the thermocycler. In addition, all bands clearly appeared to be amplified by FTA card DNA extraction in the multiplex PCR assay from the ground beef sample, suggesting that an FTA card could be a viable sampling approach for rapid and simple DNA extraction to reduce time and labour and therefore may have practical use for the food industry. Two multiplex polymerase chain reaction (PCR) assays were optimized for discrimination of six non-O157 Shiga toxin-producing Escherichia coli (STEC) and identification of their major virulence genes within a single reaction, simultaneously. This study also determined the successful ability of the FTA™ card as an alternative to commercial DNA extraction method for conducting multiplex STEC PCR assays. The FTA™ card combined with multiplex PCR holds promise for the food industry by offering a simple and rapid DNA sample method for reducing time, cost and labour for detection of STEC in

  15. Safety and Feasibility of Quantitative Multiplexed Cytokine Analysis From Office-Based Vitreous Aspiration.

    Science.gov (United States)

    Ghodasra, Devon H; Fante, Ryan; Gardner, Thomas W; Langue, Michael; Niziol, Leslie M; Besirli, Cagri; Cohen, Steven R; Dedania, Vaidehi S; Demirci, Hakan; Jain, Nieraj; Jayasundera, K Thiran; Johnson, Mark W; Kalyani, Partho S; Rao, Rajesh C; Zacks, David N; Sundstrom, Jeffrey M

    2016-06-01

    The goals of this study were to evaluate the safety of office-based vitreous sampling, and determine the utility of these samples with multiplex cytokine analysis. Vitreous samples were collected from office-based needle aspiration and the rate of adverse events during follow-up was reviewed. The vitreous cytokine concentrations in a subset of patients with diabetic macular edema (DME) were analyzed using a 42 plex-cytokine bead array. These results were compared with vitreous cytokine concentrations in proliferative diabetic retinopathy (PDR) and controls (macular hole, epiretinal membrane, symptomatic vitreous floaters) from pars plana vitrectomy. An adequate volume of vitreous fluid (100-200 μL) was obtained in 52 (88%) of 59 office-based sampling attempts. The average length of follow-up was 300 days (range, 42-926 days). There were no complications, including cataract, retinal tear or detachment, and endophthalmitis. Two patients (3%) had posterior vitreous detachments within 3 months. Vitreous cytokine concentrations were measured in 44 patients: 14 controls, 13 with DME, and 17 with PDR. The concentration of ADAM11, CXCL-10, IL-8, and PDGF-A were higher in PDR compared with controls and DME. The concentration of IL-6 was higher in PDR compared with controls, but not compared with DME. Office-based vitreous aspiration is safe and yields high-quality samples for multiplex vitreous cytokine analysis. Significant elevations of vitreous cytokines were found in PDR compared with DME and controls, including the novel finding of elevated ADAM11. As such, office-based aspiration is a safe and effective means to identify vitreous factors associated with vitreoretinal disease.

  16. Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living Amoeba Naegleria in the Environment

    Science.gov (United States)

    Pélandakis, Michel; Pernin, Pierre

    2002-01-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  17. Available number of multiplexed holograms based on signal-to-noise ratio analysis in reflection-type holographic memory using three-dimensional speckle-shift multiplexing.

    Science.gov (United States)

    Nishizaki, Tatsuya; Matoba, Osamu; Nitta, Kouichi

    2014-09-01

    The recording properties of three-dimensional speckle-shift multiplexing in reflection-type holographic memory are analyzed numerically. Three-dimensional recording can increase the number of multiplexed holograms by suppressing the cross-talk noise from adjacent holograms by using depth-direction multiplexing rather than in-plane multiplexing. Numerical results indicate that the number of multiplexed holograms in three-layer recording can be increased by 1.44 times as large as that of a single-layer recording when an acceptable signal-to-noise ratio is set to be 2 when NA=0.43 and the thickness of the recording medium is 0.5 mm.

  18. Multiplex Solid-Phase PCR for Rapid Detection and Identification of Salmonella spp. at Sub-species

    DEFF Research Database (Denmark)

    Cao, Cuong; Høgberg, Jonas; Wolff, Anders

    -PCR gel electrophoresis. The method will be useful for development of point-of-care devices for rapid detection and identification of Salmonella spp. A solid-phase PCR for rapid detection and identification of S. enteritidis, S. typhimurium and S. dublin is developed. The method offers advantages......This study presents a solid-phase PCR (SP-PCR) for rapid detection, identification, and sub-typing of various Salmonella species, the major food-borne cause of salmonellosis. The target DNA is firstly amplified with PCR primers (one primer is labeled with fluorophores) in the liquid phase...... by the liquid phase primer thus generating new templates for the SP-PCR. After the reaction, PCR products labeled with fluorophores remain attached to the substrate and can be visualized directly by fluorescence readout devices. Using this method, S. enteritidis, S. typhimurium and S. dublin can be detected...

  19. Fourier optics analysis of phase-mask-based path-length-multiplexed optical coherence tomography.

    Science.gov (United States)

    Yin, Biwei; Dwelle, Jordan; Wang, Bingqing; Wang, Tianyi; Feldman, Marc D; Rylander, Henry G; Milner, Thomas E

    2015-11-01

    Optical coherence tomography (OCT) is an imaging technique that constructs a depth-resolved image by measuring the optical path-length difference between broadband light backscattered from a sample and a reference surface. For many OCT sample arm optical configurations, sample illumination and backscattered light detection share a common path. When a phase mask is placed in the sample path, features in the detected signal are observed, which suggests that an analysis of a generic common path OCT imaging system is warranted. In this study, we present a Fourier optics analysis using a Fresnel diffraction approximation of an OCT system with a path-length-multiplexing element (PME) inserted in the sample arm optics. The analysis may be generalized for most phase-mask-based OCT systems. A radial-angle-diverse PME is analyzed in detail, and the point spread function, coherent transfer function, sensitivity of backscattering angular diversity detection, and signal formation in terms of sample spatial frequency are simulated and discussed. The analysis reveals important imaging features and application limitations of OCT imaging systems with a phase mask in the sample path optics.

  20. Development of a novel forensic STR multiplex for ancestry analysis and extended identity testing.

    Science.gov (United States)

    Phillips, Chris; Fernandez-Formoso, Luis; Gelabert-Besada, Miguel; Garcia-Magariños, Manuel; Santos, Carla; Fondevila, Manuel; Carracedo, Angel; Lareu, Maria Victoria

    2013-04-01

    There is growing interest in developing additional DNA typing techniques to provide better investigative leads in forensic analysis. These include inference of genetic ancestry and prediction of common physical characteristics of DNA donors. To date, forensic ancestry analysis has centered on population-divergent SNPs but these binary loci cannot reliably detect DNA mixtures, common in forensic samples. Furthermore, STR genotypes, forming the principal DNA profiling system, are not routinely combined with forensic SNPs to strengthen frequency data available for ancestry inference. We report development of a 12-STR multiplex composed of ancestry informative marker STRs (AIM-STRs) selected from 434 tetranucleotide repeat loci. We adapted our online Bayesian classifier for AIM-SNPs: Snipper, to handle multiallele STR data using frequency-based training sets. We assessed the ability of the 12-plex AIM-STRs to differentiate CEPH Human Genome Diversity Panel populations, plus their informativeness combined with established forensic STRs and AIM-SNPs. We found combining STRs and SNPs improves the success rate of ancestry assignments while providing a reliable mixture detection system lacking from SNP analysis alone. As the 12 STRs generally show a broad range of alleles in all populations, they provide highly informative supplementary STRs for extended relationship testing and identification of missing persons with incomplete reference pedigrees. Lastly, mixed marker approaches (combining STRs with binary loci) for simple ancestry inference tests beyond forensic analysis bring advantages and we discuss the genotyping options available. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Rapid Analysis Model: Reducing Analysis Time without Sacrificing Quality.

    Science.gov (United States)

    Lee, William W.; Owens, Diana

    2001-01-01

    Discusses the performance technology design process and the fact that the analysis phase is often being eliminated to speed up the process. Proposes a rapid analysis model that reduces time needed for analysis and still ensures more successful value-added solutions that focus on customer satisfaction. (LRW)

  2. Rapid Identification of Pathogens from Positive Blood Cultures by Multiplex PCR using the FilmArray System

    Science.gov (United States)

    Blaschke, Anne J.; Heyrend, Caroline; Byington, Carrie L.; Fisher, Mark A.; Barker, Elizabeth; Garrone, Nicholas F.; Thatcher, Stephanie A.; Pavia, Andrew T.; Barney, Trenda; Alger, Garrison D.; Daly, Judy A.; Ririe, Kirk M.; Ota, Irene; Poritz, Mark A.

    2012-01-01

    Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Inc., Salt Lake City, UT) Blood Culture (BC) panel can identify > 25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 hour. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 of 92 pathogens (91%) covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven MRSA and VRE. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture. PMID:22999332

  3. Rapid Genetic Analysis in Congenital Hyperinsulinism

    DEFF Research Database (Denmark)

    Christesen, Henrik Thybo; Brusgaard, Klaus; Alm, Jan

    2007-01-01

    with a paternal germline ABCC8 or KCNJ11 mutation and a focal loss of maternal chromosome 11p15, whereas a maternal mutation, or homozygous/compound heterozygous ABCC8 and KCNJ11 mutations predict diffuse-type disease. However, genotyping usually takes too long to be helpful in the absence of a founder mutation....... One patient had a paternal KCNJ11 mutation and focal disease confirmed by positron emission tomography scan and biopsies. One patient had a de novo heterozygous ABBC8 mutation and unexplained diffuse disease confirmed by positron emission tomography scan and biopsies. CONCLUSION: A rapid analysis...

  4. Detection of viable enterotoxin-producing Bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex PCR.

    Science.gov (United States)

    Zhang, Zhihong; Feng, Lixia; Xu, Hengyi; Liu, Chengwei; Shah, Nagendra P; Wei, Hua

    2016-02-01

    Bacillus cereus is responsible for several outbreaks of foodborne diseases due to its emetic toxin and enterotoxin. Enterotoxins, cytotoxin K (CytK), nonhemolytic enterotoxin (Nhe), and hemolysin BL (Hbl), have been recorded in several diarrheal cases due to food poisoning from B. cereus. The objective of this study was to develop a rapid and accurate method that combines multiplex PCR with propidium monoazide to selectively detect viable cells of enterotoxin-producing B. cereus in milk powder, noodles, and rice, and investigate the distribution of enterotoxins in 62 strains of B. cereus in Jiangxi province, China. The specificity of primers of 3 enterotoxins (i.e., cytK, nheA, and hblD) of B. cereus was verified by inclusivity and exclusivity tests using single PCR. Upon optimization of multiplex PCR conditions, it was found that the detection limit of viable cells was 10(2) cfu/mL of B. cereus in pure culture. By enrichment for 3 or 4 h and propidium monoazide pretreatment, a protocol for detection of viable cells as low as 2.2×10(1) cfu/g in spiked food (e.g., milk powder, noodles, and rice) was established and proved valid even under the interference of non-Bacillus cereus at as high as 10(5) cfu/g. Moreover, the protocol based on multiplex PCR for detection was applied for the analysis of distribution of toxin gene of B. cereus, and the results showed a regional feature for toxin gene distribution, indicating that potential toxigenicity of B. cereus should be evaluated further. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Rapid and Simultaneous Detection of Mycobacterium tuberculosis Complex and Beijing/W Genotype in Sputum by an Optimized DNA Extraction Protocol and a Novel Multiplex Real-Time PCR ▿

    Science.gov (United States)

    Leung, Eric T. Y.; Zheng, L.; Wong, Rity Y. K.; Chan, Edward W. C.; Au, T. K.; Chan, Raphael C. Y.; Lui, Grace; Lee, Nelson; Ip, Margaret

    2011-01-01

    Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms. PMID:21593264

  6. A multi-center ring trial of allergen analysis using fluorescent multiplex array technology

    NARCIS (Netherlands)

    King, Eva M.; Filep, Stephanie; Smith, Bryan; Platts-Mills, Thomas; Hamilton, Robert G.; Schmechel, Detlef; Sordillo, Joanne E.; Milton, Donald; van Ree, Ronald; Krop, Esmeralda J. M.; Heederik, Dick J. J.; Metwali, Nervana; Thorne, Peter S.; Zeldin, Darryl C.; Sever, Michelle L.; Calatroni, Agustin; Arbes, Samuel J.; Mitchell, Herman E.; Chapman, Martin D.

    2013-01-01

    Consistent performance of allergen assays is essential to ensure reproducibility of exposure assessments for investigations of asthma and occupational allergic disease. This study evaluated intra- and inter-laboratory reproducibility of a fluorescent multiplex array, which simultaneously measures

  7. Accuracy analysis for triangulation and tracking based on time-multiplexed structured light.

    Science.gov (United States)

    Wagner, Benjamin; Stüber, Patrick; Wissel, Tobias; Bruder, Ralf; Schweikard, Achim; Ernst, Floris

    2014-08-01

    The authors' research group is currently developing a new optical head tracking system for intracranial radiosurgery. This tracking system utilizes infrared laser light to measure features of the soft tissue on the patient's forehead. These features are intended to offer highly accurate registration with respect to the rigid skull structure by means of compensating for the soft tissue. In this context, the system also has to be able to quickly generate accurate reconstructions of the skin surface. For this purpose, the authors have developed a laser scanning device which uses time-multiplexed structured light to triangulate surface points. The accuracy of the authors' laser scanning device is analyzed and compared for different triangulation methods. These methods are given by the Linear-Eigen method and a nonlinear least squares method. Since Microsoft's Kinect camera represents an alternative for fast surface reconstruction, the authors' results are also compared to the triangulation accuracy of the Kinect device. Moreover, the authors' laser scanning device was used for tracking of a rigid object to determine how this process is influenced by the remaining triangulation errors. For this experiment, the scanning device was mounted to the end-effector of a robot to be able to calculate a ground truth for the tracking. The analysis of the triangulation accuracy of the authors' laser scanning device revealed a root mean square (RMS) error of 0.16 mm. In comparison, the analysis of the triangulation accuracy of the Kinect device revealed a RMS error of 0.89 mm. It turned out that the remaining triangulation errors only cause small inaccuracies for the tracking of a rigid object. Here, the tracking accuracy was given by a RMS translational error of 0.33 mm and a RMS rotational error of 0.12°. This paper shows that time-multiplexed structured light can be used to generate highly accurate reconstructions of surfaces. Furthermore, the reconstructed point sets can be

  8. DNA Sudoku—harnessing high-throughput sequencing for multiplexed specimen analysis

    Science.gov (United States)

    Erlich, Yaniv; Chang, Kenneth; Gordon, Assaf; Ronen, Roy; Navon, Oron; Rooks, Michelle; Hannon, Gregory J.

    2009-01-01

    Next-generation sequencers have sufficient power to analyze simultaneously DNAs from many different specimens, a practice known as multiplexing. Such schemes rely on the ability to associate each sequence read with the specimen from which it was derived. The current practice of appending molecular barcodes prior to pooling is practical for parallel analysis of up to many dozen samples. Here, we report a strategy that permits simultaneous analysis of tens of thousands of specimens. Our approach relies on the use of combinatorial pooling strategies in which pools rather than individual specimens are assigned barcodes. Thus, the identity of each specimen is encoded within the pooling pattern rather than by its association with a particular sequence tag. Decoding the pattern allows the sequence of an original specimen to be inferred with high confidence. We verified the ability of our encoding and decoding strategies to accurately report the sequence of individual samples within a large number of mixed specimens in two ways. First, we simulated data both from a clone library and from a human population in which a sequence variant associated with cystic fibrosis was present. Second, we actually pooled, sequenced, and decoded identities within two sets of 40,000 bacterial clones comprising approximately 20,000 different artificial microRNAs targeting Arabidopsis or human genes. We achieved greater than 97% accuracy in these trials. The strategies reported here can be applied to a wide variety of biological problems, including the determination of genotypic variation within large populations of individuals. PMID:19447965

  9. A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase systems.

    Science.gov (United States)

    Bras, Eduardo J S; Soares, Ruben R G; Azevedo, Ana M; Fernandes, Pedro; Arévalo-Rodríguez, Miguel; Chu, Virginia; Conde, João P; Aires-Barros, M Raquel

    2017-09-15

    Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20μL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient (K) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule (K=2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system.

    Science.gov (United States)

    Blaschke, Anne J; Heyrend, Caroline; Byington, Carrie L; Fisher, Mark A; Barker, Elizabeth; Garrone, Nicholas F; Thatcher, Stephanie A; Pavia, Andrew T; Barney, Trenda; Alger, Garrison D; Daly, Judy A; Ririe, Kirk M; Ota, Irene; Poritz, Mark A

    2012-12-01

    Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Multiplex Minisequencing of the HBB Gene: A Rapid Strategy to Confirm the Most Frequent β-Thalassemia Mutations in the Tunisian Population.

    Science.gov (United States)

    Ben Charfeddine, Ilhem; Ben Lazreg, Taheni; M'sakni, Ahlem; Amara, Abdelbasset; Mlika, Adnène; Chaïeb, Anouar; Hlel, Khalid; Zouari, Noura; Zbidi, Faïza; Bouguila, Jihène; Soyah, Najla; Ayedi, Abdelkarim; Ben Hamouda, Hechmi; Abroug, Saoussen; Boughamoura, Lamia; Saad, Ali; Gribaa, Moez

    2015-01-01

    The β hemoglobinopathies [β-thalassemia (β-thal) and structural hemoglobin (Hb) variants such as Hb S (HBB: c.20A > T) and Hb E (HBB: c.79G > A)] are among the most common inherited diseases worldwide. In Tunisia, due to the high prevalence of consanguineous marriages, the recurrent risk of this disease is high. The average prevalence of hemoglobinopathies is 4.48%, reaching 12.50% in some focus regions. The molecular investigations on thalassemia contributed to establishing the spectrum of mutations in the Tunisian population. The total number of HBB gene mutations identified was 24. The two most frequent mutations, codon 39 (C > T) (HBB: c.118C > T) and IVS-I-110 (G > A) (HBB: c.93-21G > A) accounted for 70.0% of the total encountered β-thal cases. These two mutations together with IVS-I-2 (T > G) (HBB: c.92 + 2T > G) and the Hb S variant account for more than 90.0% of all HBB genetic variants in Tunisia. Thus, developing rapid, inexpensive and reliable mutation-specific molecular diagnostic assays targeting our Tunisian populations is our aim to facilitate routine detection of hemoglobinopathies. In this report, we describe the successful application of the multiplex minisequencing assay as an alternative strategy for genetic diagnosis of HBB gene disorders in Tunisia.

  12. Cytokine profiles as markers of disease severity in sepsis: a multiplex analysis.

    Science.gov (United States)

    Bozza, Fernando A; Salluh, Jorge I; Japiassu, André M; Soares, Marcio; Assis, Edson F; Gomes, Rachel N; Bozza, Marcelo T; Castro-Faria-Neto, Hugo C; Bozza, Patrícia T

    2007-01-01

    The current shortage of accurate and readily available, validated biomarkers of disease severity in sepsis is an important limitation when attempting to stratify patients into homogeneous groups, in order to study pathogenesis or develop therapeutic interventions. The aim of the present study was to determine the cytokine profile in plasma of patients with severe sepsis by using a multiplex system for simultaneous detection of 17 cytokines. This was a prospective cohort study conducted in four tertiary hospitals. A total of 60 patients with a recent diagnosis of severe sepsis were included. Plasma samples were collected for measurement of cytokine concentrations. A multiplex analysis was performed to evaluate levels of 17 cytokines (IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, interferon-gamma, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein-1 and tumour necrosis factor-alpha). Cytokine concentrations were related to the presence of severe sepsis or septic shock, the severity and evolution of organ failure, and early and late mortality. Concentrations of IL-1 beta, IL-6, IL-7, IL-8, IL-10, IL-13, interferon-gamma, MCP-1 and tumour necrosis factor-alpha were significantly higher in septic shock patients than in those with severe sepsis. Cytokine concentrations were associated with severity and evolution of organ dysfunction. With regard to the severity of organ dysfunction on day 1, IL-8 and MCP-1 exhibited the best correlation with Sequential Organ Failure Assessment score. In addition, IL-6, IL-8 and G-CSF concentrations during the first 24 hours were predictive of worsening organ dysfunction or failure of organ dysfunction to improve on day three. In terms of predicting mortality, the cytokines IL-1 beta, IL-4, IL-6, IL-8, MCP-1 and G-CSF had good accuracy for predicting early mortality (sepsis severity

  13. Rapid and sensitive suspension array for multiplex detection of organophosphorus pesticides and carbamate pesticides based on silica–hydrogel hybrid microbeads

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xuan [Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu (China); Mu, Zhongde; Shangguan, Fengqi [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu (China); Liu, Ran; Pu, Yuepu [Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu (China); Yin, Lihong, E-mail: lhyin@seu.edu.cn [Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu (China)

    2014-05-01

    Highlights: • Silica–hydrogel hybrid microbeads were used to develop suspension array. • The results in detecting pesticides agree well with those from LC–MS/MS. • The method showed the good capability for multiplex analysis of pesticides residues. - Abstract: A technique for multiplex detection of organophosphorus pesticides and carbamate pesticides has been developed using a suspension array based on silica–hydrogel hybrid microbeads (SHHMs). The main advantage of SHHMs, which consist of both silica and hydrogel materials, is that they not only could be distinguished by their characteristic reflection peak originating from the stop-band of the photonic crystal but also have low non-specific adsorption of proteins. Using fluorescent immunoassay, the LODs for fenitrothion, chlorpyrifos-methyl, fenthion, carbaryl and metolcarb were measured to be 0.02 ng/mL, 0.012 ng/mL, 0.04 ng/mL, 0.05 ng/mL and 0.1 ng/mL, respectively, all of which are much lower than the maximum residue limits, as reported in the European Union pesticides database. All the determination coefficients for these five pesticides were greater than 0.99, demonstrating excellent correlations. The suspension array was specific and had no significant cross-reactivity with other chemicals. The results for the detection of pesticide residues collected from agricultural samples using this method agree well with those from liquid chromatography–tandem mass spectrometry. Our results showed that this simple method is suitable for simultaneous detection of these five pesticides residues in fruits and vegetables.

  14. Rapid virological diagnosis of central nervous system infections by use of a multiplex reverse transcription-PCR DNA microarray.

    Science.gov (United States)

    Leveque, Nicolas; Van Haecke, Adrien; Renois, Fanny; Boutolleau, David; Talmud, Deborah; Andreoletti, Laurent

    2011-11-01

    Viruses are the main etiological cause of central nervous system (CNS) infections. A rapid molecular diagnosis is recommended to improve the therapeutic management of patients. The aim of this study was to evaluate the performances of a DNA microarray, the Clart Entherpex kit (Genomica, Coslada, Spain), allowing the rapid and simultaneous detection of 9 DNA and RNA neurotropic viruses: herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and the human enteroviruses (HEVs). This evaluation was performed with 28 samples from the European proficiency panels (Quality Control for Molecular Diagnostics [QCMD]; Glasgow, Scotland) and then with 78 cerebrospinal fluid (CSF) specimens. The majority of the QCMD results obtained by the DNA microarray were similar to those recorded by the overall QCMD participants. The main discrepant results were observed for low concentrations of HSV-2 and HEVs. From the clinical samples, the kit detected 27 of the 28 herpesvirus CNS infections and all of the 30 HEV-positive CSF samples. No false-positive result was observed among the 20 virus-negative CSF samples. The clinical sensitivity, specificity, and negative and positive predictive values of the assay were 98.3, 100, 95.2, and 100%, respectively, when the results were compared to those of commercially available PCR assays. Interestingly, HHV-7 was detected in 11 (37%) of the 30 HEV-positive CSF samples from children suffering from aseptic meningitis causing significantly longer lengths of stay at the hospital than infection with HEVs alone (2.4 versus 1.4 days; P = 0.038). In conclusion, this preliminary study showed that this DNA microarray could be a valuable molecular diagnostic tool for single and mixed DNA and RNA virus infections of the CNS.

  15. Molecular analysis and test of linkage between the FMR-I gene and infantile autism in multiplex families

    Energy Technology Data Exchange (ETDEWEB)

    Hallmayer, J.; Pintado, E.; Lotspeich, L.; Spiker, D.; Kraemer, H.C.; Lee Wong, D.; Lin, A.; Herbert, J.; Cavalli-Sforza, L.L.; Ciaranello, R.D. [Stanford Univ., CA (United States)] [and others

    1994-11-01

    Approximately 2%-5% of autistic children show cytogenetic evidence of the fragile X syndrome. This report tests whether infantile autism in multiplex autism families arises from an unusual manifestion of the fragile X syndrome. This could arise either by expansion of the (CGG)n trinucleotide repeat in FMR-1 or from a mutation elsewhere in the gene. We studied 35 families that met stringent criteria for multiplex autism. Amplification of the trinucleotide repeat and analysis of methylation status were performed in 79 autistic children and in 31 of their unaffected siblings by Southern blot analysis. No examples of amplified repeats were seen in the autistic or control children or in their parents or grandparents. We next examined the hypothesis that there was a mutation elsewhere in the FMR-1 gene, by linkage analysis in 32 of these families. We tested four different dominant models and a recessive model. Linkage to FMR-1 could be excluded (lod score between -24 and -62) in all models by using probes DXS548, FRAXAC1, and FRAXAC2 and the CGG repeat itself. Tests for heterogeneity in this sample were negative, and the occurrence of positive lod scores in this data set could be attributed to chance. Analysis of the data by the affected-sib method also did not show evidence for linkage of any marker to autism. These results enable us to reject the hypothesis that multiplex autism arises from expansion of the (CGG)n trinucleotide repeat in FMR-1. Further, because the overall lod scores for all probes in all models tested were highly negative, linkage to FMR-1 can also be ruled out in multiplex autistic families. 35 refs., 2 figs., 5 tabs.

  16. Motion Analysis Based on Invertible Rapid Transform

    Directory of Open Access Journals (Sweden)

    J. Turan

    1999-06-01

    Full Text Available This paper presents the results of a study on the use of invertible rapid transform (IRT for the motion estimation in a sequence of images. Motion estimation algorithms based on the analysis of the matrix of states (produced in the IRT calculation are described. The new method was used experimentally to estimate crowd and traffic motion from the image data sequences captured at railway stations and at high ways in large cities. The motion vectors may be used to devise a polar plot (showing velocity magnitude and direction for moving objects where the dominant motion tendency can be seen. The experimental results of comparison of the new motion estimation methods with other well known block matching methods (full search, 2D-log, method based on conventional (cross correlation (CC function or phase correlation (PC function for application of crowd motion estimation are also presented.

  17. Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes.

    Science.gov (United States)

    Yu, Clinton; Huszagh, Alexander; Viner, Rosa; Novitsky, Eric J; Rychnovsky, Scott D; Huang, Lan

    2016-10-18

    Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pairwise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely, QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and PPIs at the proteome scale in future studies.

  18. Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

    OpenAIRE

    Tian, Huan; Cao, Liching; Tan, Yuping; Williams, Stephen; Chen, Lili; Matray, Tracy; Chenna, Ahmed; Moore, Sean; Hernandez, Vincent; Xiao, Vivian; Tang, Mengxiang; Singh, Sharat

    2004-01-01

    We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader® mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach w...

  19. Functional Multiplex PageRank

    Science.gov (United States)

    Iacovacci, Jacopo; Rahmede, Christoph; Arenas, Alex; Bianconi, Ginestra

    2016-10-01

    Recently it has been recognized that many complex social, technological and biological networks have a multilayer nature and can be described by multiplex networks. Multiplex networks are formed by a set of nodes connected by links having different connotations forming the different layers of the multiplex. Characterizing the centrality of the nodes in a multiplex network is a challenging task since the centrality of the node naturally depends on the importance associated to links of a certain type. Here we propose to assign to each node of a multiplex network a centrality called Functional Multiplex PageRank that is a function of the weights given to every different pattern of connections (multilinks) existent in the multiplex network between any two nodes. Since multilinks distinguish all the possible ways in which the links in different layers can overlap, the Functional Multiplex PageRank can describe important non-linear effects when large relevance or small relevance is assigned to multilinks with overlap. Here we apply the Functional Page Rank to the multiplex airport networks, to the neuronal network of the nematode C. elegans, and to social collaboration and citation networks between scientists. This analysis reveals important differences existing between the most central nodes of these networks, and the correlations between their so-called pattern to success.

  20. Multiplexed Component Analysis to Identify Genes Contributing to the Immune Response during Acute SIV Infection.

    Directory of Open Access Journals (Sweden)

    Iraj Hosseini

    Full Text Available Immune response genes play an important role during acute HIV and SIV infection. Using an SIV macaque model of AIDS and CNS disease, our overall goal was to assess how the expression of genes associated with immune and inflammatory responses are longitudinally changed in different organs or cells during SIV infection. To compare RNA expression of a panel of 88 immune-related genes across time points and among three tissues - spleen, mesenteric lymph nodes (MLN and peripheral blood mononuclear cells (PBMC - we designed a set of Nanostring probes. To identify significant genes during acute SIV infection and to investigate whether these genes are tissue-specific or have global roles, we introduce a novel multiplexed component analysis (MCA method. This combines multivariate analysis methods with multiple preprocessing methods to create a set of 12 "judges"; each judge emphasizes particular types of change in gene expression to which cells could respond, for example, the absolute or relative size of expression change from baseline. Compared to bivariate analysis methods, our MCA method improved classification rates. This analysis allows us to identify three categories of genes: (a consensus genes likely to contribute highly to the immune response; (b genes that would contribute highly to the immune response only if certain assumptions are met - e.g. that the cell responds to relative expression change rather than absolute expression change; and (c genes whose contribution to immune response appears to be modest. We then compared the results across the three tissues of interest; some genes are consistently highly-contributing in all tissues, while others are specific for certain tissues. Our analysis identified CCL8, CXCL10, CXCL11, MxA, OAS2, and OAS1 as top contributing genes, all of which are stimulated by type I interferon. This suggests that the cytokine storm during acute SIV infection is a systemic innate immune response against viral

  1. An integrated system of ABO typing and multiplex STR testing for forensic DNA analysis.

    Science.gov (United States)

    Jiang, Xianhua; He, Juan; Jia, Fei; Shen, Hongying; Zhao, Jinling; Chen, Chuguang; Bai, Liping; Liu, Feng; Hou, Guangwei; Guo, Faye

    2012-12-01

    A new amplification system for ABO and STR genotyping in a single reaction has been successfully developed. Two types of information can be obtained from a biological sample at one time. One is the classical information of ABO blood group typing for screening suspects and the other is STR information for individual identification. The system allows for the simultaneous detection of 15 autosomal STR loci (containing all CODIS STR loci as well as Penta D and Penta E), six ABO genotypes (O/O, B/B, A/A, A/O, A/B, and B/O) and the gender-determining locus Amelogenin. Primers are designed so that the amplicons are distributed ranging from 75bp to 500bp within a four-dye fluorescent design, leaving a fourth dye for the internal size standard. With 30 cycles, the results showed that the optimal amount of DNA template for this multiplex ranges from 250pg to 2ng and the lowest detection threshold is 125pg (as low as 63pg for ABO loci). For the DNA template outside the optimal detection range, we could adjust the number of cycles to obtain the robust profiles. Mixture studies showed that over 83% of minor alleles were detected at 1:9 ratios. The full profiles were still observed when 4ng of degraded DNA was digested by DNase I and 1ng undegraded DNA was added to 40μM haematin. Polymerase chain reaction (PCR)-based conditions including the concentrations of primers, magnesium and the Taq polymerase as well as volume, cycle numbers and annealing temperature were examined and optimised. In addition, the system was validated by 364 bloodstain samples and 32 common casework samples. According to the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, our system demonstrates good detection performance and is an ideal tool for forensic DNA typing with potential application. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. High-throughput multiplex single-nucleotide polymorphism analysis for red cell and platelet antigen genotypes.

    Science.gov (United States)

    Denomme, G A; Van Oene, M

    2005-05-01

    Transfusion recipients who become alloimmunized to red cell or platelet (PLT) antigens require antigen-negative blood to limit adverse transfusion reactions. Blood collection facilities use regulated and unregulated antibodies to phenotype blood, the cost of which can be prohibitive depending on the antisera and demand. An alternative strategy is to screen blood for these antigens with genomic DNA and the associated single-nucleotide polymorphisms (SNPs). A multiplex polymerase chain reaction (PCR)-oligonucleotide extension assay was developed with genomic DNA and a SNP genotyping platform (GenomeLab SNPstream, Beckman Coulter) to identify SNPs related to D, C/c, E, S/s, K/k, Kp(a/b), Fy(a/b), FY0 (-33 promoter silencing polymorphism), Jk(a/b), Di(a/b), and human PLT antigen (HPA)-1a/1b. A total of 372 samples were analyzed for 12 SNPs. The genotypes were compared to the blood group and PLT antigen phenotypes. Individual sample results varied from 98 to 100 percent for 11 of 12 SNPs. D was correctly identified in 292 of 296 (98.6%) D+ donors. The RHCE exon 5 E/e SNP analysis had the lowest concordance (89.5%). Thirty-three R(1)R(1) and 1 r"r were correctly identified. PCR-restriction fragment length polymorphism (RFLP) on selected samples confirmed the presence of the FY0 silencing polymorphism in nine donors. Homozygous HPA-1b/1b was identified in four donors, which was confirmed by PCR-RFLP (n = 4) and anti-HPA-1a serology (n = 2). The two HPA-1a-negative donors were recruited into the plateletpheresis program. The platform has the capacity to genotype thousands of samples per day. The suite of SNPs provides genotype data for all blood donors within 36 hours of the start of testing.

  3. Fast multiplex analysis of antibodies in complex sample matrix using the microfluidic Evalution™ platform.

    Science.gov (United States)

    Leirs, Karen; Leblebici, Pelin; Lammertyn, Jeroen; Spasic, Dragana

    2017-08-22

    Biosensor development requires comprehensive research for establishing the optimal bioassay conditions that determine the final kinetics, sensitivity and specificity. Different systems have been developed to study bioreceptor-target interactions but they often have drawbacks, such as long hands-on time, low throughput, high sample consumption and high cost. In this work, the potential of the novel microfluidic Evalution™ platform has been evaluated for developing sandwich-based assays in a fast and high-throughput fashion. An immunoassay for the detection of influenza A nucleoprotein was used as a model system. Exploiting the platform's unique features, various typical bioassay parameters (e.g. aspecific binding between assay components, different reagent concentrations and incubation times) were tested for three capture antibodies in a simple and fast manner (2 runs of 80 min). The selected conditions, giving the highest signal-to-noise ratio, were directly employed on the same platform to detect nucleoprotein in buffer and nasopharyngeal swabs. Two antibodies with a higher dissociation constant (Ab11 and Ab12) required longer incubation times (60 min) for sensitive detection (limit of detection (LOD) of 0.48 and 0.26 ng mL-1, respectively) compared to an antibody with lower dissociation constant (LOD of 0.04 ng mL-1 for Ab66 within 30 min). Moreover, one antibody (Ab12) showed limited capacity to capture nucleoprotein directly in sample matrix. The obtained results were in accordance with previous studies performed on an ELISA and SPR platform with the same antibodies. This positions the Evalution™ platform as a reliable platform for fast and multiplex analysis of antibodies' performance both in buffer and complex sample matrices. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Molecular analysis of miscarriage products using multiplex ligation-dependent probe amplification (MLPA): alternative to conventional karyotype analysis.

    Science.gov (United States)

    Kim, Ji Won; Lyu, Sang Woo; Sung, Se Ra; Sung, Sae Ra; Park, Ji Eun; Cha, Dong Hyun; Yoon, Tae Ki; Ko, Jung Jae; Shim, Sung Han

    2015-02-01

    The present study aims to evaluate whether multiplex ligation-dependent probe amplification (MLPA) technique with subtelomeric probes is to be an alternative method of routine G-banding chromosome analysis from pregnancy loss. A review of 5 years (from 2005 to 2009) of karyotype for products of conception (POCs) was carried out. From June 2010 to June 2012, MLPA was performed in parallel with karyotype analysis on 347 miscarriages. Karyotyped miscarriages served as controls in this blinded study. Abnormal results were confirmed by fluorescence in situ hybridization. A review of 5 years of karyotype results for POCs indicated that 11.46 % of cases failed to karyotyping. In the study periods, MLPA results were successfully obtained from all cases including 51 (14.7 %) culture failed cases, chromosomal abnormalities were detected in 27 (52.9 %) of cases which failed to grow or could not be cultivated. It took 3 weeks by conventional karyotyping, but it required at least 24 h and at most a week by MLPA from tissue sampling to final reporting. 47 cases showed discordant results between karyotyping and MLPA because of maternal cell contamination, polyploidy, mosaicism, or balanced translocation. MLPA technique is relatively low cost, less labor intensive and reduces waiting time with high accuracy compared with conventional cytogenetic analysis. Therefore, MLPA can be the first approach for chromosome analysis from pregnancy loss.

  5. Cluster analysis of multiplex ligation-dependent probe amplification data in choroidal melanoma.

    Science.gov (United States)

    Caines, Rhydian; Eleuteri, Antonio; Kalirai, Helen; Fisher, Anthony C; Heimann, Heinrich; Damato, Bertil E; Coupland, Sarah E; Taktak, Azzam F G

    2015-01-01

    To determine underlying correlations in multiplex ligation-dependent probe amplification (MLPA) data and their significance regarding survival following treatment of choroidal melanoma (CM). MLPA data were available for 31 loci across four chromosomes (1p, 3, 6, and 8) in tumor material obtained from 602 patients with CM treated at the Liverpool Ocular Oncology Center (LOOC) between 1993 and 2012. Data representing chromosomes 3 and 8q were analyzed in depth since their association with CM patient survival is well-known. Unsupervised k-means cluster analysis was performed to detect latent structure in the data set. Principal component analysis (PCA) was also performed to determine the intrinsic dimensionality of the data. Survival analyses of the identified clusters were performed using Kaplan-Meier (KM) and log-rank statistical tests. Correlation with largest basal tumor diameter (LTD) was investigated. Chromosome 3: A two-cluster (bimodal) solution was found in chromosome 3, characterized by centroids at unilaterally normal probe values and unilateral deletion. There was a large, significant difference in the survival characteristics of the two clusters (log-rank, pdefinition of 8q gain, and in conjunction with the chromosome 3 clusters, three prognostic groups were identified: chromosomes 3 and 8q both normal, either chromosome 3 or 8q abnormal, and both chromosomes 3 and 8q abnormal. KM analysis showed 5-year survival figures of approximately 97%, 80%, and 30% for these prognostic groups, respectively (log-rank, p<0.001). All MLPA probes within both chromosomes were significantly correlated with each other (Spearman, p<0.001). Within chromosome 3, the strong correlation between the MLPA variables and the uniform coefficients from the PCA indicates a lack of evidence for a signature gene that might account for the bimodality we observed. We hypothesize that the two clusters we found correspond to binary underlying states of complete monosomy or disomy 3 and that

  6. Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses

    Directory of Open Access Journals (Sweden)

    Deising Holger B

    2011-01-01

    Full Text Available Abstract Background The toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples. Results We took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024 groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide

  7. Computational tradeoffs in multiplex PCR assay design for SNP genotyping

    Directory of Open Access Journals (Sweden)

    Cantor Charles

    2005-07-01

    Full Text Available Abstract Background Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield. Results This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube, coverage (the % of SNP whose associated primers are actually assigned to one of several available tube, and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criterial. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels. Conclusion The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs

  8. Sum-rate analysis of spectrum sharing spatial multiplexing MIMO systems with zero-forcing and multiuser diversity

    KAUST Repository

    Yang, Liang

    2013-06-01

    This paper considers a multiuser spectrum sharing (SS) multiple-input multiple-output (MIMO) system with zero-forcing (ZF) operating in a Rayleigh fading environment. We provide an asymptotic sum-rate analysis to investigate the effects of different parameters on the multiuser diversity gain. For a ZF SS spatial multiplexing system with scheduling, the asymptotic sum-rate scales like Nt log2(Q(Nt Np√K - 1)/N t), where Np denotes the number of antennas of primary receiver, Q is the interference temperature, and K represents the number of secondary transmitters. © 2013 IEEE.

  9. Cytokine profiles as markers of disease severity in sepsis: a multiplex analysis

    OpenAIRE

    Bozza, Fernando A; Salluh, Jorge I; Japiassu, André M; Soares, Marcio; Assis, Edson F; Gomes, Rachel N; Bozza, Marcelo T; Castro-Faria-Neto, Hugo C; Bozza, Patrícia T

    2007-01-01

    Introduction The current shortage of accurate and readily available, validated biomarkers of disease severity in sepsis is an important limitation when attempting to stratify patients into homogeneous groups, in order to study pathogenesis or develop therapeutic interventions. The aim of the present study was to determine the cytokine profile in plasma of patients with severe sepsis by using a multiplex system for simultaneous detection of 17 cytokines. Methods This was a prospective cohort s...

  10. Development of genomic microsatellite multiplex PCR using dye-labeled universal primer and its validation in pedigree analysis of Pacific oyster ( Crassostrea gigas)

    Science.gov (United States)

    Liu, Ting; Li, Qi; Song, Junlin; Yu, Hong

    2017-02-01

    There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.

  11. Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice.

    Science.gov (United States)

    Fehlings, M; Chakarov, S; Simoni, Y; Sivasankar, B; Ginhoux, F; Newell, E W

    2018-02-01

    Antigen-specific T cells play a crucial role for the host protective immunity against viruses and other diseases. The use of mass cytometry together with a combinatorial multiplex tetramer staining has successfully been applied for probing and characterization of multiple antigen-specific CD8 + T cells in human blood samples. The present study shows that this approach can also be used to rapidly assess the magnitude of influenza-specific CD8 + T cell epitope dominance across lymph nodes and lungs in a murine model of a highly pathological influenza infection. Moreover, we show feasibility of extending this approach to include concurrent identification of virus-specific CD4 + T cells. By using a double coding approach, we probed for five influenza-specific MHCI-peptide complexes as well as one influenza-specific MHCII-peptide complex in the presence of irrelevant control peptides and show that this approach is capable of tracking antigen-specific T cells across individual lymph nodes and lungs. The simultaneous staining with 26 surface maker molecules further facilitated an in-depth characterization of T cells reacting with influenza epitopes and revealed tissue specific phenotypic differences between CD4 + T cells targeting the same pathogenic epitope. In conclusion, this approach provides the possibility for a rapid and comprehensive analysis of antigen-specific CD8 + and CD4 + T cells in different disease settings that might be advantageous for subsequent vaccine formulation strategies. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Accelerated genome engineering through multiplexing.

    Science.gov (United States)

    Bao, Zehua; Cobb, Ryan E; Zhao, Huimin

    2016-01-01

    Throughout the biological sciences, the past 15 years have seen a push toward the analysis and engineering of biological systems at the organism level. Given the complexity of even the simplest organisms, though, to elicit a phenotype of interest often requires genotypic manipulation of several loci. By traditional means, sequential editing of genomic targets requires a significant investment of time and labor, as the desired editing event typically occurs at a very low frequency against an overwhelming unedited background. In recent years, the development of a suite of new techniques has greatly increased editing efficiency, opening up the possibility for multiple editing events to occur in parallel. Termed as multiplexed genome engineering, this approach to genome editing has greatly expanded the scope of possible genome manipulations in diverse hosts, ranging from bacteria to human cells. The enabling technologies for multiplexed genome engineering include oligonucleotide-based and nuclease-based methodologies, and their application has led to the great breadth of successful examples described in this review. While many technical challenges remain, there also exists a multiplicity of opportunities in this rapidly expanding field. © 2015 Wiley Periodicals, Inc.

  13. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number

    Directory of Open Access Journals (Sweden)

    Dehghan fatemeh

    2014-05-01

    Conclusions: Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN.

  14. MicroRNA expression analysis and Multiplex ligation-dependent probe amplification in metastatic and non-metastatic uveal melanoma

    DEFF Research Database (Denmark)

    Larsen, Ann-Cathrine; Holst, Line; Kaczkowski, Bogumil

    2014-01-01

    Purpose: To determine the association of microRNA expression and chromosomal changes with metastasis and survival in uveal melanoma (UM). Methods: Thirty-six patients with UM were selected based on the metastatic status, and clinicopathological data were collected. Multiplex ligation-dependent pr......Purpose: To determine the association of microRNA expression and chromosomal changes with metastasis and survival in uveal melanoma (UM). Methods: Thirty-six patients with UM were selected based on the metastatic status, and clinicopathological data were collected. Multiplex ligation...... regression analysis were performed to determine the association with metastasis and survival. Results: Metastasis and metastatic death occurred in 20 patients, two patients died of other causes and one patient of unknown causes. A significant association between increasing size category (p = 0.002, log...... regression analysis), mixed (p = 0.029) and epithelioid (p = 0.0058) cell types, chromosome 3 loss (p 0.014) and 8q gain (p = 0.010) were significant prognosticators for a poor survival. Hierarchical clustering divided the UM into three groups based on microRNA expression. The clusters showed no association...

  15. Denoising analysis of spatial pixel multiplex coded spectrometer with Hadamard H-matrix

    Science.gov (United States)

    Yue, Jiang; Han, Jing; Li, Long; Bai, Lian-fa

    2018-01-01

    We present a multiplex coded Hadamard transform spectrometer with which Hadamard H-matrix is employed to encode the entrance pixels. Specifically, the Hadamard H-matrix is divided into two coding matrices, H+ and H-; here H- =(H - J) / 2 and H+ =(H + J) / 2, where each element of J equals 1. We analyzed its denoising capability and compared with the ordinary Hadamard spectrometer with cyclic-S matrix coded and slit spectrometers. The simulation and experiment show that the proposed Hadamard spectrometer with H-matrix outperforms Hadamard transform spectrometer with cyclic-S matrix, gaining about 2 dB of the signal-to-noise ratio.

  16. Queueing analysis of synchronous time division multiplexing with individual source buffering

    Science.gov (United States)

    Clare, L.

    1982-01-01

    Synchronous time division multiplexing is analyzed. Packets of information arrive at the system as a compound Poisson process, and are transmitted only during individual periodic intervals. Packet arrivals are blocked (lost) if the system has a finite capacity and is congested. Using the theory of semiregenerative processes, the distribution of the number of packets in the system (system size) is found. This nonstationary distribution is used to determine the complete system behavior, including the delay distributions, the blocking probability, and the density of the system size at arrival instants. Numerical examples illustrate applications of the results given.

  17. On-Chip integration of sample pretreatment and Multiplex polymerase chain reaction (PCR) for DNA analysis

    DEFF Research Database (Denmark)

    Brivio, Monica; Snakenborg, Detlef; Søgaard, E.

    2008-01-01

    In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a custom-made thermocycler (Figure 1) and includes a simple...... and efficient method for switching the liquid flow between the PT and PCR chamber. Purification of human genomic DNA from EDTA-treated blood and multiplex PCR were successfully carried out on-chip using the developed lab-on-a-chip system....

  18. Analysis of hybrid subcarrier multiplexing of OCDMA based on single photodiode detection

    Science.gov (United States)

    Ahmad, N. A. A.; Junita, M. N.; Aljunid, S. A.; Rashidi, C. B. M.; Endut, R.

    2017-11-01

    This paper analyzes the performance of subcarrier multiplexing (SCM) of spectral amplitude coding optical code multiple access (SAC-OCDMA) by applying Recursive Combinatorial (RC) code based on single photodiode detection (SPD). SPD is used in the receiver part to reduce the effect of multiple access interference (MAI) which contributes as a dominant noise in incoherent SAC-OCDMA systems. Results indicate that the SCM OCDMA network performance could be improved by using lower data rates and higher number of weight. Total number of users can also be enhanced by adding lower data rates and higher number of subcarriers.

  19. [Application of combined chromosome karyotype analysis and multiplex ligation probe amplification technique for the prenatal diagnosis of fetal abnormalities].

    Science.gov (United States)

    Jiang, Nan; Yu, Dongyi

    2016-12-10

    To assess the value of combined chromosome karyotype analysis and multiplex ligation probe amplification (MLPA) assay for the prenatal diagnosis of fetuses with abnormalities detected by ultrasonography. With informed consent obtained, 72 pregnant women with ultrasound detected fetal structural abnormalities underwent percutaneous umbilical cord blood sampling. Routine karyotype analysis and MLPA assay were used to detect potential chromosomal deletions and duplications. Five cases were found with an abnormal karyotype. In addition, the MLPA has detected 2 chromosomal microdeletions and 1 microduplication. Together the two methods have yielded a detection rate of 11.11%. For fetal abnormalities revealed by ultrasonography, combined karyotype analysis and MLPA assay can provide a better option for its efficiency and simplicity.

  20. Finite-SNR Diversity-Multiplexing Tradeoff via Asymptotic Analysis of Large MIMO Systems

    CERN Document Server

    Loyka, Sergey

    2010-01-01

    Diversity-multiplexing tradeoff (DMT) was characterized asymptotically (SNR-> infinity) for i.i.d. Rayleigh fading channel by Zheng and Tse [1]. The SNR-asymptotic DMT overestimates the finite-SNR one [2]. This paper outlines a number of additional limitations and difficulties of the DMT framework and discusses their implications. Using the recent results on the size-asymptotic (in the number of antennas) outage capacity distribution, the finite-SNR, size-asymptotic DMT is derived for a broad class of fading distributions. The SNR range over which the finite-SNR DMT is accurately approximated by the SNR-asymptotic one is characterized. The multiplexing gain definition is shown to affect critically this range and thus should be carefully selected, so that the SNR-asymptotic DMT is an accurate approximation at realistic SNR values and thus has operational significance to be used as a design criteria. The finite SNR diversity gain is shown to decrease with correlation and power imbalance in a broad class of fadi...

  1. Microfluidic Wheatstone bridge for rapid sample analysis.

    Science.gov (United States)

    Tanyeri, Melikhan; Ranka, Mikhil; Sittipolkul, Natawan; Schroeder, Charles M

    2011-12-21

    We developed a microfluidic analogue of the classic Wheatstone bridge circuit for automated, real-time sampling of solutions in a flow-through device format. We demonstrate precise control of flow rate and flow direction in the "bridge" microchannel using an on-chip membrane valve, which functions as an integrated "variable resistor". We implement an automated feedback control mechanism in order to dynamically adjust valve opening, thereby manipulating the pressure drop across the bridge and precisely controlling fluid flow in the bridge channel. At a critical valve opening, the flow in the bridge channel can be completely stopped by balancing the flow resistances in the Wheatstone bridge device, which facilitates rapid, on-demand fluid sampling in the bridge channel. In this article, we present the underlying mechanism for device operation and report key design parameters that determine device performance. Overall, the microfluidic Wheatstone bridge represents a new and versatile method for on-chip flow control and sample manipulation.

  2. Multiplex PCR provides a low-cost alternative to DNA probe methods for rapid identification of Mycobacterium avium and Mycobacterium intracellulare.

    OpenAIRE

    Cousins, D; Francis, B; Dawson, D

    1996-01-01

    A multiplex PCR designed to differentiate Mycobacterium tuberculosis complex organisms from M. avium and M. intracellulare was used to test 105 isolates identified by DNA probe methods as M. avium, M. intracellulare, or M. avium complex type X. The multiple PCR correctly identified 33 of 34 isolates identified by commercial probe methods as M. avium and all 51 isolates identified as M. intracellulare. The 20 isolates identified as M. avium complex type X by probe were identified as Mycobacter...

  3. Electrochemical sensor for multiplex screening of genetically modified DNA: identification of biotech crops by logic-based biomolecular analysis.

    Science.gov (United States)

    Liao, Wei-Ching; Chuang, Min-Chieh; Ho, Ja-An Annie

    2013-12-15

    Genetically modified (GM) technique, one of the modern biomolecular engineering technologies, has been deemed as profitable strategy to fight against global starvation. Yet rapid and reliable analytical method is deficient to evaluate the quality and potential risk of such resulting GM products. We herein present a biomolecular analytical system constructed with distinct biochemical activities to expedite the computational detection of genetically modified organisms (GMOs). The computational mechanism provides an alternative to the complex procedures commonly involved in the screening of GMOs. Given that the bioanalytical system is capable of processing promoter, coding and species genes, affirmative interpretations succeed to identify specified GM event in terms of both electrochemical and optical fashions. The biomolecular computational assay exhibits detection capability of genetically modified DNA below sub-nanomolar level and is found interference-free by abundant coexistence of non-GM DNA. This bioanalytical system, furthermore, sophisticates in array fashion operating multiplex screening against variable GM events. Such a biomolecular computational assay and biosensor holds great promise for rapid, cost-effective, and high-fidelity screening of GMO. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging.

    Science.gov (United States)

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-09-22

    We report a fast non-iterative lifetime data analysis method for the Fourier multiplexed frequency-sweeping confocal FLIM (Fm-FLIM) system [Opt. Express 22, 10221 (2014)]. The new method, named R-method, allows fast multi-channel lifetime image analysis in the system's FPGA data processing board. Experimental tests proved that the performance of the R-method is equivalent to that of single-exponential iterative fitting, and its sensitivity is well suited for time-lapse FLIM-FRET imaging of live cells, for example cyclic adenosine monophosphate (cAMP) level imaging with GFP-Epac-mCherry sensors. With the R-method and its FPGA implementation, multi-channel lifetime images can now be generated in real time on the multi-channel frequency-sweeping FLIM system, and live readout of FRET sensors can be performed during time-lapse imaging.

  5. Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles.

    Science.gov (United States)

    Wang, Hye-Young; Kim, Seoyong; Kim, Jungho; Park, Soon Deok; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; PReal-methicillin-resistant Staphylococcusaureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.

  6. Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platform (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    Energy Technology Data Exchange (ETDEWEB)

    Tremblay, Julien

    2012-06-01

    Julien Tremblay from DOE JGI presents "Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platorm" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  7. Discrimination of chemical warfare simulants via multiplex coherent anti-Stokes Raman scattering and multivariate statistical analysis

    Science.gov (United States)

    Brady, John J.; Farrell, Mikella E.; Pellegrino, Paul M.

    2014-02-01

    Multiplex coherent anti-Stokes Raman scattering (MCARS) is used to detect several chemical warfare simulants, such as dimethyl methylphosphonate and 2-chloroethyl ethyl sulfide, with high specificity. The spectral bandwidth of the femtosecond laser pulse used in these studies is sufficient to coherently and simultaneously drive all the vibrational modes in the molecule of interest. Evidence shows that MCARS is capable of overcoming common sensitivity limitations of spontaneous Raman scattering, thus allowing for the detection of the target material in milliseconds with standard, uncooled universal serial bus spectrometers as opposed to seconds with cooled, intensified CCD-based spectrometers. In addition, the obtained MCARS spectrum of the investigated sample provides multiple unique signatures. These signatures are used in an off-line multivariate statistical analysis allowing for the material's discrimination with high fidelity.

  8. Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

    Science.gov (United States)

    Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

    Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

  9. Rapid Exploitation and Analysis of Documents

    Energy Technology Data Exchange (ETDEWEB)

    Buttler, D J; Andrzejewski, D; Stevens, K D; Anastasiu, D; Gao, B

    2011-11-28

    Analysts are overwhelmed with information. They have large archives of historical data, both structured and unstructured, and continuous streams of relevant messages and documents that they need to match to current tasks, digest, and incorporate into their analysis. The purpose of the READ project is to develop technologies to make it easier to catalog, classify, and locate relevant information. We approached this task from multiple angles. First, we tackle the issue of processing large quantities of information in reasonable time. Second, we provide mechanisms that allow users to customize their queries based on latent topics exposed from corpus statistics. Third, we assist users in organizing query results, adding localized expert structure over results. Forth, we use word sense disambiguation techniques to increase the precision of matching user generated keyword lists with terms and concepts in the corpus. Fifth, we enhance co-occurrence statistics with latent topic attribution, to aid entity relationship discovery. Finally we quantitatively analyze the quality of three popular latent modeling techniques to examine under which circumstances each is useful.

  10. Automated wholeslide analysis of multiplex-brightfield IHC images for cancer cells and carcinoma-associated fibroblasts

    Science.gov (United States)

    Lorsakul, Auranuch; Andersson, Emilia; Vega Harring, Suzana; Sade, Hadassah; Grimm, Oliver; Bredno, Joerg

    2017-03-01

    Multiplex-brightfield immunohistochemistry (IHC) staining and quantitative measurement of multiple biomarkers can support therapeutic targeting of carcinoma-associated fibroblasts (CAF). This paper presents an automated digitalpathology solution to simultaneously analyze multiple biomarker expressions within a single tissue section stained with an IHC duplex assay. Our method was verified against ground truth provided by expert pathologists. In the first stage, the automated method quantified epithelial-carcinoma cells expressing cytokeratin (CK) using robust nucleus detection and supervised cell-by-cell classification algorithms with a combination of nucleus and contextual features. Using fibroblast activation protein (FAP) as biomarker for CAFs, the algorithm was trained, based on ground truth obtained from pathologists, to automatically identify tumor-associated stroma using a supervised-generation rule. The algorithm reported distance to nearest neighbor in the populations of tumor cells and activated-stromal fibroblasts as a wholeslide measure of spatial relationships. A total of 45 slides from six indications (breast, pancreatic, colorectal, lung, ovarian, and head-and-neck cancers) were included for training and verification. CK-positive cells detected by the algorithm were verified by a pathologist with good agreement (R2=0.98) to ground-truth count. For the area occupied by FAP-positive cells, the inter-observer agreement between two sets of ground-truth measurements was R2=0.93 whereas the algorithm reproduced the pathologists' areas with R2=0.96. The proposed methodology enables automated image analysis to measure spatial relationships of cells stained in an IHC-multiplex assay. Our proof-of-concept results show an automated algorithm can be trained to reproduce the expert assessment and provide quantitative readouts that potentially support a cutoff determination in hypothesis testing related to CAF-targeting-therapy decisions.

  11. A rapid genotyping method for an obligate fungal pathogen, Puccinia striiformis f.sp. tritici, based on DNA extraction from infected leaf and Multiplex PCR genotyping

    Directory of Open Access Journals (Sweden)

    Enjalbert Jérôme

    2011-07-01

    Full Text Available Abstract Background Puccinia striiformis f.sp. tritici (PST, an obligate fungal pathogen causing wheat yellow/stripe rust, a serious disease, has been used to understand the evolution of crop pathogen using molecular markers. However, numerous questions regarding its evolutionary history and recent migration routes still remains to be addressed, which need the genotyping of a large number of isolates, a process that is limited by both DNA extraction and genotyping methods. To address the two issues, we developed here a method for direct DNA extraction from infected leaves combined with optimized SSR multiplexing. Findings We report here an efficient protocol for direct fungal DNA extraction from infected leaves, avoiding the costly and time consuming step of spore multiplication. The genotyping strategy we propose, amplified a total of 20 SSRs in three Multiplex PCR reactions, which were highly polymorphic and were able to differentiate different PST populations with high efficiency and accuracy. Conclusion These two developments enabled a genotyping strategy that could contribute to the development of molecular epidemiology of yellow rust disease, both at a regional or worldwide scale.

  12. Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Lan, Ruiting; Xu, Huaqing; Ma, Aijing; Li, Dongxun; Dai, Hang; Yuan, Xuejiao; Xu, Jianguo; Ye, Changyun

    2015-07-01

    Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  13. Development of a multiplexed microsphere PCR for rapid, culture-free detection and Gram-typing of bacteria in human blood samples.

    Science.gov (United States)

    Liang, Fang; Browne, Daniel J; Gray, Megan J; Gartlan, Kate H; Smith, David D; Barnard, Ross; Hill, Geoff R; Corrie, Simon Robert; Markey, Kate

    2018-01-19

    Blood stream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected blood stream infection remain centered around blood culture (highly variable timing, hours to days), and empiric use of broad-spectrum antibiotics is often employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g. combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives), however current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5-hour time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labelled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labelled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.

  14. Rapid detection and identification of human hookworm infections through high resolution melting (HRM analysis.

    Directory of Open Access Journals (Sweden)

    Romano Ngui

    Full Text Available BACKGROUND: Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR coupled with high resolution melting-curve (HRM analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. METHODS: Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2 of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. CONCLUSION: The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

  15. Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis.

    Science.gov (United States)

    Ngui, Romano; Lim, Yvonne A L; Chua, Kek Heng

    2012-01-01

    Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

  16. The multiplex dependency structure of financial markets

    CERN Document Server

    Musmeci, Nicoló; Aste, Tomaso; Di Matteo, Tiziana; Latora, Vito

    2016-01-01

    We propose here a multiplex network approach to investigate simultaneously different types of dependency in complex data sets. In particular, we consider multiplex networks made of four layers corresponding respectively to linear, non-linear, tail, and partial correlations among a set of financial time series. We construct the sparse graph on each layer using a standard network filtering procedure, and we then analyse the structural properties of the obtained multiplex networks. The study of the time evolution of the multiplex constructed from financial data uncovers important changes in intrinsically multiplex properties of the network, and such changes are associated with periods of financial stress. We observe that some features are unique to the multiplex structure and would not be visible otherwise by the separate analysis of the single-layer networks corresponding to each dependency measure.

  17. The Multiplex Dependency Structure of Financial Markets

    Directory of Open Access Journals (Sweden)

    Nicolò Musmeci

    2017-01-01

    Full Text Available We propose here a multiplex network approach to investigate simultaneously different types of dependency in complex datasets. In particular, we consider multiplex networks made of four layers corresponding, respectively, to linear, nonlinear, tail, and partial correlations among a set of financial time series. We construct the sparse graph on each layer using a standard network filtering procedure, and we then analyse the structural properties of the obtained multiplex networks. The study of the time evolution of the multiplex constructed from financial data uncovers important changes in intrinsically multiplex properties of the network, and such changes are associated with periods of financial stress. We observe that some features are unique to the multiplex structure and would not be visible otherwise by the separate analysis of the single-layer networks corresponding to each dependency measure.

  18. Towards a pathogenic Escherichia coli detection platform using multiplex SYBR®Green Real-time PCR methods and high resolution melting analysis.

    Directory of Open Access Journals (Sweden)

    Dafni-Maria Kagkli

    Full Text Available Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2 producing E. coli (VTEC or STEC respectively have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA has requested the monitoring of the "top-five" serogroups (O26, O103, O111, O145 and O157 most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae in the case of VTEC, or aggregative protein (aggR, in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform.

  19. Air segmented amplitude modulated multiplexed flow analysis with software-based phase recognition: determination of phosphate ion.

    Science.gov (United States)

    Ogusu, Takeshi; Uchimoto, Katsuya; Takeuchi, Masaki; Tanaka, Hideji

    2014-01-01

    Amplitude modulated multiplexed flow analysis (AMMFA) has been improved by introducing air segmentation and software-based phase recognition. Sample solutions, the flow rates of which are respectively varied at different frequencies, are merged. Air is introduced to the merged liquid stream in order to limit the dispersion of analytes within each liquid segment separated by air bubbles. The stream is led to a detector with no physical deaeration. Air signals are distinguished from liquid signals through the analysis of detector output signals, and are suppressed down to the level of liquid signals. Resulting signals are smoothed based on moving average computation. Thus processed signals are analyzed by fast Fourier transform. The analytes in the samples are respectively determined from the amplitudes of the corresponding wave components obtained. The developed system has been applied to the simultaneous determinations of phosphate ions in water samples by a Malachite Green method. The linearity of the analytical curve (0.0-31.0 μmol dm(-3)) is good (r(2)>0.999) and the detection limit (3.3 σ) at the modulation period of 30s is 0.52 μmol dm(-3). Good recoveries around 100% have been obtained for phosphate ions spiked into real water samples. © 2013 Elsevier B.V. All rights reserved.

  20. Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice.

    Directory of Open Access Journals (Sweden)

    Hong-Jhang Chen

    Full Text Available Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD, a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877, 1.71% (15/877, and 2.62% (23/877 of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood-brain barrier (BBB (Alb, Fga, and Trf, suppressed excitotoxicity (Grm5, Gnai, and Gdi, and enhanced energy metabolism (Bdh, thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3 and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke.

  1. Simultaneous confirmatory analysis of different transgenic maize (zea mays) lines using multiplex polymerase chain reaction-restriction analysis and capillary gel electrophoresis with laser induced fluorescence detection.

    Science.gov (United States)

    García-Cañas, Virginia; Cifuentes, Alejandro

    2008-09-24

    A novel analytical procedure based on the combination of multiplex PCR, restriction analysis, and CGE-LIF to unambiguosly and simultaneously confirm the presence of multiple lines of genetically modified corn is proposed. This methodology is based on the amplification of event-specific DNA regions by multiplex PCR using 6-FAM-labeled primers. Subsequently, PCR products are digested by a mixture containing specific restriction endonucleases. Thus, restriction endonucleases selectively recognize DNA target sequences contained in the PCR products and cleave the double-stranded DNA at a given cleavage site. Next, the restriction digest is analyzed by CGE-LIF corroborating the length of the expected restriction fragments, confirming (or not) the existence of GMOs. For accurate size determination of the DNA fragments by CGE-LIF a special standard DNA mixture was produced in this laboratory for calibration. The suitability of this mixture for size determination of labeled DNA fragments is also demonstrated. The usefulness of the proposed methodology is demonstrated through the simultaneous detection and confirmatory analysis of samples containing 0.5% of GA21 and MON863 maize plus an endogenous gene of maize as control.

  2. 2D Gel-Based Multiplexed Proteomic Analysis during Larval Development and Metamorphosis of the Biofouling Polychaete Tubeworm Hydroides elegans

    KAUST Repository

    Zhang, Yu

    2010-09-03

    Larval settlement and metamorphosis of a common biofouling polychaete worm, Hydroides elegans, involve remarkable structural and physiological changes during this pelagic to sessile habitat shift. The endogenous protein molecules and post-translational modifications that drive this larval transition process are not only of interest to ecologists but also to the antifouling paint industry, which aims to control the settlement of this biofouling species on man-made structures (e.g., ship hulls). On the basis of our recent proteomic studies, we hypothesize that rapid larval settlement of H. elegans could be mediated through changes in phosphorylation status of proteins rather than extensive de novo synthesis of proteins. To test this hypothesis, 2D gel-based multiplexed proteomics technology was used to monitor the changes in protein expression and phosphorylation status during larval development and metamorphosis of H. elegans. The protein expression profiles of larvae before and after they reached competency to attach and metamorphose were similar in terms of major proteins, but the percentage of phosphorylated proteins increased from 41% to 49% after competency. Notably, both the protein and phosphoprotein profiles of the metamorphosed individuals (adult) were distinctly different from that of the larvae, with only 40% of the proteins phosphorylated in the adult stage. The intensity ratio of all phosphoprotein spots to all total protein spots was also the highest in the competent larval stage. Overall, our results indicated that the level of protein phosphorylation might play a crucial role in the initiation of larval settlement and metamorphosis. © 2010 American Chemical Society.

  3. Comparative analysis of acon- Plasmodium falciparum rapid malaria ...

    African Journals Online (AJOL)

    Comparative analysis of acon- Plasmodium falciparum rapid malaria diagnostic test with routine microscopy among school children and pregnant women in a rural community in Enugu State, ... The following parameters were determined: intrinsic validity, predictive values, species diagnostic power and logistic factors.

  4. Rapid Automatized Naming and Reading Performance: A Meta-Analysis

    Science.gov (United States)

    Araújo, Susana; Reis, Alexandra; Petersson, Karl Magnus; Faísca, Luís

    2015-01-01

    Evidence that rapid naming skill is associated with reading ability has become increasingly prevalent in recent years. However, there is considerable variation in the literature concerning the magnitude of this relationship. The objective of the present study was to provide a comprehensive analysis of the evidence on the relationship between rapid…

  5. Rapid inorganic ion analysis using quantitative microchip capillary electrophoresis

    NARCIS (Netherlands)

    Vrouwe, E.X.; Lüttge, Regina; Olthuis, Wouter; van den Berg, Albert

    2006-01-01

    Rapid quantitative microchip capillary electrophoresis (CE) for online monitoring of drinking water enabling inorganic ion separation in less than 15s is presented. Comparing cationic and anionic standards at different concentrations the analysis of cationic species resulted in non-linear

  6. The FilmArray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens.

    Science.gov (United States)

    Babady, N Esther

    2013-11-01

    The FilmArray Respiratory Panel (RP) (BioFire(™) Diagnostics, Inc., Salt Lake City, UT, USA) is the first multiplex molecular panel cleared by the US FDA for the detection of both bacterial and viral respiratory pathogens in nasopharygeal swabs. The FilmArray RP targets 20 pathogens including 17 viruses and subtypes and three bacteria, and is performed with minimal sample manipulation. The FilmArray RP has a fully automated sample-to-answer workflow with a turn-around-time of approximately 1 h. The reported sensitivity and specificity of the assay ranges from 80 to 100 and 100%, respectively, with the sensitivity for the adenovirus as low as 46%. A new version of the FilmArray RP assay (version 1.7) with improved sensitivity for the adenovirus was released in 2013. The performance characteristics and simplified workflow have allowed its implementation in a wide range of laboratories. The FilmArray RP has changed the diagnostic landscape and will have a significant impact on the care of patients with respiratory tract infection.

  7. Rapid detection of aneuploidy and unbalanced chromosomal rearrangements by subtelomeric multiplex ligation-dependent probe amplification in fetuses with congenital heart disease.

    Science.gov (United States)

    Wang, Jing; Liu, Zhen; Liu, Hongqian; Li, Nana; Li, Shengli; Chen, Xinlin; Lin, Yuan; Wang, He; Zhu, Jun; Liu, Shanling

    2013-01-01

    To validate multiplex ligation-dependent probe amplification (MLPA) with subtelomeric probe mixes as a tool for diagnosis of aneuploidy and unbalanced terminal chromosomal rearrangements in fetuses with congenital heart disease. A prospective study of 117 fetuses found to have structural heart defects by ultrasound at 17-40 weeks' gestation. MLPA with P036E and P070B probe mixes was performed and compared to traditional karyotyping by cell culture and to findings of quantitative fluorescence-polymerase chain reaction (QF-PCR). MLPA was able to define the fetal karyotype in 99% of cases whereas cell culture only defined the karyotype in 64% of cases. Consequently, the overall number of chromosomal abnormalities that were detected increased. The majority of these affected chromosomes, 21, 18, 13, X or Y, were also confirmed by QF-PCR. Two (5%) cases had atypical aneuploidy that was confirmed by MLPA but not by QF-PCR. In 4 cases, structural rearrangements or mosaicism were not detected by MLPA. Subtelomeric MLPA may be a valuable adjunct to QF-PCR and/or conventional cytogenetics for the investigation of chromosomal abnormalities in fetuses with congenital heart disease. Copyright © 2013 S. Karger AG, Basel.

  8. Rapid molecular characterization of Acinetobacter baumannii clones with rep-PCR and evaluation of carbapenemase genes by new multiplex PCR in Hospital District of Helsinki and Uusimaa.

    Directory of Open Access Journals (Sweden)

    Tanja Pasanen

    Full Text Available Multidrug-resistant Acinetobacter baumannii (MDRAB is an increasing problem worldwide. Prevalence of carbapenem resistance in Acinetobacter spp. due to acquired carbapenemase genes is not known in Finland. The purpose of this study was to examine prevalence and clonal spread of multiresistant A. baumannii group species, and their carbapenemase genes. A total of 55 Acinetobacter isolates were evaluated with repetitive PCR (DiversiLab to analyse clonality of isolates, in conjunction with antimicrobial susceptibility profile for ampicillin/sulbactam, colistin, imipenem, meropenem, rifampicin and tigecycline. In addition, a new real-time PCR assay, detecting most clinically important carbapenemase genes just in two multiplex reactions, was developed. The assay detects genes for KPC, VIM, IMP, GES-1/-10, OXA-48, NDM, GIM-1, SPM-1, IMI/NMC-A, SME, CMY-10, SFC-1, SIM-1, OXA-23-like, OXA-24/40-like, OXA-58 and ISAbaI-OXA-51-like junction, and allows confident detection of isolates harbouring acquired carbapenemase genes. There was a time-dependent, clonal spread of multiresistant A. baumannii strongly correlating with carbapenamase gene profile, at least in this geographically restricted study material. The new carbapenemase screening assay was able to detect all the genes correctly suggesting it might be suitable for epidemiologic screening purposes in clinical laboratories.

  9. A multiplex polymerase chain reaction based method for rapid identification of two species of the genus Scolytus Geoffroy (Col: Curculionidae: Scolytinae in Iran

    Directory of Open Access Journals (Sweden)

    S. Amini

    2016-04-01

    Full Text Available Molecular identification is going to be more widespread in taxonomic studies of insects when traditional tools are problematic and time consuming. Identification of bark beetles, as one of the most important pests of forests, based on morphological characteristics is difficult because of their small size and morphological similarities. In the current study, species-specific primers were desi gned to identify two most abundant and morphologically similar bark beetle species Scolytus ensifer Eichhoff 1881 and S. ecksteini Butovitsch 1929, both found on Ulmus minor Miller in north of Iran. These species-specific primers successfully produced a fragment size with 318 bp and 465 bp of mitochondrial cytochrome oxidase 1 (CO1 gene in S. ensifer and S. ecksteini respectively. The results revealed tha t the multiplex polymerase chain reaction using the species-specific primers could amplify a unique band to distinguish these two species so confirmed this method as a convenient and quick tool to identify those two bark beetle species.

  10. Development of a rapid multiplex PCR assay to genotype Pasteurella multocida strains by use of the lipopolysaccharide outer core biosynthesis locus.

    Science.gov (United States)

    Harper, Marina; John, Marietta; Turni, Conny; Edmunds, Mark; St Michael, Frank; Adler, Ben; Blackall, P J; Cox, Andrew D; Boyce, John D

    2015-02-01

    Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Bacterial Nanoscale Cultures for Phenotypic Multiplexed Antibiotic Susceptibility Testing

    Science.gov (United States)

    Weibull, Emilie; Antypas, Haris; Kjäll, Peter; Brauner, Annelie; Andersson-Svahn, Helene

    2014-01-01

    An optimal antimicrobial drug regimen is the key to successful clinical outcomes of bacterial infections. To direct the choice of antibiotic, access to fast and precise antibiotic susceptibility profiling of the infecting bacteria is critical. We have developed a high-throughput nanowell antibiotic susceptibility testing (AST) device for direct, multiplexed analysis. By processing in real time the optical recordings of nanoscale cultures of reference and clinical uropathogenic Escherichia coli strains with a mathematical algorithm, the time point when growth shifts from lag phase to early logarithmic phase (Tlag) was identified for each of the several hundreds of cultures tested. Based on Tlag, the MIC could be defined within 4 h. Heatmap presentation of data from this high-throughput analysis allowed multiple resistance patterns to be differentiated at a glance. With a possibility to enhance multiplexing capacity, this device serves as a high-throughput diagnostic tool that rapidly aids clinicians in prescribing the optimal antibiotic therapy. PMID:24989602

  12. Rapid RNA-ligand interaction analysis through high-information content conformational and stability landscapes

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Nathan J. [National Inst. of Health (NIH), Bethesda, MD (United States); Inglese, James [National Inst. of Health (NIH), Bethesda, MD (United States); Ferré-D’Amaré, Adrian R. [National Inst. of Health (NIH), Bethesda, MD (United States)

    2015-12-07

    The structure and biological properties of RNAs are a function of changing cellular conditions, but comprehensive, simultaneous investigation of the effect of multiple interacting environmental variables is not easily achieved. We have developed an efficient, high-throughput method to characterize RNA structure and thermodynamic stability as a function of multiplexed solution conditions using Förster resonance energy transfer (FRET). In a single FRET experiment using conventional quantitative PCR instrumentation, 19,400 conditions of MgCl2, ligand and temperature are analysed to generate detailed empirical conformational and stability landscapes of the cyclic diguanylate (c-di-GMP) riboswitch. This method allows rapid comparison of RNA structure modulation by cognate and non-cognate ligands. Landscape analysis reveals that kanamycin B stabilizes a non-native, idiosyncratic conformation of the riboswitch that inhibits c-di-GMP binding. Our research demonstrates that allosteric control of folding, rather than direct competition with cognate effectors, is a viable approach for pharmacologically targeting riboswitches and other structured RNA molecules.

  13. A lab-on-a-chip system integrating tissue sample preparation and multiplex RT-qPCR for gene expression analysis in point-of-care hepatotoxicity assessment.

    Science.gov (United States)

    Lim, Geok Soon; Chang, Joseph S; Lei, Zhang; Wu, Ruige; Wang, Zhiping; Cui, Kemi; Wong, Stephen

    2015-10-21

    A truly practical lab-on-a-chip (LOC) system for point-of-care testing (POCT) hepatotoxicity assessment necessitates the embodiment of full-automation, ease-of-use and "sample-in-answer-out" diagnostic capabilities. To date, the reported microfluidic devices for POCT hepatotoxicity assessment remain rudimentary as they largely embody only semi-quantitative or single sample/gene detection capabilities. In this paper, we describe, for the first time, an integrated LOC system that is somewhat close to a practical POCT hepatotoxicity assessment device - it embodies both tissue sample preparation and multiplex real-time RT-PCR. It features semi-automation, is relatively easy to use, and has "sample-in-answer-out" capabilities for multiplex gene expression analysis. Our tissue sample preparation module incorporating both a microhomogenizer and surface-treated paramagnetic microbeads yielded high purity mRNA extracts, considerably better than manual means of extraction. A primer preloading surface treatment procedure and the single-loading inlet on our multiplex real-time RT-PCR module simplify off-chip handling procedures for ease-of-use. To demonstrate the efficacy of our LOC system for POCT hepatotoxicity assessment, we perform a preclinical animal study with the administration of cyclophosphamide, followed by gene expression analysis of two critical protein biomarkers for liver function tests, aspartate transaminase (AST) and alanine transaminase (ALT). Our experimental results depict normalized fold changes of 1.62 and 1.31 for AST and ALT, respectively, illustrating up-regulations in their expression levels and hence validating their selection as critical genes of interest. In short, we illustrate the feasibility of multiplex gene expression analysis in an integrated LOC system as a viable POCT means for hepatotoxicity assessment.

  14. A laser ablation ICP-MS based method for multiplexed immunoblot analysis: applications to manganese-dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.).

    Science.gov (United States)

    de Bang, Thomas Christian; Petersen, Jørgen; Pedas, Pai Rosager; Rogowska-Wrzesinska, Adelina; Jensen, Ole Noerregaard; Schjoerring, Jan Kofod; Jensen, Poul Erik; Thelen, Jay J; Husted, Søren

    2015-08-01

    Manganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody-based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn-dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA-ICP-MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA-ICP-MS provides an analytical platform with high throughput appropriate for screening large collections of plants. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  15. Rapid structural analysis of nanomaterials in aqueous solutions

    Science.gov (United States)

    Ryuzaki, Sou; Tsutsui, Makusu; He, Yuhui; Yokota, Kazumichi; Arima, Akihide; Morikawa, Takanori; Taniguchi, Masateru; Kawai, Tomoji

    2017-04-01

    Rapid structural analysis of nanoscale matter in a liquid environment represents innovative technologies that reveal the identities and functions of biologically important molecules. However, there is currently no method with high spatio-temporal resolution that can scan individual particles in solutions to gain structural information. Here we report the development of a nanopore platform realizing quantitative structural analysis for suspended nanomaterials in solutions with a high z-axis and xy-plane spatial resolution of 35.8 ± 1.1 and 12 nm, respectively. We used a low thickness-to-diameter aspect ratio pore architecture for achieving cross sectional areas of analyte (i.e. tomograms). Combining this with multiphysics simulation methods to translate ionic current data into tomograms, we demonstrated rapid structural analysis of single polystyrene (Pst) beads and single dumbbell-like Pst beads in aqueous solutions.

  16. Rapid Detection of Bloodstream Pathogens in Liver Transplantation Patients With FilmArray Multiplex Polymerase Chain Reaction Assays: Comparison With Conventional Methods.

    Science.gov (United States)

    Otlu, B; Bayindir, Y; Ozdemir, F; Ince, V; Cuglan, S; Hopoglu, M; Yakupogullari, Y; Kizilkaya, C; Kuzucu, C; Isık, B; Yilmaz, S

    2015-01-01

    Bloodstream infection (BSI) is an important concern in transplant patients. Early intervention with appropriate antimicrobial therapy is critical to better clinical outcome; however, there is significant delay when conventional identification methods are used. We aimed to determine the diagnostic performance of the FilmArray Blood Culture Identification Panel, a recently approved multiplex polymerase chain reaction assay detecting 24 BSI pathogens and 3 resistance genes, in comparison with the performances of conventional identification methods in liver transplant (LT) patients. A total of 52 defined sepsis episodes (signal-positive by blood culture systems) from 45 LT patients were prospectively studied. The FilmArray successfully identified 37 of 39 (94.8%) bacterial and 3 of 3 (100%) yeast pathogens in a total of 42 samples with microbial growth, failing to detect only 2 of 39 (5.1%) bacterial pathogens that were not covered by the test panel. The FilmArray could also detect additional pathogens in 3 samples that had been reported as having monomicrobial growth, and it could detect Acinetobacter baumannii in 2 samples suspected of skin flora contamination. The remaining 8 blood cultures showing a positive signal but yielding no growth were also negative by this assay. Results of MecA, KPC, and VanA/B gene detection were in high accordance. The FilmArray produced results with significantly shorter turnaround times (1.33 versus 36.2, 23.6, and 19.5 h; P FilmArray appeared as a reliable alternative diagnostic method with the potential to mitigate problems with protracted diagnosis of the BSI pathogens in LT patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Multiplex ligation-dependent probe amplification (MLPA) in prenatal diagnosis-experience of a large series of rapid testing for aneuploidy of chromosomes 13, 18, 21, X, and Y.

    Science.gov (United States)

    Gerdes, Tommy; Kirchhoff, Maria; Lind, Anne-Marie; Vestergaard Larsen, Gitte; Kjaergaard, Susanne

    2008-12-01

    Multiplex ligation-dependent probe amplification (MLPA) is a relatively new method for rapid prenatal diagnosis of common aneuploidies, and larger series to evaluate its performance remain to be reported. A total of 2400 prenatal chorionic villus samples (CVS) and 1525 prenatal samples of amniotic fluids (AF) were analyzed using a commercial MLPA kit (SALSA P095) for aneuploidy of chromosomes 13, 18, 21, X, and Y, and subsequent G-banding. MLPA gave conclusive results in 2330 (97.1%) CVS and 1417 (92.9%) AF samples. MLPA and G-banding showed concordant results except for five CVS and two AF. These were acceptable differences, as MLPA is not expected to detect all cases of mosaicism or partial deletions. MLPA gave inconclusive results for 19 (0.79%) CVS and 20 (1.31%) AF samples in which mosaicism, triploidy, contamination by maternal cells, or structural abnormalities were suspected by MLPA. Finally, 30 (1.97%) AF were discarded because of maternal blood staining, and 51 (2.1%) CVS and 58 (3.8%) AF were discarded because of technical problems. The data presented confirm that MLPA is a rapid, simple and reliable method for large scale testing for nonmosaic aneuploidy of chromosomes 13, 18, 21, X, or Y in trypsin-digested CVS and in AF. Copyright (c) 2008 John Wiley & Sons, Ltd.

  18. Development of a multiplex real-time PCR for the rapid detection of the predominant beta-lactamase genes CTX-M, SHV, TEM and CIT-type AmpCs in Enterobacteriaceae.

    Directory of Open Access Journals (Sweden)

    Nicole Roschanski

    Full Text Available Beta-lactamase resistant bacteria and especially ESBL producing Enterobacteriaceae are an increasing problem worldwide. For this reason a major interest in efficient and reliable methods for rapid screening of high sample numbers is recognizable. Therefore, a multiplex real-time PCR was developed to detect the predominant class A beta-lactamase genes blaCTX-M, blaSHV, blaTEM and CIT-type AmpCs in a one-step reaction. A set of 114 Enterobacteriaceae containing previously identified resistance gene subtypes and in addition 20 undefined animal and environmental isolates were used for the validation of this assay. To confirm the accessibility in variable settings, the real-time runs were performed analogous in two different laboratories using different real-time cyclers. The obtained results showed complete accordance between the real-time data and the predetermined genotypes. Even if sequence analyses are further necessary for a comprehensive characterization, this method was proofed to be reliable for rapid screening of high sample numbers and therefore could be an important tool for e. g. epidemiological purposes or support infection control measures.

  19. Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay.

    Science.gov (United States)

    Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

    2016-02-17

    The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 10(4) CFU mL(-1) or 10(5) CFU mL(-1) for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R(2)) of 0.916-0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥ 80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

  20. Charging YOYO-1 on capillary wall for online DNA intercalation and integrating this approach with multiplex PCR and bare narrow capillary-hydrodynamic chromatography for online DNA analysis.

    Science.gov (United States)

    Chen, Huang; Zhu, Zaifang; Lu, Joann Juan; Liu, Shaorong

    2015-02-03

    Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary-hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions.

  1. Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis

    Science.gov (United States)

    2016-01-01

    Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions. PMID:25555111

  2. Detection, analysis and clinical validation of chromosomal aberrations by multiplex ligation-dependent probe amplification in chronic leukemia.

    Directory of Open Access Journals (Sweden)

    Adam Abdool

    Full Text Available Current diagnostic screening strategies based on karyotyping or fluorescent in situ hybridization (FISH for detection of chromosomal abnormalities in chronic lymphocytic leukemia (CLL are laborious, time-consuming, costly, and have limitations in resolution. Multiplex ligation-dependent probe amplification (MLPA can simultaneously detect copy number changes of multiple loci in one simple PCR reaction, making it an attractive alternative to FISH. To enhance the clinical robustness and further harness MLPA technology for routine laboratory operations, we have developed and validated a protocol for comprehensive, automatic data analysis and interpretation. A training set of 50 normal samples was used to establish reference ranges for each individual probe, for the calling of statistically significant copy number changes. The maximum normal ranges of 2 and 3 standard deviations (SD are distributed between 0.82 and 1.18 (Mean ± 2SD, 95% CI, P = 0.05, and between 0.73 and 1.27 (Mean ± 3SD, 99% CI, P = 0.01, respectively. We found an excellent correlation between MLPA and FISH with 93.6% concordance (P<0.0001 from a testing cohort of 100 clinically suspected CLL cases. MLPA analyses done on 94/100 patients showed sensitivity and specificity of 94.2% and 92.9%, respectively. MLPA detected additional copy number gains on 18q21.1 and chromosome 19, and novel micro-deletions at 19q13.43 and 19p13.2 loci in six samples. Three FISH-failed samples were tested positive by MLPA, while three 13q- cases with a low percentage of leukemia cells (7%, 12% and 19% were not detected by MLPA. The improved CLL MLPA represents a high-throughput, accurate, cost-effective and user-friendly platform that can be used as a first-line screening test in a clinical laboratory.

  3. Barcoded DNA-tag reporters for multiplex cis-regulatory analysis.

    Directory of Open Access Journals (Sweden)

    Jongmin Nam

    Full Text Available Cis-regulatory DNA sequences causally mediate patterns of gene expression, but efficient experimental analysis of these control systems has remained challenging. Here we develop a new version of "barcoded" DNA-tag reporters, "Nanotags" that permit simultaneous quantitative analysis of up to 130 distinct cis-regulatory modules (CRMs. The activities of these reporters are measured in single experiments by the NanoString RNA counting method and other quantitative procedures. We demonstrate the efficiency of the Nanotag method by simultaneously measuring hourly temporal activities of 126 CRMs from 46 genes in the developing sea urchin embryo, otherwise a virtually impossible task. Nanotags are also used in gene perturbation experiments to reveal cis-regulatory responses of many CRMs at once. Nanotag methodology can be applied to many research areas, ranging from gene regulatory networks to functional and evolutionary genomics.

  4. Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

    Science.gov (United States)

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Lauffenburger, Doug A; Chen, Chia-Hung

    2015-02-21

    Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.

  5. Multiplex ligation-dependent probe amplification (MLPA) assay for blood group genotyping, copy number quantification, and analysis of RH variants

    NARCIS (Netherlands)

    Veldhuisen, Barbera; van der Schoot, C. E.; de Haas, Masja

    2015-01-01

    The blood group multiplex ligation-dependent probe amplification (MLPA) is a comprehensive assay, developed for genotyping the majority of clinically relevant blood group antigens in both patients and donors. The MLPA is an easy method to apply and only requires a thermal cycler and capillary

  6. Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates.

    Science.gov (United States)

    Ullal, Adeeti V; Peterson, Vanessa; Agasti, Sarit S; Tuang, Suan; Juric, Dejan; Castro, Cesar M; Weissleder, Ralph

    2014-01-15

    Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.

  7. Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station.

    Directory of Open Access Journals (Sweden)

    Macarena Parra

    Full Text Available The International Space Station (ISS National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for

  8. Etiological Analysis of Fungal Keratitis and Rapid Identification of Predominant Fungal Pathogens.

    Science.gov (United States)

    He, Dan; Hao, Jilong; Gao, Song; Wan, Xue; Wang, Wanting; Shan, Qiushi; Wang, Li

    2016-02-01

    Fungal keratitis is a worldwide-distributed refractory and potentially blinding ocular infection caused by various fungi. It is necessary to investigate the etiological and epidemiological characteristics of this disease and establish a rapid and specific pathogenic identification method. Here, we isolated and identified fungal pathogens of 275 patients with presumed fungal keratitis from Jilin Province, China, and conducted statistical analyses of epidemiological information. The positive rate of fungal culture was 72.0 %. Fusarium sp. was the most common genus among 210 fungal isolates. The predominant species were Fusarium solani, Aspergillus fumigatus, and Candida glabrata, which accounted for over 50 % of the isolated organisms. Corneal trauma and previous use of drugs were the most important predisposing factors. In addition, a multiplex polymerase chain reaction (PCR) was designed with species-specific primers of the three species that could identify them with amplicons of approximately 330 bp from F. solani, 275 bp from A. fumigatus, and 230 bp from C. glabrata. Additionally, PCR with fungal universal primers and multiplex PCR were performed using DNA prepared by an improved DNA extraction method from corneal scrapings. With this method, fungal pathogens from corneal scrapings could be specifically and rapidly identified within 8 h. The culture-independent rapid identification of corneal scrapings may have great significance for the early diagnosis and treatment of fungal keratitis.

  9. RAPID ANALYSIS OF EMERGENCY URINE AND WATER SAMPLES

    Energy Technology Data Exchange (ETDEWEB)

    Maxwell, S

    2007-02-26

    There is a need for fast, reliable methods for the determination of actinides and Sr-89/90 analysis on environmental and bioassay samples in response to an emergency radiological incident. The SRS (Savannah River Site) Environmental Bioassay Laboratory participated in the National Institute of Standards and Technology Radiochemistry Intercomparison Program (NRIP-06) and analyzed water and urine samples within 8 hours of receipt. The SRS Environmental Laboratory was the only lab that participated in the program that analyzed these samples for both actinides and Sr-89/90 within the requested 8 hour turnaround time. A new, rapid actinide and strontium 89/90 separation method was used for both urine and water samples. This method uses stacked TEVA Resin{reg_sign}, TRU Resin{reg_sign} and Sr-Resin{reg_sign} cartridges from Eichrom Technologies (Darien, IL, USA) that allows the rapid separation of plutonium (Pu), neptunium (Np), uranium (U), and americium (Am), curium (Cm) and thorium (Th) using a single multi-stage column combined with alpha spectrometry. By using vacuum box cartridge technology and stacked cartridges with rapid flow rates, sample preparation time was minimized. This paper discusses the technology and conditions employed for both water and urine samples and presents the SRS performance data on the NRIP-06 samples.

  10. Rapid automatic keyword extraction for information retrieval and analysis

    Science.gov (United States)

    Rose, Stuart J [Richland, WA; Cowley,; E, Wendy [Richland, WA; Crow, Vernon L [Richland, WA; Cramer, Nicholas O [Richland, WA

    2012-03-06

    Methods and systems for rapid automatic keyword extraction for information retrieval and analysis. Embodiments can include parsing words in an individual document by delimiters, stop words, or both in order to identify candidate keywords. Word scores for each word within the candidate keywords are then calculated based on a function of co-occurrence degree, co-occurrence frequency, or both. Based on a function of the word scores for words within the candidate keyword, a keyword score is calculated for each of the candidate keywords. A portion of the candidate keywords are then extracted as keywords based, at least in part, on the candidate keywords having the highest keyword scores.

  11. Mach-Zehnder interferometer (MZI) point-of-care system for rapid multiplexed detection of microRNAs in human urine specimens.

    Science.gov (United States)

    Liu, Qing; Shin, Yong; Kee, Jack Sheng; Kim, Kyoung Woo; Mohamed Rafei, Siti Rafeah; Perera, Agampodi Promoda; Tu, Xiaoguang; Lo, Guo-Qiang; Ricci, Estelle; Colombel, Marc; Chiong, Edmund; Thiery, Jean Paul; Park, Mi Kyoung

    2015-09-15

    MicroRNAs have been identified as promising biomarkers for human diseases. The development of a point-of-care (POC) test for the disease-associated miRNAs would be especially beneficial, since miRNAs are unexpectedly well preserved in various human specimens, including urine. Here, we present the Mach-Zehnder interferometer-miRNA detection system capable of detecting multiple miRNAs in clinical urine samples rapidly and simultaneously in a label-free and real-time manner. Through measurement of the light phase change, the MZI sensor provides an optical platform for fast profiling of small molecules with improved accuracy. We demonstrate that this system could specifically detect target miRNAs (miR-21, and let-7a), and even identify the single nucleotide polymorphism of the let-7 family of miRNAs from synthetic and cell line samples. The clinical applicability of this system is confirmed by simultaneously detecting two types of miRNAs in urine samples of bladder cancer patients in a single reaction, with a detection time of 15 min. The POC system can be expanded to detect a number of miRNAs of different species and should be useful for a variety of clinical applications requiring at or near the site of patient care. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. SIMS: a hybrid method for rapid conformational analysis.

    Directory of Open Access Journals (Sweden)

    Bryant Gipson

    Full Text Available Proteins are at the root of many biological functions, often performing complex tasks as the result of large changes in their structure. Describing the exact details of these conformational changes, however, remains a central challenge for computational biology due the enormous computational requirements of the problem. This has engendered the development of a rich variety of useful methods designed to answer specific questions at different levels of spatial, temporal, and energetic resolution. These methods fall largely into two classes: physically accurate, but computationally demanding methods and fast, approximate methods. We introduce here a new hybrid modeling tool, the Structured Intuitive Move Selector (sims, designed to bridge the divide between these two classes, while allowing the benefits of both to be seamlessly integrated into a single framework. This is achieved by applying a modern motion planning algorithm, borrowed from the field of robotics, in tandem with a well-established protein modeling library. sims can combine precise energy calculations with approximate or specialized conformational sampling routines to produce rapid, yet accurate, analysis of the large-scale conformational variability of protein systems. Several key advancements are shown, including the abstract use of generically defined moves (conformational sampling methods and an expansive probabilistic conformational exploration. We present three example problems that sims is applied to and demonstrate a rapid solution for each. These include the automatic determination of "active" residues for the hinge-based system Cyanovirin-N, exploring conformational changes involving long-range coordinated motion between non-sequential residues in Ribose-Binding Protein, and the rapid discovery of a transient conformational state of Maltose-Binding Protein, previously only determined by Molecular Dynamics. For all cases we provide energetic validations using well

  13. Rapid Prototyping Integrated With Nondestructive Evaluation and Finite Element Analysis

    Science.gov (United States)

    Abdul-Aziz, Ali; Baaklini, George Y.

    2001-01-01

    Most reverse engineering approaches involve imaging or digitizing an object then creating a computerized reconstruction that can be integrated, in three dimensions, into a particular design environment. Rapid prototyping (RP) refers to the practical ability to build high-quality physical prototypes directly from computer aided design (CAD) files. Using rapid prototyping, full-scale models or patterns can be built using a variety of materials in a fraction of the time required by more traditional prototyping techniques (refs. 1 and 2). Many software packages have been developed and are being designed to tackle the reverse engineering and rapid prototyping issues just mentioned. For example, image processing and three-dimensional reconstruction visualization software such as Velocity2 (ref. 3) are being used to carry out the construction process of three-dimensional volume models and the subsequent generation of a stereolithography file that is suitable for CAD applications. Producing three-dimensional models of objects from computed tomography (CT) scans is becoming a valuable nondestructive evaluation methodology (ref. 4). Real components can be rendered and subjected to temperature and stress tests using structural engineering software codes. For this to be achieved, accurate high-resolution images have to be obtained via CT scans and then processed, converted into a traditional file format, and translated into finite element models. Prototyping a three-dimensional volume of a composite structure by reading in a series of two-dimensional images generated via CT and by using and integrating commercial software (e.g. Velocity2, MSC/PATRAN (ref. 5), and Hypermesh (ref. 6)) is being applied successfully at the NASA Glenn Research Center. The building process from structural modeling to the analysis level is outlined in reference 7. Subsequently, a stress analysis of a composite cooling panel under combined thermomechanical loading conditions was performed to validate

  14. Structure of triadic relations in multiplex networks

    Science.gov (United States)

    Cozzo, Emanuele; Kivelä, Mikko; De Domenico, Manlio; Solé-Ribalta, Albert; Arenas, Alex; Gómez, Sergio; Porter, Mason A.; Moreno, Yamir

    2015-07-01

    Recent advances in the study of networked systems have highlighted that our interconnected world is composed of networks that are coupled to each other through different ‘layers’ that each represent one of many possible subsystems or types of interactions. Nevertheless, it is traditional to aggregate multilayer networks into a single weighted network in order to take advantage of existing tools. This is admittedly convenient, but it is also extremely problematic, as important information can be lost as a result. It is therefore important to develop multilayer generalizations of network concepts. In this paper, we analyze triadic relations and generalize the idea of transitivity to multiplex networks. By focusing on triadic relations, which yield the simplest type of transitivity, we generalize the concept and computation of clustering coefficients to multiplex networks. We show how the layered structure of such networks introduces a new degree of freedom that has a fundamental effect on transitivity. We compute multiplex clustering coefficients for several real multiplex networks and illustrate why one must take great care when generalizing standard network concepts to multiplex networks. We also derive analytical expressions for our clustering coefficients for ensemble averages of networks in a family of random multiplex networks. Our analysis illustrates that social networks have a strong tendency to promote redundancy by closing triads at every layer and that they thereby have a different type of multiplex transitivity from transportation networks, which do not exhibit such a tendency. These insights are invisible if one only studies aggregated networks.

  15. Rapid and in-depth analysis for seismic risk evaluation

    Science.gov (United States)

    Dwi Astuti, Novi; Anta Alvita, Meli; Sangadji, Senot; Rahmadi, AP; Purwanto, Edy

    2017-11-01

    The high public demand on housing in urban areas requires the government of Indonesia to adopt a policy of encouraging the development of vertical housing. Cilacap has been allocated Rusunawa (low-income apartment) development in 2006. Evident from some earthquakes occurrence in recent years, however, Cilacap may be seen as an earthquake prone region which posing some risk to this type of vertical structures. The Appropriate strategy should be performed to evaluate the seismic risks of this local government owned four stories low-income apartment. This paper demonstrates two tier evaluation strategy; rapid evaluation and in-depth analysis and compares both results. First evaluation was conducted by means of Building Rapid Visual Screening (RVS) of FEMA 154 of the Rusunawa block A and B. The result was used further to calculate seismic risk score (SR) which exhibit the probability of the building damage given the Maximum Considered Earthquake (MCER) that will occur during the Rusunawa service life. The in-depth analysis was conducted by developing fragility function expressed in the form of fragility curves for the Rusunawa. The fragility shows the probability that certain damage states will be exceeded given the intensity of earthquakes which will occur during building service life. The fragility was developed as lognormal curves in which the building response to earthquake input was analyzed by means of pushover.

  16. Novel pan-genomic analysis approach in target selection for multiplex PCR identification and detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia complex species: a proof-of-concept study.

    Science.gov (United States)

    Ho, Chi-Chun; Lau, Candy C Y; Martelli, Paolo; Chan, San-Yuen; Tse, Cindy W S; Wu, Alan K L; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

    2011-03-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified.

  17. Novel Pan-Genomic Analysis Approach in Target Selection for Multiplex PCR Identification and Detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia Complex Species: a Proof-of-Concept Study▿

    Science.gov (United States)

    Ho, Chi-Chun; Lau, Candy C. Y.; Martelli, Paolo; Chan, San-Yuen; Tse, Cindy W. S.; Wu, Alan K. L.; Yuen, Kwok-Yung; Lau, Susanna K. P.; Woo, Patrick C. Y.

    2011-01-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified. PMID:21177905

  18. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis

    National Research Council Canada - National Science Library

    Hanson, Erin K; Ballantyne, Jack

    2014-01-01

    .... However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically...

  19. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis

    OpenAIRE

    Erin K. Hanson; Jack Ballantyne

    2014-01-01

    Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based appro...

  20. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis

    National Research Council Canada - National Science Library

    Hanson, Erin K; Ballantyne, Jack

    2013-01-01

    .... However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically...

  1. Rapid identification of methicillin-resistant Staphylococcus aureus transmission in hospitals by use of phage-derived open reading frame typing enhanced by multiplex PCR and reverse line blot assay.

    Science.gov (United States)

    O'Sullivan, Matthew V N; Kong, Fanrong; Sintchenko, Vitali; Gilbert, Gwendolyn L

    2010-08-01

    The relatively high-level clonality of methicillin-resistant Staphylococcus aureus (MRSA) and its frequent high-level endemicity in nosocomial settings complicate the development of methods for rapid subtyping of MRSA strains that are capable of identifying person-to-person transmission in hospitals. Phage-derived open reading frame (PDORF) typing is an MRSA typing method targeting mobile genetic elements that was recently described and evaluated using a geographically restricted set of isolates. The objective of this study was to develop a multiplex PCR-reverse line blot (mPCR/RLB) assay for PDORF typing and to test its applicability on a broad range of isolates and in an environment where MRSA is highly endemic. The 16 targets were identified using a 23-primer-pair mPCR/RLB assay with two probes for each target. The method was evaluated using 42 MRSA reference strains, including those representing major international clones, and 35 isolates from episodes of suspected nosocomial transmission. In vivo stability was explored using 81 isolate pairs. Pulsed-field gel electrophoresis (PFGE) and spa typing were performed for comparison. Among the 42 reference strains, there were 33 PFGE subtypes, 30 PDORF types, and 22 spa types. Simpson's index of diversity was 0.987, 0.971, and 0.926 for PFGE subtyping, PDORF typing, and spa typing, respectively. Typing of clinical isolates by PDORF typing and PFGE demonstrated concordant results. mPCR/RLB-based PDORF typing has similar discriminatory power to that of PFGE, can assist in tracking MRSA transmission events in a setting of high-level endemicity, and has the advantage of being a high-throughput technique.

  2. A method for rapid similarity analysis of RNA secondary structures

    Directory of Open Access Journals (Sweden)

    Liu Na

    2006-11-01

    Full Text Available Abstract Background Owing to the rapid expansion of RNA structure databases in recent years, efficient methods for structure comparison are in demand for function prediction and evolutionary analysis. Usually, the similarity of RNA secondary structures is evaluated based on tree models and dynamic programming algorithms. We present here a new method for the similarity analysis of RNA secondary structures. Results Three sets of real data have been used as input for the example applications. Set I includes the structures from 5S rRNAs. Set II includes the secondary structures from RNase P and RNase MRP. Set III includes the structures from 16S rRNAs. Reasonable phylogenetic trees are derived for these three sets of data by using our method. Moreover, our program runs faster as compared to some existing ones. Conclusion The famous Lempel-Ziv algorithm can efficiently extract the information on repeated patterns encoded in RNA secondary structures and makes our method an alternative to analyze the similarity of RNA secondary structures. This method will also be useful to researchers who are interested in evolutionary analysis.

  3. Rapid Analysis, Self-Calibrating Array for Air Monitoring

    Science.gov (United States)

    Homer, Margie L.; Shevade, Abhijit V.; Lara, Liana; Huerta, Ramon; Vergara, Alexander; Muezzinoglua, Mehmet K.

    2012-01-01

    Human space missions have critical needs for monitoring and control for life support systems. These systems have monitoring needs that include feedback for closed loop processes and quality control for environmental factors. Sensors and monitoring technologies assure that the air environment and water supply for the astronaut crew habitat fall within acceptable limits, and that the life support system is functioning properly and efficiently. The longer the flight duration and the more distant the destination, the more critical it becomes to have carefully monitored and automated control systems for life support. Past experiments with the JPL ENose have demonstrated a lifetime of the sensor array, with the software, of around 18 months. The lifetime of the calibration, for some analytes, was as long as 24 months. We are working on a sensor array and new algorithms that will include sensor response time in the analysis. The preliminary array analysis for two analytes shows that the analysis time, of an event, can be dropped from 45 minutes to less than10 minutes and array training time can be cut substantially. We will describe the lifetime testing of an array and show lifetime data on individual sensors. This progress will lead to more rapid identification of analytes, and faster training time of the array.

  4. Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR.

    Science.gov (United States)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Hung, Tran Quang; Wolff, Anders; Bang, Dang Duong

    2017-04-01

    Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011 molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

  5. Multiplex PageRank.

    Directory of Open Access Journals (Sweden)

    Arda Halu

    Full Text Available Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation.

  6. Multiplex PageRank.

    Science.gov (United States)

    Halu, Arda; Mondragón, Raúl J; Panzarasa, Pietro; Bianconi, Ginestra

    2013-01-01

    Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation.

  7. [Electromyography Analysis of Rapid Eye Movement Sleep Behavior Disorder].

    Science.gov (United States)

    Nakano, Natsuko; Kinoshita, Fumiya; Takada, Hiroki; Nakayama, Meiho

    2018-01-01

    Polysomnography (PSG), which records physiological phenomena including brain waves, breathing status, and muscle tonus, is useful for the diagnosis of sleep disorders as a gold standard. However, measurement and analysis are complex for several specific sleep disorders, such as rapid eye movement (REM) sleep behavior disorder (RBD). Usually, brain waves during REM sleep indicate an awakening pattern under relaxed conditions of skeletal and antigravity muscles. However, these muscles are activated during REM sleep when patients suffer from RBD. These activated muscle movements during REM, so-called REM without atonia (RWA) recorded by PSG, may be related to a neurodegenerative disease such as Parkinson's disease. Thus, careful analysis of RWA is significant not only physically, but also clinically. Commonly, manual viewing measurement analysis of RWA is time-consuming. Therefore, quantitative studies on RWA are rarely reported. A software program, developed from Microsoft Office Excel ® , was used to semiautomatically analyze the RWA ratio extracted from PSG to compare with manual viewing measurement analysis. In addition, a quantitative muscle tonus study was carried out to evaluate the effect of medication on RBD patients. Using this new software program, we were able to analyze RWA on the same cases in approximately 15 min as compared with 60 min in the manual viewing measurement analysis. This software program can not only quantify RWA easily but also identify RWA waves for either phasic or tonic bursts. We consider that this software program will support physicians and scientists in their future research on RBD. We are planning to offer this software program for free to physicians and scientists.

  8. Multiplex tokamak power plant

    Energy Technology Data Exchange (ETDEWEB)

    Dabiri, A.E.

    1986-07-01

    The concept of multiplexing for a fusion power core as an option for producing power is explored. Superconducting, as well as normal magnet, coils in either first or second stability regimes are considered. The results show that multiplex plants with superconducting magnets operating in the second stability regime could be competitive with the single-unit plants in some unit sizes. The key issues that impact the expected benefits of multiplexing must be investigated further. These are factory fabrication, economy of scale, the extent of equipment sharing, inherent safety, maintainability, and utility load management.

  9. Multiplexed magnetic nanoparticle-antibody conjugates (MNPs-ABS) based prognostic detection of ovarian cancer biomarkers, CA-125, β-2M and ApoA1 using fluorescence spectroscopy with comparison of surface plasmon resonance (SPR) analysis.

    Science.gov (United States)

    Pal, Manoj K; Rashid, Mohammad; Bisht, Manisha

    2015-11-15

    A multiplexed MNPs-Abs based fluorescence spectroscopic system in analysis of serum biomarkers; CA-125, β2-M and ApoA1 for the early detection of ovarian cancer was first time proposed. The lowest detection limits measured in multiplexed setup were 0.26 U/mL, 0.55 ng/mL and 7.7 ng/mL respectively for CA-125, β2-M and ApoA1. A comparative real sample analysis of healthy normal (Control), benign and ovarian cancer patients with SPR has also been done to validate the process. Moreover CA-125 detection only confirms 50-60% of early stage disease. This multiplexed system achieved sensitivity and specificity up to 94% and 98% respectively to distinguish early stage ovarian cancer patients from healthy individuals. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

    KAUST Repository

    Harrison, H.B.

    2014-04-24

    Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo-Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co-amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent-offspring relationships in natural populations, with over 99.6% accuracy in parent-offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28-96% of loci. The multiplex PCRs developed here provide a reliable and cost-effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species. 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

  11. The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae, Lamiaceae, Rosaceae.

    Science.gov (United States)

    Sytar, Oksana; Bruckova, Klaudia; Hunkova, Elena; Zivcak, Marek; Konate, Kiessoun; Brestic, Marian

    2015-01-16

    The aim of our research work was to quantify total flavonoid contents in the leaves of 13 plant species family Asteraceae, 8 representatives of family Lamiaceae and 9 plant species belonging to family Rosaceae, using the multiplex fluorimetric sensor. Fluorescence was measured using optical fluorescence apparatus Multiplex(R) 3 (Force-A, France) for non-destructive flavonoids estimation. The content of total flavonoids was estimated by FLAV index (expressed in relative units), that is deduced from flavonoids UV absorbing properties. Among observed plant species, the highest amount of total flavonoids has been found in leaves of Helianthus multiflorus (1.65 RU) and Echinops ritro (1.27 RU), Rudbeckia fulgida (1.13 RU) belonging to the family Asteraceae. Lowest flavonoid content has been observed in the leaves of marigold (Calendula officinalis) (0.14 RU) also belonging to family Asteraceae. The highest content of flavonoids among experimental plants of family Rosaceae has been estimated in the leaves of Rosa canina (1.18 RU) and among plant species of family Lamiaceae in the leaves of Coleus blumei (0.90 RU). This research work was done as pre-screening of flavonoids content in the leaves of plant species belonging to family Asteraceae, Lamiaceae and Rosaceae. Results indicated that statistically significant differences (P > 0.05) in flavonoids content were observed not only between families, but also among individual plant species within one family.

  12. Rapid and precise genotyping of porcine microsatellites.

    Science.gov (United States)

    Yue, G H; Beeckmann, P; Bartenschlager, H; Moser, G; Geldermann, H

    1999-11-01

    Microsatellites are useful markers for genetic mapping and linkage analysis because they are highly polymorphic, abundant in genomes and relatively easily scored with polymerase chain reaction (PCR). A rapid genotyping system for microsatellites was developed, which included multiplex PCRs, multiple use of Hydrolink gels, automated fluorescent detection of fragments on an A.L.F. DNA sequencer, automatic assignment of alleles to each locus and verification of genotypes with a self-developed computer program "Fragtest". Eight multiplex PCRs have been developed to genotype 29 microsatellites for genetic and quantitative trait loci (QTL) mapping on pig chromosomes 6, 7, 12 and 13. Three to six microsatellites could be amplified in one multiplex PCR. Each multiplex reaction required only different concentrations of each pair of primers and a low concentration of dNTP (100 microM). A dNTP concentration of 100 microM proved to be optimal for the coamplification of microsatellites under the concentration of 1.5 mM MgCl2. Using four internal size standards added in each sample, the 5% Hydrolink gel could subsequently be used up to five times (total running time of 500 min) on the A.L.F. automated sequencer without significant loss of resolution and precision of fragment length analysis. Automatic assignment of alleles on each locus using "Fragtest" significantly increased the efficiency and precision of the genotyping. This system is thus a rapid, cheap, and highly discriminating genotyping system.

  13. Software Tools for Design of Reagents for Multiplex Genetic Analyses

    OpenAIRE

    Stenberg, Johan

    2006-01-01

    Methods using oligonucleotide probes are powerful tools for the analysis of nucleic acids. During recent years, many such methods have been developed that enable the simultaneous interrogation of multiple qualities of a sample. Many of these multiplexing techniques share common limitations. This thesis discusses new developments to overcome the problems of multiplex amplification of genomic sequences and design of sets of oligonucleotide probes for multiplex genetic analyses. A novel molecula...

  14. Multiplex tests to identify gastrointestinal bacteria, viruses and parasites in people with suspected infectious gastroenteritis: a systematic review and economic analysis.

    Science.gov (United States)

    Freeman, Karoline; Mistry, Hema; Tsertsvadze, Alexander; Royle, Pam; McCarthy, Noel; Taylor-Phillips, Sian; Manuel, Rohini; Mason, James

    2017-04-01

    Gastroenteritis is a common, transient disorder usually caused by infection and characterised by the acute onset of diarrhoea. Multiplex gastrointestinal pathogen panel (GPP) tests simultaneously identify common bacterial, viral and parasitic pathogens using molecular testing. By providing test results more rapidly than conventional testing methods, GPP tests might positively influence the treatment and management of patients presenting in hospital or in the community. To systematically review the evidence for GPP tests [xTAG ® (Luminex, Toronto, ON, Canada), FilmArray (BioFire Diagnostics, Salt Lake City, UT, USA) and Faecal Pathogens B (AusDiagnostics, Beaconsfield, NSW, Australia)] and to develop a de novo economic model to compare the cost-effectiveness of GPP tests with conventional testing in England and Wales. Multiple electronic databases including MEDLINE, EMBASE, Web of Science and the Cochrane Database were searched from inception to January 2016 (with supplementary searches of other online resources). Eligible studies included patients with acute diarrhoea; comparing GPP tests with standard microbiology techniques; and patient, management, test accuracy or cost-effectiveness outcomes. Quality assessment of eligible studies used tailored Quality Assessment of Diagnostic Accuracy Studies-2, Consolidated Health Economic Evaluation Reporting Standards and Philips checklists. The meta-analysis included positive and negative agreement estimated for each pathogen. A de novo decision tree model compared patients managed with GPP testing or comparable coverage with patients managed using conventional tests, within the Public Health England pathway. Economic models included hospital and community management of patients with suspected gastroenteritis. The model estimated costs (in 2014/15 prices) and quality-adjusted life-year losses from a NHS and Personal Social Services perspective. Twenty-three studies informed the review of clinical evidence (17 xTAG, four

  15. Budget impact analysis of a breast rapid diagnostic unit.

    Science.gov (United States)

    Elmi, M; Hussein, H; Nofech-Mozes, S; Curpen, B; Leahey, A; Look Hong, N

    2017-06-01

    The Odette Cancer Centre's recent implementation of a rapid diagnostic unit (rdu) for breast lesions has significantly decreased wait times to diagnosis. However, the economic impact of the unit remains unknown. This project defined the development and implementation costs and the operational costs of a breast rdu in a tertiary care facility. From an institutional perspective, a budget impact analysis identified the direct costs associated with the breast rdu. A base-case model was also used to calculate the cost per patient to achieve a diagnosis. Sensitivity analyses computed costs based on variations in key components. Costs are adjusted to 2015 valuations using health care-specific consumer price indices and are reported in Canadian dollars. Initiation cost for the rdu was $366,243. The annual operational cost for support staff was $111,803. The average per-patient clinical cost for achieving a diagnosis was $770. Sensitivity analyses revealed that, if running at maximal institutional capacity, the total annual clinical cost for achieving a diagnosis could range between $136,080 and $702,675. Establishment and maintenance of a breast rdu requires significant investment to achieve reductions in time to diagnosis. Expenditures ought to be interpreted in the context of institutional patient volumes and trade-offs in patient-centred outcomes, including lessened patient anxiety and possibly shorter times to definitive treatment. Our study can be used as a resource-planning tool for future rdus in health care systems wishing to improve diagnostic efficiency.

  16. Laser capture microdissection and multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in human kidney disease.

    Directory of Open Access Journals (Sweden)

    Ray Wilkinson

    Full Text Available Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD but emerging evidence is now linking many forms of acute kidney disease (AKD with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate "real time" gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-β in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.

  17. 2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting

    Directory of Open Access Journals (Sweden)

    Evelo Chris T

    2011-03-01

    Full Text Available Abstract Background Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation. Methods Both a non-targeted (classical 2D-electrophoresis combined with mass spectrometry as well as a targeted proteomic approach (multiplex immunoassay were applied to investigate how fasting for 36 h, as compared to 12 h, affects the proteome of platelets, peripheral blood mononuclear cells (PBMC, plasma, urine and saliva collected from ten healthy volunteers. Results Between-subject variability was highest in the plasma proteome and lowest in the PBMC proteome. Random Forests analysis performed on the entire dataset revealed that changes in the level of the RhoGDI2 protein in PBMC and plasma ApoA4 levels were the two most obvious biomarkers of an extended fasting. Random Forests (RF analysis of the multiplex immunoassay data revealed leptin and MMP-3 as biomarkers for extended fasting. However, high between-subject variability may have masked the extended fasting effects in the proteome of the biofluids and blood cells. Conclusions Identification of significantly changed proteins in biofluids and blood cells using a non-targeted approach, together with the outcome of targeted analysis revealed both known and novel markers for a 36 h fasting period, including the cellular proteins RhoGDI2 and CLIC1, and plasma proteins ApoA4, leptin and MMP-3. The PBMC proteome exhibited the lowest between-subject variability and therefore these cells appear to represent the best biosamples for biomarker discovery in human nutrigenomics.

  18. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    Science.gov (United States)

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  19. A new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clones.

    Science.gov (United States)

    Martins, Natacha; Picão, Renata Cristina; Cerqueira-Alves, Morgana; Uehara, Aline; Barbosa, Lívia Carvalho; Riley, Lee W; Moreira, Beatriz Meurer

    2016-08-01

    A collection of 163 Acinetobacter baumannii isolates detected in a large Brazilian hospital, was potentially related with the dissemination of four clonal complexes (CC): 113/79, 103/15, 109/1 and 110/25, defined by University of Oxford/Institut Pasteur multilocus sequence typing (MLST) schemes. The urge of a simple multiplex-PCR scheme to specify these clones has motivated the present study. The established trilocus sequence-based typing (3LST, for ompA, csuE and blaOXA-51-like genes) multiplex-PCR rapidly identifies international clones I (CC109/1), II (CC118/2) and III (CC187/3). Thus, the system detects only one (CC109/1) out of four main CC in Brazil. We aimed to develop an alternative multiplex-PCR scheme to detect these clones, known to be present additionally in Africa, Asia, Europe, USA and South America. MLST, performed in the present study to complement typing our whole collection of isolates, confirmed that all isolates belonged to the same four CC detected previously. When typed by 3LST-based multiplex-PCR, only 12% of the 163 isolates were classified into groups. By comparative sequence analysis of ompA, csuE and blaOXA-51-like genes, a set of eight primers was designed for an alternative multiplex-PCR to distinguish the five CC 113/79, 103/15, 109/1, 110/25 and 118/2. Study isolates and one CC118/2 isolate were blind-tested with the new alternative PCR scheme; all were correctly clustered in groups of the corresponding CC. The new multiplex-PCR, with the advantage of fitting in a single reaction, detects five leading A. baumannii clones and could help preventing the spread in healthcare settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Communicability reveals a transition to coordinated behavior in multiplex networks

    Science.gov (United States)

    Estrada, Ernesto; Gómez-Gardeñes, Jesús

    2014-04-01

    We analyze the flow of information in multiplex networks by means of the communicability function. First, we generalize this measure from its definition from simple graphs to multiplex networks. Then, we study its relevance for the analysis of real-world systems by studying a social multiplex where information flows using formal-informal channels and an air transportation system where the layers represent different air companies. Accordingly, the communicability, which is essential for the good performance of these complex systems, emerges at a systemic operation point in the multiplex where the performance of the layers operates in a coordinated way very differently from the state represented by a collection of unconnected networks.

  1. A multiplex GC-MS/MS technique for the sensitive and quantitative single-run analysis of acidic phytohormones and related compounds, and its application to Arabidopsis thaliana.

    Science.gov (United States)

    Müller, Axel; Düchting, Petra; Weiler, Elmar W

    2002-11-01

    A highly sensitive and accurate multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique is reported for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid. The optimized setup allows the routine processing and analysis of up to 60 plant samples of between 20 and 200 mg of fresh weight per day. The protocol was designed and the equipment used was chosen to facilitate implementation of the method into other laboratories and to provide access to state-of-the-art analytical tools for the acidic phytohormones and related signalling molecules. Whole-plant organ-distribution maps for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid were generated for Arabidopsis thaliana (L.) Heynh. For leaves of A. thaliana, a spatial resolution of hormone quantitation down to approximately 2 mm(2) was achieved.

  2. A laser ablation ICP-MS based method for multiplexed immunoblot analysis: applications to manganese-dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.)

    DEFF Research Database (Denmark)

    de Bang, Thomas Christian; Petersen, Jørgen; Pedas, Pai Rosager

    2015-01-01

    Manganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we...... analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC......-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn...

  3. Analysis of Rapid Acquisition Processes to Fulfill Future Urgent Needs

    Science.gov (United States)

    2015-12-01

    UAVs , the DOD defines UAVs as “powered aerial vehicle that does not carry a human operator; use aerodynamic forces to provide lift; can be autonomously...25 B. UAV ...25 1. UAV Background ...............................................................................25 2. Rapid Acquisition in UAV

  4. A new multiplex polymerase chain reaction assay for the identification a panel of bacteria involved in bacteremia

    Directory of Open Access Journals (Sweden)

    Hossein Fazzeli

    2013-01-01

    Conclusions: The presented multiplex PCR offers a rapid and accurate molecular diagnostic tool for simultaneous detection of some pathogenic microorganisms. The IC exists in the multiplex PCR accompanied by other primers in the system, can serve as a simple, cost- effective internal control for the multiplex PCR assay.

  5. Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS).

    Science.gov (United States)

    Yang, Yao; Sebra, Robert; Pullman, Benjamin S; Qiao, Wanqiong; Peter, Inga; Desnick, Robert J; Geyer, C Ronald; DeCoteau, John F; Scott, Stuart A

    2015-05-06

    DNA methylation has essential roles in transcriptional regulation, imprinting, X chromosome inactivation and other cellular processes, and aberrant CpG methylation is directly involved in the pathogenesis of human imprinting disorders and many cancers. To address the need for a quantitative and highly multiplexed bisulfite sequencing method with long read lengths for targeted CpG methylation analysis, we developed single-molecule real-time bisulfite sequencing (SMRT-BS). Optimized bisulfite conversion and PCR conditions enabled the amplification of DNA fragments up to ~1.5 kb, and subjecting overlapping 625-1491 bp amplicons to SMRT-BS indicated high reproducibility across all amplicon lengths (r=0.972) and low standard deviations (≤0.10) between individual CpG sites sequenced in triplicate. Higher variability in CpG methylation quantitation was correlated with reduced sequencing depth, particularly for intermediately methylated regions. SMRT-BS was validated by orthogonal bisulfite-based microarray (r=0.906; 42 CpG sites) and second generation sequencing (r=0.933; 174 CpG sites); however, longer SMRT-BS amplicons (>1.0 kb) had reduced, but very acceptable, correlation with both orthogonal methods (r=0.836-0.897 and r=0.892-0.927, respectively) compared to amplicons less than ~1.0 kb (r=0.940-0.951 and r=0.948-0.963, respectively). Multiplexing utility was assessed by simultaneously subjecting four distinct CpG island amplicons (702-866 bp; 325 CpGs) and 30 hematological malignancy cell lines to SMRT-BS (average depth of 110X), which identified a spectrum of highly quantitative methylation levels across all interrogated CpG sites and cell lines. SMRT-BS is a novel, accurate and cost-effective targeted CpG methylation method that is amenable to a high degree of multiplexing with minimal clonal PCR artifacts. Increased sequencing depth is necessary when interrogating longer amplicons (>1.0 kb) and the previously reported bisulfite sequencing PCR bias towards

  6. In vivo multiplex quantitative analysis of 3 forms of alpha melanocyte stimulating hormone in pituitary of prolyl endopeptidase deficient mice

    Directory of Open Access Journals (Sweden)

    Perroud Bertrand

    2009-06-01

    Full Text Available Abstract Background In vitro reactions are useful to identify putative enzyme substrates, but in vivo validation is required to identify actual enzyme substrates that have biological meaning. To investigate in vivo effects of prolyl endopeptidase (PREP, a serine protease, on alpha melanocyte stimulating hormone (α-MSH, we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of α-MSH. Methods Using Multiple Reaction Monitoring (MRM, we analyzed peptide transitions to quantify three different forms of α-MSH. Transitions were first confirmed using standard peptides. Samples were then analyzed by mass spectrometry using a triple quadrupole mass spectrometer, after elution from a reverse phase C18 column by a gradient of acetonitrile. Results We first demonstrate in vitro that PREP digests biological active alpha melanocyte stimulating hormone (α-MSH1–13, by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (α-MSH1–12 product – the 12 residues α-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of α-MSH: the deacetylated α-MSH1–13, the acetylated α-MSH1–13 and the truncated form α-MSH1–12. For this experiment, we used a mouse model (PREP-GT in which the serine protease, prolyl endopeptidase, is deficient due to a genetrap insertion. Here we report that the ratio between acetylated α-MSH1–13 and α-MSH1–12 is significantly increased (P-value = 0.015, N = 6 in the pituitaries of PREP-GT mice when compared to wild type littermates. In addition no significant changes were revealed in the relative level of α-MSH1–13 versus the deacetylated α-MSH1–13. These results combined with the demonstration that PREP digests α-MSH1–13 in vitro, strongly suggest that α-MSH1–13 is an in vivo substrate of PREP. Conclusion The multiplex targeted quantitative peptidomics technique we

  7. How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis by RAPD and multiplex-endonuclease genotyping approach.

    Science.gov (United States)

    Claes, F; Agbo, E C; Radwanska, M; Te Pas, M F W; Baltz, T; De Waal, D T; Goddeeris, B M; Claassen, E; Büscher, P

    2003-05-01

    The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response.

  8. A multiplexed quantum memory.

    Science.gov (United States)

    Lan, S-Y; Radnaev, A G; Collins, O A; Matsukevich, D N; Kennedy, T A; Kuzmich, A

    2009-08-03

    A quantum repeater is a system for long-distance quantum communication that employs quantum memory elements to mitigate optical fiber transmission losses. The multiplexed quantum memory (O. A. Collins, S. D. Jenkins, A. Kuzmich, and T. A. B. Kennedy, Phys. Rev. Lett. 98, 060502 (2007)) has been shown theoretically to reduce quantum memory time requirements. We present an initial implementation of a multiplexed quantum memory element in a cold rubidium gas. We show that it is possible to create atomic excitations in arbitrary memory element pairs and demonstrate the violation of Bell's inequality for light fields generated during the write and read processes.

  9. Rapid economic analysis of northern hardwood stand improvement options

    Science.gov (United States)

    William B. Leak

    1980-01-01

    Data and methodology are provided for projecting basal area, diameter, volumes, and values by product for northern hardwood stands, and for determining the rate of return on stand improvement investments. The method is rapid, requires a minimum amount of information, and should prove useful for on-the-ground economic analyses.

  10. Cytokine and chemokine profiles of aqueous humor and serum in horses with uveitis measured using multiplex bead immunoassay analysis.

    Science.gov (United States)

    Curto, Elizabeth; Messenger, Kristen M; Salmon, Jacklyn H; Gilger, Brian C

    2016-12-01

    To determine whether horses with clinically diagnosed Equine Recurrent Uveitis (ERU) and those with Leptospirosis infection have a specific cytokine profile in their aqueous humor (AH) and serum that differs from horses with uveitis secondary to other ocular inflammatory processes and from horses with normal eyes. Twenty-five client-owned horses with uveitis that were presented to the North Carolina State University Ophthalmology Service, and four University-owned horses without history or clinical signs of ocular disease. Samples of AH and serum were obtained from horses with ERU (n=13), acute or non-recurrent uveitis (UV; n=7), uveitis secondary to infectious keratitis (IK; n=5), and normal eyes (N; n=4). Cytokine levels in AH and serum were quantified using a multiplex bead immunoassay. Leptospiral antibody titers in serum and AH and PCR for Leptospiral DNA in AH were performed. In the AH of horses with ERU, increased levels of IL-1a, IL-4, IL-6, IL-8, IL-12p70, FGF-2, G-CSF, and RANTES were measured compared to UV, IK and N eyes, but the differences were not significant. However, IL-10 was significantly higher in ERU eyes compared to IK and N (P=0.029; 0.013), and IP-10 in ERU eyes was significantly higher than in UV and N (P=0.004). Furthermore, MCP-1 was significantly higher in ERU than N (P=0.04). In the serum, increased levels of IL-1a, IL-4, IL-6, IL-8, IL-12p70, fractalkine, and G-CSF were measured in horses with ERU, but the levels were not significantly higher than those observed in UV, IK, or N horses. However, serum IP-10 levels in horses with ERU were significantly higher than in UV and N horses (P=0.005) and MCP-1 levels were significantly higher in ERU than N (P=0.03). Horses with marked ocular inflammation had significantly higher serum levels of G-CSF, IL-1a, fractalkine, IL-13, IL-4, IL-17a, IL-12p70, IFN-γ, and MCP-1. Elevated IL-10 in AH was significantly associated with disease chronicity, both overall and in ERU eyes (P=0.049), and in

  11. Longitudinal study of a heteroplasmic 3460 Leber hereditary optic neuropathy family by multiplexed primer-extension analysis and nucleotide sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, S.S.; Fahy, E. [Applied Genetics, San Diego, CA (United States); Bodis-Wollner, I. [State Univ. of New York College of Optometry, New York, NY (United States)] [and others

    1996-02-01

    Nucleotide-sequencing and multiplexed primer-extension assays have been used to quantitate the mutant-allele frequency in 14 maternal relatives, spanning three generations, from a family that is heteroplasmic for the primary Leber hereditary optic neuropathy (LHON) mutation at nucleotide 3460 of the mitochondrial genome. There was excellent agreement between the values that were obtained with the two different methods. The longitudinal study shows that the mutant-allele frequency was constant within individual family members over a sampling period of 3.5 years. Second, although there was an overall increase in the mutant-allele frequency in successive generations, segregation in the direction of the mutant allele was not invariant, and there was one instance in which there was a significant decrease in the frequency from parent to offspring. From these two sets of results, and from previous studies of heteroplasmic LHON families, we conclude that there is no evidence for a marked selective pressure that determines the replication, segregation, or transmission of primary LHON mutations to white blood cells and platelets. Instead, the mtDNA molecules are most likely to replicate and segregate under conditions of random drift at the cellular level. Finally, the pattern of transmission in this maternal lineage is compatible with a developmental bottleneck model in which the number of mitochondrial units of segregation in the female germ line is relatively small in relation to the number of mtDNA molecules within a cell. However, this is not an invariant pattern for humans, and simple models of mitochondrial gene transmission are inappropriate at the present time. 37 refs., 4 figs., 1 tab.

  12. Multiplex PCR Analysis of 17 (11 Novels) STR Markers Linked to Six Autosomal Recessive Intellectual Disability Genes in Iranian Population.

    Science.gov (United States)

    Shirin, Ghadami; Maryam, Abiri; Farideh, Zonozi Rishsefid; Javad, Tavakkoly-Bazzaz; Sirous, Zeinali

    2016-01-01

    Non-syndromic autosomal recessive intellectual disability (NS-ARID) is a heterogeneous neurodevelopmental disease. More research is needed to study the NS-ARID genes. Using STR markers linked to a specific gene, we can perform homozygosity mapping and prenatal diagnosis. One approach to investigate the NS-ARID genes in large families is homozygosity mapping. In this study, we surveyed allele frequency and allele heterozygosity of 17 NS-ARID STR markers linked to the six NS-ARID genes in Iranian population. The test group consisted of 120 unrelated healthy individuals. STR markers were designed using SERV software. Also, genotyping was done using multiplex PCR. Data was analyzed by Gene Mapper software. Allele frequency and observed heterozygosity rates were estimated using PowerStatV12. Deviation from the Hardy-Weinberg equilibrium (HWE) was performed based on the Exact test. Out of 17 STR loci, 11 were novel. In total, 166 alleles were detected for the 17 markers. According to our study, the D9MAN1B1SD7.4, D19S872, D6GRIK2SI1.7, D19TECRSDI.8, D1ST3GAL3SIO.5, and D14ZC3H14SD5.3 STR loci were found to be the most informative and polymorphic STR markers for MAN1B1, ZNF526, GRIK2, TECR, ST3GAL3, and ZC3H14 genes, respectively. We also performed other statistical analyses on these STR markers and found that all of the 17 STRs were polymorphic and met the Hardy-Weinberg equilibrium. Finding novel STRs, with high allele heterozygosity, is one of the most significant outcomes of the present study. These findings can be useful for homozygosity mapping, PGD, and PND practices for the NS-ARID.

  13. Multiplex editing system

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to a multiplex editing system. The system allows multiple editing of nucleic acid sequences such as genomic sequences, such as knockins of genes of interest in a genome, knockouts of genomic sequences and/or allele replacement. Also provided herein are a method...... for editing nucleic acids and a cell comprising a stably integrated endonuclease....

  14. Arthrogryposis multiplex congenita

    DEFF Research Database (Denmark)

    Linnet, Karen Markussen; Balslev, Thomas; Møller-Madsen, Bjarne

    2015-01-01

    Arthrogryposis multiplex congenita (AMC) is a sign rather than a diagnosis. It implies contractures in multiple body areas and occurs in 1:3,000-5,000 live births. Primary aetiologies include neuropathic, myopathic, metabolic, end plate and vascular disorder affecting the developing foetus...

  15. [Rapid analysis of the radionuclides plutonium and americium-241 in soils].

    Science.gov (United States)

    Egorov, A V; Klochkova, N V

    2009-01-01

    The paper shows it possible to perform a rapid analysis of the isotopes of plutomium and americium-241. The basis of the developed rapid analysis is X-spectrometric determination of the amount of plutonium isotopes and gamma-spectrometric determination of the radionuclide 241AM.

  16. Extracting information from multiplex networks.

    Science.gov (United States)

    Iacovacci, Jacopo; Bianconi, Ginestra

    2016-06-01

    Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ̃(S) for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science.

  17. Evaluating Robustness and Sensitivity of the NanoString Technologies nCounter Platform to Enable Multiplexed Gene Expression Analysis of Clinical Samples.

    Science.gov (United States)

    Veldman-Jones, Margaret H; Brant, Roz; Rooney, Claire; Geh, Catherine; Emery, Hollie; Harbron, Chris G; Wappett, Mark; Sharpe, Alan; Dymond, Michael; Barrett, J Carl; Harrington, Elizabeth A; Marshall, Gayle

    2015-07-01

    Analysis of clinical trial specimens such as formalin-fixed paraffin-embedded (FFPE) tissue for molecular mechanisms of disease progression or drug response is often challenging and limited to a few markers at a time. This has led to the increasing importance of highly multiplexed assays that enable profiling of many biomarkers within a single assay. Methods for gene expression analysis have undergone major advances in biomedical research, but obtaining a robust dataset from low-quality RNA samples, such as those isolated from FFPE tissue, remains a challenge. Here, we provide a detailed evaluation of the NanoString Technologies nCounter platform, which provides a direct digital readout of up to 800 mRNA targets simultaneously. We tested this system by examining a broad set of human clinical tissues for a range of technical variables, including sensitivity and limit of detection to varying RNA quantity and quality, reagent performance over time, variability between instruments, the impact of the number of fields of view sampled, and differences between probe sequence locations and overlapping genes across CodeSets. This study demonstrates that Nanostring offers several key advantages, including sensitivity, reproducibility, technical robustness, and utility for clinical application. ©2015 American Association for Cancer Research.

  18. MLPAinter for MLPA interpretation: an integrated approach for the analysis, visualisation and data management of Multiplex Ligation-dependent Probe Amplification

    Directory of Open Access Journals (Sweden)

    Morreau Hans

    2010-01-01

    Full Text Available Abstract Background Multiplex Ligation-Dependent Probe Amplification (MLPA is an application that can be used for the detection of multiple chromosomal aberrations in a single experiment. In one reaction, up to 50 different genomic sequences can be analysed. For a reliable work-flow, tools are needed for administrative support, data management, normalisation, visualisation, reporting and interpretation. Results Here, we developed a data management system, MLPAInter for MLPA interpretation, that is windows executable and has a stand-alone database for monitoring and interpreting the MLPA data stream that is generated from the experimental setup to analysis, quality control and visualisation. A statistical approach is applied for the normalisation and analysis of large series of MLPA traces, making use of multiple control samples and internal controls. Conclusions MLPAinter visualises MLPA data in plots with information about sample replicates, normalisation settings, and sample characteristics. This integrated approach helps in the automated handling of large series of MLPA data and guarantees a quick and streamlined dataflow from the beginning of an experiment to an authorised report.

  19. Multiplexing 200 spatial modes with a single hologram

    Science.gov (United States)

    Rosales-Guzmán, Carmelo; Bhebhe, Nkosiphile; Mahonisi, Nyiku; Forbes, Andrew

    2017-11-01

    The on-demand tailoring of light's spatial shape is of great relevance in a wide variety of research areas. Computer-controlled devices, such as spatial light modulators (SLMs) or digital micromirror devices, offer a very accurate, flexible and fast holographic means to this end. Remarkably, digital holography affords the simultaneous generation of multiple beams (multiplexing), a tool with numerous applications in many fields. Here, we provide a self-contained tutorial on light beam multiplexing. Through the use of several examples, the readers will be guided step by step in the process of light beam shaping and multiplexing. Additionally, we provide a quantitative analysis on the multiplexing capabilities of SLMs to assess the maximum number of beams that can be multiplexed on a single SLM, showing approximately 200 modes on a single hologram.

  20. Automated Methods for Multiplexed Pathogen Detection

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2005-09-01

    Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead

  1. Steatocystoma multiplex: A review

    Directory of Open Access Journals (Sweden)

    Manahel Mahmood AlSabbagh

    2016-07-01

    Full Text Available Steatocystoma multiplex is an uncommon benign disorder typically manifests as multiple asymptomatic intradermal cysts. It can be sporadic, familial or a part of a syndrome. Rare variants can be easily misdiagnosed. Histologically, it appears as an epithelium-lined cystic wall associated with sebaceous lobules. Treatment is needed if symptomatic or for cosmetic issues with the best results reported following radiofrequency, laser therapy and minimally invasive procedures.

  2. Multiplex Ligation-Dependent Probe Amplification Analysis of GATA4 Gene Copy Number Variations in Patients with Isolated Congenital Heart Disease

    Directory of Open Access Journals (Sweden)

    Valentina Guida

    2010-01-01

    Full Text Available GATA4 mutations are found in patients with different isolated congenital heart defects (CHDs, mostly cardiac septal defects and tetralogy of Fallot. In addition, GATA4 is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role of GATA4 copy number variations (CNVs in non-syndromic CHDs. To explore the possible occurrence of GATA4 gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA a cohort of 161 non-syndromic patients with cardiac anomalies previously associated with GATA4 gene mutations. The patients were mutation-negative for GATA4, NKX2.5, and FOG2 genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution of GATA4 gene CNVs in CHD pathogenesis.

  3. A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq.

    Science.gov (United States)

    Wang, Lin; Si, Yaqing; Dedow, Lauren K; Shao, Ying; Liu, Peng; Brutnell, Thomas P

    2011-01-01

    The emergence of NextGen sequencing technology has generated much interest in the exploration of transcriptomes. Currently, Illumina Inc. (San Diego, CA) provides one of the most widely utilized sequencing platforms for gene expression analysis. While Illumina reagents and protocols perform adequately in RNA-sequencing (RNA-seq), alternative reagents and protocols promise a higher throughput at a much lower cost. We have developed a low-cost and robust protocol to produce Illumina-compatible (GAIIx and HiSeq2000 platforms) RNA-seq libraries by combining several recent improvements. First, we designed balanced adapter sequences for multiplexing of samples; second, dUTP incorporation in 2(nd) strand synthesis was used to enforce strand-specificity; third, we simplified RNA purification, fragmentation and library size-selection steps thus drastically reducing the time and increasing throughput of library construction; fourth, we included an RNA spike-in control for validation and normalization purposes. To streamline informatics analysis for the community, we established a pipeline within the iPlant Collaborative. These scripts are easily customized to meet specific research needs and improve on existing informatics and statistical treatments of RNA-seq data. In particular, we apply significance tests for determining differential gene expression and intron retention events. To demonstrate the potential of both the library-construction protocol and data-analysis pipeline, we characterized the transcriptome of the rice leaf. Our data supports novel gene models and can be used to improve current rice genome annotation. Additionally, using the rice transcriptome data, we compared different methods of calculating gene expression and discuss the advantages of a strand-specific approach to detect bona-fide anti-sense transcripts and to detect intron retention events. Our results demonstrate the potential of this low cost and robust method for RNA-seq library construction and

  4. Multiplex Ligation-Dependent Probe Amplification Technique for Copy Number Analysis on Small Amounts of DNA Material

    DEFF Research Database (Denmark)

    Sørensen, Karina; Andersen, Paal; Larsen, Lars

    2008-01-01

    analysis on small amounts of DNA. Six patients with congenital adrenal hyperplasia (CAH) were used in this study. DNA was extracted from both whole blood and DBSS and subjected to MLPA analysis using normal and modified probes. Results were analyzed using GeneMarker and manual Excel analysis. A total...

  5. A multiplex chemiluminescent biosensor for type B-fumonisins and aflatoxin B1 quantitative detection in maize flour.

    Science.gov (United States)

    Zangheri, Martina; Di Nardo, Fabio; Anfossi, Laura; Giovannoli, Cristina; Baggiani, Claudio; Roda, Aldo; Mirasoli, Mara

    2015-01-07

    A multiplex chemiluminescent biosensor for simple, rapid and ultrasensitive on-site quantification of aflatoxin B1 and type B-fumonisins in maize samples has been developed. The biosensor integrates a multiplex indirect competitive lateral flow immunoassay (LFIA) based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a lensless "contact" imaging configuration. The developed assay requires a simple extraction of the analytes from maize flour samples followed by their detection with a 30 min assay time. The use of chemiluminescence detection allowed accurate and objective analytes quantification, enabling simultaneous detection of type B-fumonisins and aflatoxin B1 down to 6 μg kg(-1) and 1.5 μg kg(-1), respectively, thus fulfilling the standards imposed by the legislation of European Union. Maize flour samples spiked with both analytes were subjected to multiplex analysis obtaining recoveries ranging from 80 to 115% and the coefficient of variation below 20%. Finally, analysis of naturally contaminated maize samples resulted in a good agreement between CL-LFIA and a validated confirmatory HPLC-UV and commercial ELISA kit, obtaining recoveries in the range 88-120%. The proposed CL-LFIA protocol is rapid, inexpensive, easy-to-use, and fit for the purpose of rapid screening of mycotoxins in maize flour.

  6. Rapid All-Sky Transient Discovery and Analysis with Evryscope

    Science.gov (United States)

    Corbett, Henry T.; Law, Nicholas; Fors, Octavi; Ratzloff, Jeff; Goeke, Erin; Howard, Ward S.

    2018-01-01

    The Evryscope is an array of 24 small telescopes on a common mount, capable of observing the entire visible sky down to g' ~ 16 with a two-minute cadence. Each exposure covers 8000 square degrees over 691 MPix and requires minimal readout time, providing 97% continuous coverage of the night sky. The system's large field of view and rapid cadence enable exploration of a previously inaccessible parameter space of bright and fast transients, including nearby microlensing events, supernovae, and kilonovae GW counterparts. The first instrument, located at CTIO in Chile, was deployed in mid-2015 and is currently in production creating multi-year light curves with percent-level precision. A second identical system is on track for deployment at Mount Laguna Observatory in California in early 2018. Once operational, the two sites will provide simultaneous two-color photometry over a 4000 square degree overlapping region accessible to both instruments, operating as a combined discovery and follow-up network for transient phenomena on all nearby stars and many nearby galaxies. I will present recent science results from the Evryscope and an overview of our data reduction pipeline.

  7. A multiplex PCR assay for the detection and quantification of Sclerotinia sclerotiorum and Botrytis cinerea.

    Science.gov (United States)

    Reich, J D; Alexander, T W; Chatterton, S

    2016-05-01

    Traditional culture methods for identifying the plant fungal pathogens Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow and laborious. The goal of this study was to develop a multiplex real-time PCR (qPCR) assay to detect and quantify DNA from S. sclerotiorum and B. cinerea. A primer set (SsIGS_5) for S. sclerotiorum was designed that targeted the intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the assay increased its specificity: when the primer/probe set was tested against 21 fungal species (35 strains), amplification was detected from all S. sclerotiorum strains and no other species. For qPCR, the SsIGS_5 primer and probe set exhibited a linear range from 7·0 ng to 0·07 pg target DNA (R(2)  = 0·99). SsIGS_5 was then multiplexed with a previously published primer/probe set for B. cinerea to develop a high-throughput method for the detection and quantification of DNA from both pathogens. When multiplexed, the sensitivity and specificity of both assays were not different from individual qPCR reactions. The multiplex assay is currently being used to detect and quantify S. sclerotiorum and B. cinerea DNA from aerosol samples collected in commercial seed alfalfa fields. A primer and probe set for the quantification of Sclerotinia sclerotiorum DNA in a PCR assay was developed. The probe-based nature of this assay signifies an improvement over previous assays for this species by allowing multiplex reactions while maintaining high sensitivity. The primer/probe set was used in a multiplex real-time PCR assay for the quantification of S. sclerotiorum and Botrytis cinerea DNA, enabling rapid analysis of environmental samples. In crops susceptible to both pathogens, this multiplex assay can be used to quickly quantify the presence of each pathogen. © 2016 Her Majesty the Queen in Right of Canada © 2016 The Society for Applied Microbiology. Reproduced with the permission of the Office of the

  8. Clinical framework for next generation sequencing based analysis of treatment predictive mutations and multiplexed gene fusion detection in non-small cell lung cancer.

    Science.gov (United States)

    Lindquist, Kajsa Ericson; Karlsson, Anna; Levéen, Per; Brunnström, Hans; Reuterswärd, Christel; Holm, Karolina; Jönsson, Mats; Annersten, Karin; Rosengren, Frida; Jirström, Karin; Kosieradzki, Jaroslaw; Ek, Lars; Borg, Åke; Planck, Maria; Jönsson, Göran; Staaf, Johan

    2017-05-23

    Precision medicine requires accurate multi-gene clinical diagnostics. We describe the implementation of an Illumina TruSight Tumor (TST) clinical NGS diagnostic framework and parallel validation of a NanoString RNA-based ALK, RET, and ROS1 gene fusion assay for combined analysis of treatment predictive alterations in non-small cell lung cancer (NSCLC) in a regional healthcare region of Sweden (Scandinavia). The TST panel was clinically validated in 81 tumors (99% hotspot mutation concordance), after which 533 consecutive NSCLCs were collected during one-year of routine clinical analysis in the healthcare region (~90% advanced stage patients). The NanoString assay was evaluated in 169 of 533 cases. In the 533-sample cohort 79% had 1-2 variants, 12% >2 variants and 9% no detected variants. Ten gene fusions (five ALK, three RET, two ROS1) were detected in 135 successfully analyzed cases (80% analysis success rate). No ALK or ROS1 FISH fusion positive case was missed by the NanoString assay. Stratification of the 533-sample cohort based on actionable alterations in 11 oncogenes revealed that 66% of adenocarcinomas, 13% of squamous carcinoma (SqCC) and 56% of NSCLC not otherwise specified harbored ≥1 alteration. In adenocarcinoma, 10.6% of patients (50.3% if including KRAS) could potentially be eligible for emerging therapeutics, in addition to the 15.3% of patients eligible for standard EGFR or ALK inhibitors. For squamous carcinoma corresponding proportions were 4.4% (11.1% with KRAS) vs 2.2%. In conclusion, multiplexed NGS and gene fusion analyses are feasible in NSCLC for clinical diagnostics, identifying notable proportions of patients potentially eligible for emerging molecular therapeutics.

  9. Layer Communities in Multiplex Networks

    Science.gov (United States)

    Kao, Ta-Chu; Porter, Mason A.

    2017-08-01

    Multiplex networks are a type of multilayer network in which entities are connected to each other via multiple types of connections. We propose a method, based on computing pairwise similarities between layers and then doing community detection, for grouping structurally similar layers in multiplex networks. We illustrate our approach using both synthetic and empirical networks, and we are able to find meaningful groups of layers in both cases. For example, we find that airlines that are based in similar geographic locations tend to be grouped together in a multiplex airline network and that related research areas in physics tend to be grouped together in a multiplex collaboration network.

  10. Development of a GeXP-multiplex PCR assay for the simultaneous detection and differentiation of six cattle viruses.

    Directory of Open Access Journals (Sweden)

    Qing Fan

    Full Text Available Foot-and-mouth disease virus (FMDV, Bluetongue virus (BTV, Vesicular stomatitis Virus (VSV, Bovine viral diarrheal (BVDV, Bovine rotavirus (BRV, and Bovine herpesvirus 1 (IBRV are common cattle infectious viruses that cause a great economic loss every year in many parts of the world. A rapid and high-throughput GenomeLab Gene Expression Profiler (GeXP analyzer-based multiplex PCR assay was developed for the simultaneous detection and differentiation of these six cattle viruses. Six pairs of chimeric primers consisting of both the gene-specific primer and a universal primer were designed and used for amplification. Then capillary electrophoresis was used to separate the fluorescent labeled PCR products according to the amplicons size. The specificity of GeXP-multiplex PCR assay was examined with samples of the single template and mixed template of six viruses. The sensitivity was evaluated using the GeXP-multiplex PCR assay on serial 10-fold dilutions of ssRNAs obtained via in vitro transcription. To further evaluate the reliability, 305 clinical samples were tested by the GeXP-multiplex PCR assay. The results showed that the corresponding virus specific fragments of genes were amplified. The detection limit of the GeXP-multiplex PCR assay was 100 copies/μL in a mixed sample of ssRNAs containing target genes of six different cattle viruses, whereas the detection limit for the Gexp-mono PCR assay for a single target gene was 10 copies/μL. In detection of viruses in 305 clinical samples, the results of GeXP were consistent with simplex real-time PCR. Analysis of positive samples by sequencing demonstrated that the GeXP-multiplex PCR assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP-multiplex PCR assay is a high throughput, specific, sensitive, rapid and simple method for the detection and differentiation of six cattle viruses. It is an effective tool that can be applied for the rapid differential diagnosis

  11. A rapid analysis of plasma/serum ethylene and propylene glycol by headspace gas chromatography

    National Research Council Canada - National Science Library

    Ehlers, Alexandra; Morris, Cory; Krasowski, Matthew D

    2013-01-01

    A rapid headspace-gas chromatography (HS-GC) method was developed for the analysis of ethylene glycol and propylene glycol in plasma and serum specimens using 1,3-propanediol as the internal standard...

  12. A Rapid Aeroelastic/Aeroservoelastic Modeling, Analysis and Optimization System for Advanced Flight Vehicles Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Stirling Dynamics Inc and the University of Washington propose to develop a MATLAB toolbox for rapid aeroelastic (AE) and aeroservoelastic (ASE) modeling, analysis...

  13. An Analysis of Test And Evaluation in Rapid Acquisition Programs

    Science.gov (United States)

    2015-12-01

    Outfits DT Developmental Testing DT&E Development Test and Evaluation DTC Developmental Test Command E3 Electromagnetic Environmental Effects...by the OEM and evaluated by the Communication-Electronics Command (CECOM) Safety Office and the former Developmental Test Command ( DTC ) covering all...Force Management School AMC Army Materiel Command AOA Analysis of Alternatives AOR Area of Operation APC Acquisition Program Candidates APG Aberdeen

  14. Multiple routes transmitted epidemics on multiplex networks

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Dawei [Information Security Center, State Key Laboratory of Networking and Switching Technology, Beijing University of Posts and Telecommunications, P.O. Box 145, Beijing 100876 (China); National Engineering Laboratory for Disaster Backup and Recovery, Beijing University of Posts and Telecommunications, Beijing 100876 (China); Shandong Provincial Key Laboratory of Computer Network, Shandong Computer Science Center, Jinan 250014 (China); Li, Lixiang [Information Security Center, State Key Laboratory of Networking and Switching Technology, Beijing University of Posts and Telecommunications, P.O. Box 145, Beijing 100876 (China); National Engineering Laboratory for Disaster Backup and Recovery, Beijing University of Posts and Telecommunications, Beijing 100876 (China); Peng, Haipeng, E-mail: penghaipeng@bupt.edu.cn [Information Security Center, State Key Laboratory of Networking and Switching Technology, Beijing University of Posts and Telecommunications, P.O. Box 145, Beijing 100876 (China); National Engineering Laboratory for Disaster Backup and Recovery, Beijing University of Posts and Telecommunications, Beijing 100876 (China); Luo, Qun; Yang, Yixian [Information Security Center, State Key Laboratory of Networking and Switching Technology, Beijing University of Posts and Telecommunications, P.O. Box 145, Beijing 100876 (China); National Engineering Laboratory for Disaster Backup and Recovery, Beijing University of Posts and Telecommunications, Beijing 100876 (China)

    2014-02-01

    This letter investigates the multiple routes transmitted epidemic process on multiplex networks. We propose detailed theoretical analysis that allows us to accurately calculate the epidemic threshold and outbreak size. It is found that the epidemic can spread across the multiplex network even if all the network layers are well below their respective epidemic thresholds. Strong positive degree–degree correlation of nodes in multiplex network could lead to a much lower epidemic threshold and a relatively smaller outbreak size. However, the average similarity of neighbors from different layers of nodes has no obvious effect on the epidemic threshold and outbreak size. -- Highlights: •We studies multiple routes transmitted epidemic process on multiplex networks. •SIR model and bond percolation theory are used to analyze the epidemic processes. •We derive equations to accurately calculate the epidemic threshold and outbreak size. •ASN has no effect on the epidemic threshold and outbreak size. •Strong positive DDC leads to a lower epidemic threshold and a smaller outbreak size.

  15. Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat.

    Science.gov (United States)

    Bernardo, Amy; Wang, Shan; St Amand, Paul; Bai, Guihua

    2015-01-01

    With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat (Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat.

  16. Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat.

    Directory of Open Access Journals (Sweden)

    Amy Bernardo

    Full Text Available With the advent of next generation sequencing (NGS technologies, single nucleotide polymorphisms (SNPs have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat (Triticum aestivum L. that can be effectively used in marker-assisted selection (MAS is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP and sequence tagged site (STS markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat.

  17. Prospective evaluation of a high multiplexing real-time polymerase chain reaction array for the rapid identification and characterization of bacteria causative of nosocomial pneumonia from clinical specimens: a proof-of-concept study.

    Science.gov (United States)

    Roisin, S; Huang, T-D; de Mendonça, R; Nonhoff, C; Bogaerts, P; Hites, M; Delaere, B; Hamels, S; de Longueville, F; Glupczynski, Y; Denis, O

    2017-09-27

    The purpose of this study was evaluation of the VAPChip assay based on the "Rapid-Array-PCR-technology" which targets 13 respiratory pathogens and 24 β-lactam resistance genes directly on respiratory clinical specimens. The first step included analysis of 45 respiratory specimens in order to calibrate and determine the threshold for target genes. The second prospective step involved 85 respiratory samples from patients suspected of nosocomial pneumonia collected in two academic hospitals over an 8-month period. Results of the VAPChip assay were compared to routine methods. The first step showed a large proportion of positive signals for H. influenzae and/or S. pneumoniae. For identification, discrepancies were observed in seven samples. Thresholds were adapted and two probes were re-designed to create a new version of the cartridge. In the second phase, sensitivity and specificity of the VAPchip for bacterial identification were 72.9% and 99.1%, respectively. Seventy (82%) pathogens were correctly identified by both methods. Nine pathogens detected by the VAPChip were culture negative and 26 pathogens identified by culture were VAPChip negative. For resistance mechanisms, 11 probes were positive without identification of pathogens with an antimicrobial-susceptibility testing compatible by culture. However, the patient's recent microbiological history was able to explain most of these positive signals. The VAPChip assay simultaneously detects different pathogens and resistance mechanisms directly from clinical samples. This system seems very promising but the extraction process needs to be automated for routine implementation. This kind of rapid point-of-care automated platform permitting a syndromic approach will be the future challenge in the management of infectious diseases.

  18. Effectiveness Analysis of a Part-Time Rapid Response System During Operation Versus Nonoperation.

    Science.gov (United States)

    Kim, Youlim; Lee, Dong Seon; Min, Hyunju; Choi, Yun Young; Lee, Eun Young; Song, Inae; Park, Jong Sun; Cho, Young-Jae; Jo, You Hwan; Yoon, Ho Il; Lee, Jae Ho; Lee, Choon-Taek; Do, Sang Hwan; Lee, Yeon Joo

    2017-06-01

    To evaluate the effect of a part-time rapid response system on the occurrence rate of cardiopulmonary arrest by comparing the times of rapid response system operation versus nonoperation. Retrospective cohort study. A 1,360-bed tertiary care hospital. Adult patients admitted to the general ward were screened. Data were collected over 36 months from rapid response system implementation (October 2012 to September 2015) and more than 45 months before rapid response system implementation (January 2009 to September 2012). None. The rapid response system operates from 7 AM to 10 PM on weekdays and from 7 AM to 12 PM on Saturdays. Primary outcomes were the difference of cardiopulmonary arrest incidence between pre-rapid response system and post-rapid response system periods and whether the rapid response system operating time affects the cardiopulmonary arrest incidence. The overall cardiopulmonary arrest incidence (per 1,000 admissions) was 1.43. Although the number of admissions per month and case-mix index were increased (3,555.18 vs 4,564.72, p times (0.82 vs 0.49/1,000 admissions; p = 0.001) but remained similar during rapid response system nonoperating times (0.77 vs 0.73/1,000 admissions; p = 0.729). The implementation of a part-time rapid response system reduced the cardiopulmonary arrest incidence based on the reduction of cardiopulmonary arrest during rapid response system operating times. Further analysis of the cost effectiveness of part-time rapid response system is needed.

  19. Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP Assay for the Detection of Bacterial Meningitis Pathogens

    Directory of Open Access Journals (Sweden)

    Owen Higgins

    2018-02-01

    Full Text Available Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

  20. Preliminary Assessment of Microwave Readout Multiplexing Factor

    Energy Technology Data Exchange (ETDEWEB)

    Croce, Mark Philip [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Koehler, Katrina Elizabeth [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Rabin, Michael W. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Bennett, D. A. [National Inst. of Standards and Technology (NIST), Boulder, CO (United States); Mates, J. A. B. [National Inst. of Standards and Technology (NIST), Boulder, CO (United States); Gard, J. D. [National Inst. of Standards and Technology (NIST), Boulder, CO (United States); Becker, D. [National Inst. of Standards and Technology (NIST), Boulder, CO (United States); Schmidt, D. R. [National Inst. of Standards and Technology (NIST), Boulder, CO (United States); Ullom, J. N. [National Inst. of Standards and Technology (NIST), Boulder, CO (United States)

    2017-01-23

    Ultra-high resolution microcalorimeter gamma spectroscopy is a new non-destructive assay technology for measurement of plutonium isotopic composition, with the potential to reduce total measurement uncertainty to a level competitive with destructive analysis methods [1-4]. Achieving this level of performance in practical applications requires not only the energy resolution now routinely achieved with transition-edge sensor microcalorimeter arrays (an order of magnitude better than for germanium detectors) but also high throughput. Microcalorimeter gamma spectrometers have not yet achieved detection efficiency and count rate capability that is comparable to germanium detectors, largely because of limits from existing readout technology. Microcalorimeter detectors must be operated at low temperature to achieve their exceptional energy resolution. Although the typical 100 mK operating temperatures can be achieved with reliable, cryogen-free systems, the cryogenic complexity and heat load from individual readout channels for large sensor arrays is prohibitive. Multiplexing is required for practical systems. The most mature multiplexing technology at present is time-division multiplexing (TDM) [3, 5-6]. In TDM, the sensor outputs are switched by applying bias current to one SQUID amplifier at a time. Transition-edge sensor (TES) microcalorimeter arrays as large as 256 pixels have been developed for X-ray and gamma-ray spectroscopy using TDM technology. Due to bandwidth limits and noise scaling, TDM is limited to a maximum multiplexing factor of approximately 32-40 sensors on one readout line [8]. Increasing the size of microcalorimeter arrays above the kilopixel scale, required to match the throughput of germanium detectors, requires the development of a new readout technology with a much higher multiplexing factor.

  1. Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

    Directory of Open Access Journals (Sweden)

    Peng Xia

    2009-01-01

    Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

  2. Highspeed multiplexed heterodyne interferometry.

    Science.gov (United States)

    Isleif, Katharina-S; Gerberding, Oliver; Köhlenbeck, Sina; Sutton, Andrew; Sheard, Benjamin; Goßler, Stefan; Shaddock, Daniel; Heinzel, Gerhard; Danzmann, Karsten

    2014-10-06

    Digitally enhanced heterodyne interferometry is a metrology technique that uses pseudo-random noise codes for modulating the phase of the laser light. Multiple interferometric signals from the same beam path can thereby be isolated based on their propagation delay, allowing one to use advantageous optical layouts in comparison to classic laser interferometers. We present here a high speed version of this technique for measuring multiple targets spatially separated by only a few centimetres. This allows measurements of multiplexed signals using free beams, making the technique attractive for several applications requiring compact optical set-ups like for example space-based interferometers. In an experiment using a modulation and sampling rate of 1.25 GHz we are able to demonstrate multiplexing between targets only separated by 36 cm and we achieve a displacement measurement noise floor of up. Utilising an active clock jitter correction scheme we are also able to reduce this noise in a null measurement configuration by one order of magnitude.

  3. Multiplex Recurrence Networks

    Science.gov (United States)

    Eroglu, Deniz; Marwan, Norbert

    2017-04-01

    The complex nature of a variety of phenomena in physical, biological, or earth sciences is driven by a large number of degrees of freedom which are strongly interconnected. Although the evolution of such systems is described by multivariate time series (MTS), so far research mostly focuses on analyzing these components one by one. Recurrence based analyses are powerful methods to understand the underlying dynamics of a dynamical system and have been used for many successful applications including examples from earth science, economics, or chemical reactions. The backbone of these techniques is creating the phase space of the system. However, increasing the dimension of a system requires increasing the length of the time series in order get significant and reliable results. This requirement is one of the challenges in many disciplines, in particular in palaeoclimate, thus, it is not easy to create a phase space from measured MTS due to the limited number of available obervations (samples). To overcome this problem, we suggest to create recurrence networks from each component of the system and combine them into a multiplex network structure, the multiplex recurrence network (MRN). We test the MRN by using prototypical mathematical models and demonstrate its use by studying high-dimensional palaeoclimate dynamics derived from pollen data from the Bear Lake (Utah, US). By using the MRN, we can distinguish typical climate transition events, e.g., such between Marine Isotope Stages.

  4. Lab-on-a-Chip Multiplex Assays.

    Science.gov (United States)

    Peter, Harald; Wienke, Julia; Bier, Frank F

    2017-01-01

    Lab-on-a-chip multiplex assays allow a rapid identification of multiple parameters in an automated manner. Here we describe a lab-based preparation followed by a rapid and fully automated DNA microarray hybridization and readout in less than 10 min using the Fraunhofer in vitro diagnostics (ivD) platform to enable rapid identification of bacterial species and detection of antibiotic resistance. The use of DNA microarrays allows a fast adaptation of new biomarkers enabling the identification of different genes as well as single-nucleotide-polymorphisms (SNPs) within these genes. In this protocol we describe a DNA microarray developed for identification of Staphylococcus aureus and the mecA resistance gene.

  5. Immunocap® ISAC and microtest for multiplex allergen testing in people with difficult to manage allergic disease: A systematic review and cost analysis

    NARCIS (Netherlands)

    M. Westwood (Marie); B.L.T. Ramaekers (Bram); Lang, S. (Shona); N. Armstrong (Nigel); C. Noake; De Kock, S. (Shelley); M.A. Joore (Manuela); J.L. Severens (Hans); J. Kleijnen (Jos)

    2016-01-01

    textabstractBackground Allergy is a form of immune-mediated exaggerated sensitivity (hypersensitivity) to a substance that is either inhaled, swallowed, injected or comes into contact with the skin. Foreign substances that provoke allergies are called allergens. It has been claimed that multiplex

  6. Considerations for Task Analysis Methods and Rapid E-Learning Development Techniques

    Directory of Open Access Journals (Sweden)

    Dr. Ismail Ipek

    2014-02-01

    Full Text Available The purpose of this paper is to provide basic dimensions for rapid training development in e-learning courses in education and business. Principally, it starts with defining task analysis and how to select tasks for analysis and task analysis methods for instructional design. To do this, first, learning and instructional technologies as visions of the future were discussed. Second, the importance of task analysis methods in rapid e-learning was considered, with learning technologies as asynchronous and synchronous e-learning development. Finally, rapid instructional design concepts and e-learning design strategies were defined and clarified with examples, that is, all steps for effective task analysis and rapid training development techniques based on learning and instructional design approaches were discussed, such as m-learning and other delivery systems. As a result, the concept of task analysis, rapid e-learning development strategies and the essentials of online course design were discussed, alongside learner interface design features for learners and designers.

  7. A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

    Directory of Open Access Journals (Sweden)

    Aline Padilha Fraga

    2013-01-01

    Full Text Available Mycoplasma gallisepticum (MS and Mycoplasma synoviae (MS are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR. The complete assay (Multiplex MGMS was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR and MS (MS PCR. The results demonstrated an agreement of 100% (23 positive and 44 negative samples between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.

  8. Spatially multiplexed dark-field microspectrophotometry for nanoplasmonics

    Science.gov (United States)

    Pini, V.; Kosaka, P. M.; Ruz, J. J.; Malvar, O.; Encinar, M.; Tamayo, J.; Calleja, M.

    2016-03-01

    Monitoring the effect of the substrate on the local surface plasmon resonance (LSPR) of metallic nanoparticles is key for deepening our understanding of light-matter interactions at the nanoscale. This coupling gives rise to shifts of the LSPR as well as changes in the scattering pattern shape. The problem requires of high-throughput techniques that present both high spatial and spectral resolution. We present here a technique, referred to as Spatially Multiplexed Micro-Spectrophotometry (SMMS), able to perform polarization-resolved spectral and spatial analysis of the scattered light over large surface areas. The SMMS technique provides three orders of magnitude faster spectroscopic analysis than conventional dark-field microspectrophotometry, with the capability for mapping the spatial distribution of the scattered light intensity with lateral resolution of 40 nm over surface areas of 0.02 mm2. We show polarization-resolved dark-field spectral analysis of hundreds of gold nanoparticles deposited on a silicon surface. The technique allows determining the effect of the substrate on the LSPR of single nanoparticles and dimers and their scattering patterns. This is applied for rapid discrimination and counting of monomers and dimers of nanoparticles. In addition, the diameter of individual nanoparticles can be rapidly assessed with 1 nm accuracy.

  9. Multiplexed analysis combining distinctly-sized CdTe-MPA quantum dots and chemometrics for multiple mutually interfering analyte determination.

    Science.gov (United States)

    Bittar, Dayana B; Ribeiro, David S M; Páscoa, Ricardo N M J; Soares, José X; Rodrigues, S Sofia M; Castro, Rafael C; Pezza, Leonardo; Pezza, Helena R; Santos, João L M

    2017-11-01

    Semiconductor quantum dots (QDs) have demonstrated a great potential as fluorescent probes for heavy metals monitoring. However, their great reactivity, whose tunability could be difficult to attain, could impair selectivity yielding analytical results with poor accuracy. In this work, the combination in the same analysis of multiple QDs, each with a particular ability to interact with the analyte, assured a multi-point detection that was not only exploited for a more precise analyte discrimination but also for the simultaneous discrimination of multiple mutually interfering species, in the same sample. Three different MPA-CdTe QDs (2.5, 3.0 and 3.8nm) with a good size distribution, confirmed by the FWHM values of 48.6, 55.4 and 80.8nm, respectively, were used. Principal component analysis (PCA) and partial least squares regression (PLS) were used for fluorescence data analysis. Mixtures of two MPA-CdTe QDs, emitting at different wavelength namely 549/566, 549/634 and 566/634nm were assayed. The 549/634nm emitting QDs mixture provided the best results for the discrimination of distinct ions on binary and ternary mixtures. The obtained RMSECV and R(2)CV values for the binary mixture were good, namely, from 0.01 to 0.08mgL(-1) and from 0.74 to 0.89, respectively. Regarding the ternary mixture the RMSECV and R(2)CV values were good for Hg(II) (0.06 and 0.73mgL(-1), respectively) and Pb(II) (0.08 and 0.87mg L(-1), respectively) and acceptable for Cu(II) (0.02 and 0.51mgL(-1), respectively). In conclusion, the obtained results showed that the developed approach is capable of resolve binary and ternary mixtures of Pb (II), Hg (II) and Cu (II), providing accurate information about lead (II) and mercury (II) concentration and signaling the occurrence of Cu (II). Copyright © 2017 Elsevier B.V. All rights reserved.

  10. FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ.

    Science.gov (United States)

    Kumar, Sunil; Alibhai, Dominic; Margineanu, Anca; Laine, Romain; Kennedy, Gordon; McGinty, James; Warren, Sean; Kelly, Douglas; Alexandrov, Yuriy; Munro, Ian; Talbot, Clifford; Stuckey, Daniel W; Kimberly, Christopher; Viellerobe, Bertrand; Lacombe, Francois; Lam, Eric W-F; Taylor, Harriet; Dallman, Margaret J; Stamp, Gordon; Murray, Edward J; Stuhmeier, Frank; Sardini, Alessandro; Katan, Matilda; Elson, Daniel S; Neil, Mark A A; Dunsby, Chris; French, Paul M W

    2011-02-25

    A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ**

    Science.gov (United States)

    Kumar, Sunil; Alibhai, Dominic; Margineanu, Anca; Laine, Romain; Kennedy, Gordon; McGinty, James; Warren, Sean; Kelly, Douglas; Alexandrov, Yuriy; Munro, Ian; Talbot, Clifford; Stuckey, Daniel W; Kimberly, Christopher; Viellerobe, Bertrand; Lacombe, Francois; Lam, Eric W-F; Taylor, Harriet; Dallman, Margaret J; Stamp, Gordon; Murray, Edward J; Stuhmeier, Frank; Sardini, Alessandro; Katan, Matilda; Elson, Daniel S; Neil, Mark A A; Dunsby, Chris; French, Paul M W

    2011-01-01

    A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated. PMID:21337485

  12. Rapid biocompatibility analysis of materials via in vivo fluorescence imaging of mouse models.

    Directory of Open Access Journals (Sweden)

    Kaitlin M Bratlie

    Full Text Available BACKGROUND: Many materials are unsuitable for medical use because of poor biocompatibility. Recently, advances in the high throughput synthesis of biomaterials has significantly increased the number of potential biomaterials, however current biocompatibility analysis methods are slow and require histological analysis. METHODOLOGY/PRINCIPAL FINDINGS: Here we develop rapid, non-invasive methods for in vivo quantification of the inflammatory response to implanted biomaterials. Materials were placed subcutaneously in an array format and monitored for host responses as per ISO 10993-6: 2001. Host cell activity in response to these materials was imaged kinetically, in vivo using fluorescent whole animal imaging. Data captured using whole animal imaging displayed similar temporal trends in cellular recruitment of phagocytes to the biomaterials compared to histological analysis. CONCLUSIONS/SIGNIFICANCE: Histological analysis similarity validates this technique as a novel, rapid approach for screening biocompatibility of implanted materials. Through this technique there exists the possibility to rapidly screen large libraries of polymers in vivo.

  13. Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments

    Directory of Open Access Journals (Sweden)

    Wen Daxing

    2012-08-01

    Full Text Available Abstract Background Multiplex PCR has been successfully applied in many areas since it was first reported in 1988; however, it suffers from poor universality. Results A novel method called Universal Multiplex PCR (UM-PCR was created, which simultaneously amplifies multiple target fragments from genomic DNA. The method has two steps. First, the universal adapter-F and universal adapter-R are connected to the forward primers and the reverse primers, respectively. Hairpin structures and cross dimers of five pairs of adapter-primers are detected. Second, UM-PCR amplification is implemented using a novel PCR procedure termed “Two Rounds Mode” (three and 28–32 cycles. The first round (the first three cycles is named the “One by One Annealing Round”. The second round (28–32 cycles combines annealing with extension. In the first two cycles of the first round, primers only amplify the specific templates; there are no templates for the universal adapters. The templates of universal adapters begin to be synthesized from the second cycle of the first round, and universal adapters and primers commence full amplification from the third cycle of the first round. Conclusions UM-PCR greatly improves the universality of multiplex PCR. UM-PCR could rapidly detect the genetic purity of maize seeds. In addition, it could be applied in other areas, such as analysis of polymorphisms, quantitative assays and identifications of species.

  14. Arthrogryposis multiplex congenita.

    Science.gov (United States)

    Mennen, Ulrich; van Heest, Ann; Ezaki, Mary-Beth; Tonkin, Michael; Gericke, George

    2005-10-01

    Since much confusion exists regarding arthrogryposis multiplex congenita (AMC), the President of the IFSSH commissioned the AMC Committee to compile a report on the various aspects of this condition. This report discusses all the facets of AMC, including definition, terminology, dermographics, aetiology, classification, clinical features, management, prognosis and further studies. Manipulation of the deformities starting soon after birth improves the range of motion, which, if surgery needs to be done, makes the operation less extensive. Sometimes surgery may not even be necessary. Early one-stage corrective surgery between 3 and 12 months of age is encouraged. This results in improved function and near normal cosmetic appearance. Late, piece-meal surgery results in disappointing outcome.

  15. Multiplex families with epilepsy

    Science.gov (United States)

    Afawi, Zaid; Oliver, Karen L.; Kivity, Sara; Mazarib, Aziz; Blatt, Ilan; Neufeld, Miriam Y.; Helbig, Katherine L.; Goldberg-Stern, Hadassa; Misk, Adel J.; Straussberg, Rachel; Walid, Simri; Mahajnah, Muhammad; Lerman-Sagie, Tally; Ben-Zeev, Bruria; Kahana, Esther; Masalha, Rafik; Kramer, Uri; Ekstein, Dana; Shorer, Zamir; Wallace, Robyn H.; Mangelsdorf, Marie; MacPherson, James N.; Carvill, Gemma L.; Mefford, Heather C.; Jackson, Graeme D.; Scheffer, Ingrid E.; Bahlo, Melanie; Gecz, Jozef; Heron, Sarah E.; Corbett, Mark; Mulley, John C.; Dibbens, Leanne M.; Korczyn, Amos D.

    2016-01-01

    Objective: To analyze the clinical syndromes and inheritance patterns of multiplex families with epilepsy toward the ultimate aim of uncovering the underlying molecular genetic basis. Methods: Following the referral of families with 2 or more relatives with epilepsy, individuals were classified into epilepsy syndromes. Families were classified into syndromes where at least 2 family members had a specific diagnosis. Pedigrees were analyzed and molecular genetic studies were performed as appropriate. Results: A total of 211 families were ascertained over an 11-year period in Israel. A total of 169 were classified into broad familial epilepsy syndrome groups: 61 generalized, 22 focal, 24 febrile seizure syndromes, 33 special syndromes, and 29 mixed. A total of 42 families remained unclassified. Pathogenic variants were identified in 49/211 families (23%). The majority were found in established epilepsy genes (e.g., SCN1A, KCNQ2, CSTB), but in 11 families, this cohort contributed to the initial discovery (e.g., KCNT1, PCDH19, TBC1D24). We expand the phenotypic spectrum of established epilepsy genes by reporting a familial LAMC3 homozygous variant, where the predominant phenotype was epilepsy with myoclonic-atonic seizures, and a pathogenic SCN1A variant in a family where in 5 siblings the phenotype was broadly consistent with Dravet syndrome, a disorder that usually occurs sporadically. Conclusion: A total of 80% of families were successfully classified, with pathogenic variants identified in 23%. The successful characterization of familial electroclinical and inheritance patterns has highlighted the value of studying multiplex families and their contribution towards uncovering the genetic basis of the epilepsies. PMID:26802095

  16. Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

    Energy Technology Data Exchange (ETDEWEB)

    Lattanzio, Veronica M.T., E-mail: veronica.lattanzio@ispa.cnr.it [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy); Nivarlet, Noan [UNISENSOR S.A., Zoning industriel du Dossay, Rue du Dossay no 3, B-4020 Liege (Belgium); Lippolis, Vincenzo; Gatta, Stefania Della [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy); Huet, Anne-Catherine; Delahaut, Philippe [Centre d' Economie Rurale (CER Groupe), Rue du Point du Jour no 8, B-6900 Marloie (Belgium); Granier, Benoit [UNISENSOR S.A., Zoning industriel du Dossay, Rue du Dossay no 3, B-4020 Liege (Belgium); Visconti, Angelo [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer We developed a rapid method based on a multiplex dipstick immunoassay. Black-Right-Pointing-Pointer The assay allowed the determination of major Fusarium toxins in wheat, oats, maize. Black-Right-Pointing-Pointer We obtained cut off levels close to EU regulatory levels. - Abstract: A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 {mu}g kg{sup -1}, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 {mu}g kg{sup -1}, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins

  17. A 128 Multiplexing Factor Time-Domain SQUID Multiplexer

    Science.gov (United States)

    Prêle, D.; Voisin, F.; Piat, M.; Decourcelle, T.; Perbost, C.; Chapron, C.; Rambaud, D.; Maestre, S.; Marty, W.; Montier, L.

    2016-07-01

    A cryogenic 128:1 Time-Domain Multiplexer (TDM) has been developed for the readout of kilo-pixel Transition Edge Sensor (TES) arrays dedicated to the Q&U Bolometric Interferometer for Cosmology (QUBIC) instrument which aims to measure the B-mode polarization of the Cosmic Microwave Background. Superconducting QUantum Interference Devices (SQUIDs) are usually used to read out TESs. Moreover, SQUIDs are used to build TDM by biasing sequentially the SQUIDs connected together—one for each TES. In addition to this common technique which allows a typical 32 multiplexing factor, a cryogenic integrated circuit provides a 4:1 second multiplexing stage. This cryogenic integrated circuit is one of the original part of our TDM achieving an unprecedented 128 multiplexing factor. We present these two dimension TDM stages: topology of the SQUID multiplexer, operation of the cryogenic integrated circuit, and integration of the full system to read out a TES array dedicated to the QUBIC instrument. Flux-locked loop operation in multiplexed mode is also discussed.

  18. Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Kumar Sukhdeo

    Full Text Available Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116. Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

  19. Universal Super-Resolution Multiplexing by DNA Exchange.

    Science.gov (United States)

    Schueder, Florian; Strauss, Maximilian T; Hoerl, David; Schnitzbauer, Joerg; Schlichthaerle, Thomas; Strauss, Sebastian; Yin, Peng; Harz, Hartmann; Leonhardt, Heinrich; Jungmann, Ralf

    2017-03-27

    Super-resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20× higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time-consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super-resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

    Energy Technology Data Exchange (ETDEWEB)

    Letant, S E; .Ortiz, J I; Tammero, L; Birch, J M; Derlet, R W; Cohen, S; Manning, D; McBride, M T

    2007-04-11

    We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. This article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.

  1. Rapid Analysis of Corni fructus Using Paper Spray-Mass Spectrometry.

    Science.gov (United States)

    Guo, Yuan; Gu, Zhixin; Liu, Xuemei; Liu, Jingjing; Ma, Ming; Chen, Bo; Wang, Liping

    2017-07-01

    Paper spray-mass spectrometry (PS-MS) is a kind of ambient MS technique for the rapid analysis of samples. Corni fructus has been widely used in traditional Chinese compound preparations and healthy food. However, a number of counterfeits of Corni fructus, such as Crataegi fructus, Lycii fructus, and grape skin are illegally sold in crude herb markets. Therefore, the development of a rapid and high-throughput quality evaluation method is important for ensuring the effectiveness and safety of the crude materials of Corni fructus. To develop PS-MS chemical profiles and a semi-quantitative method of Corni fructus for quality assessment and control, and species distinction of Corni fructus. Both positive and negative ion PS-MS chemical profiles were constructed for species distinction. The statistical analysis of the chemical profiles was accomplished by principal component analysis (PCA). Rapid semi-quantitative analysis of loganin and morroniside in the extracts of Corni fructus were accomplished by PS-MS. The profiles of the Corni fructus and Crataegi fructus samples were clearly clustered into two categories. The limit of quantification (LOQ) in the semi-quantitative analysis was 6 μg/mL and 5.6 μg/mL for loganin and morroniside, respectively. PS-MS is a simple, rapid, and high-throughput method for the quality control and species distinction of Corni fructus. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  2. A multiplex PCR for detection of six viruses in ducks.

    Science.gov (United States)

    Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

    2017-10-01

    In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10 2 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

  3. Navigability of multiplex temporal network

    Science.gov (United States)

    Wang, Yan; Song, Qiao-Zhen

    2017-01-01

    Real world complex systems have multiple levels of relationships and in many cases, they need to be modeled as multiplex networks where the same nodes can interact with each other in different layers, such as social networks. However, social relationships only appear at prescribed times so the temporal structures of edge activations can also affect the dynamical processes located above them. To consider both factors are simultaneously, we introduce multiplex temporal networks and propose three different walk strategies to investigate the concurrent dynamics of random walks and the temporal structure of multiplex networks. Thus, we derive analytical results for the multiplex centrality and coverage function in multiplex temporal networks. By comparing them with the numerical results, we show how the underlying topology of the layers and the walk strategy affect the efficiency when exploring the networks. In particular, the most interesting result is the emergence of a super-diffusion process, where the time scale of the multiplex is faster than that of both layers acting separately.

  4. A parylene-based dual channel microelectrophoresis system for rapid mutation detection via heteroduplex analysis

    NARCIS (Netherlands)

    Sukas, S.; Erson, Ayse Elif; Sert, Cuneyt; Kulah, Haluk

    2008-01-01

    A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 μm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing

  5. A novel microfluidic system for the rapid analysis of protein thermal stability.

    Science.gov (United States)

    Yang, Xin; Liu, Jia; Xie, Ye Lei; Wang, Yang; Ying, Hong; Wu, Qiong; Huang, Wei; Jenkins, Gareth

    2014-06-07

    We describe a simple microfluidic device for the rapid analysis of protein thermal stability using a novel imaging method. The change in UV absorption upon thermal denaturation or aggregation of proteins is used to get a spatial image of proteins' folding or aggregation state along a linear temperature gradient.

  6. Rapid Identification and Characterization of Formulated Protein Products by Raman Spectroscopy Coupled with Discriminant Analysis.

    Science.gov (United States)

    Cao, Xiaolin; Zhou, Dan; Loussaert, James A; Meriage, David S; Levine, Joseph D; Gabrielson, John P; Wen, Zai-Qing

    2016-01-01

    The rapid identification of protein drug products for packaging and receiving can significantly reduce disposition cycle time, and thereby improve the efficiency and productivity of the supply chain to better meet the needs of patients. In this feasibility study, we demonstrate a novel methodology that combines Raman spectroscopy with discriminant analysis that can be used for rapid identification or verification of finished products. With this methodology, Raman spectra of formulated therapeutic proteins were collected non-invasively with the samples either in a quartz cuvette or in the original glass vials, and analyzed without subtraction of buffer or placebo solutions. The algorithm used for the discriminant analysis was Mahalanobis distance by principal component analysis with residuals. In addition to product identification, the methodology has the potential to be used for characterizing formulated proteins when exposed to external stresses based on the changes of Mahalanobis distances. The rapid identification of protein drug products for packaging and receiving can significantly reduce disposition cycle time, and thereby improve the efficiency and productivity of the supply chain. In this study, we demonstrate a novel methodology that combines Raman spectroscopy with discriminant analysis to rapidly identify formulated proteins non-invasively. © PDA, Inc. 2016.

  7. Rapid Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Perlin

    2005-08-14

    Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleic acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe development

  8. Robust SNP genotyping by multiplex PCR and arrayed primer extension

    Directory of Open Access Journals (Sweden)

    Podder Mohua

    2008-01-01

    Full Text Available Abstract Background Arrayed primer extension (APEX is a microarray-based rapid minisequencing methodology that may have utility in 'personalized medicine' applications that involve genetic diagnostics of single nucleotide polymorphisms (SNPs. However, to date there have been few reports that objectively evaluate the assay completion rate, call rate and accuracy of APEX. We have further developed robust assay design, chemistry and analysis methodologies, and have sought to determine how effective APEX is in comparison to leading 'gold-standard' genotyping platforms. Our methods have been tested against industry-leading technologies in two blinded experiments based on Coriell DNA samples and SNP genotype data from the International HapMap Project. Results In the first experiment, we genotyped 50 SNPs across the entire 270 HapMap Coriell DNA sample set. For each Coriell sample, DNA template was amplified in a total of 7 multiplex PCRs prior to genotyping. We obtained good results for 41 of the SNPs, with 99.8% genotype concordance with HapMap data, at an automated call rate of 94.9% (not including the 9 failed SNPs. In the second experiment, involving modifications to the initial DNA amplification so that a single 50-plex PCR could be achieved, genotyping of the same 50 SNPs across each of 49 randomly chosen Coriell DNA samples allowed extremely robust 50-plex genotyping from as little as 5 ng of DNA, with 100% assay completion rate, 100% call rate and >99.9% accuracy. Conclusion We have shown our methods to be effective for robust multiplex SNP genotyping using APEX, with 100% call rate and >99.9% accuracy. We believe that such methodology may be useful in future point-of-care clinical diagnostic applications where accuracy and call rate are both paramount.

  9. Multiplexed lasing in tissues

    Science.gov (United States)

    Chen, Yu-Cheng; Chen, Qiushu; Fan, Xudong

    2017-02-01

    Biolasers are an emerging technology for next generation biochemical detection and clinical applications. Progress has recently been made to achieve lasing from biomolecules and single living cells. Tissues, which consist of cells embedded in extracellular matrix, mimic more closely the actual complex biological environment in a living body and therefore are of more practical significance. Here, we developed a highly versatile tissue laser platform, in which tissues stained with fluorophores are sandwiched in a high-Q Fabry-Pérot microcavity. Distinct lasing emissions from muscle and adipose tissues stained respectively with fluorescein isothiocyanate (FITC) and boron-dipyrromethene (BODIPY), and hybrid muscle/adipose tissue with dual-staining were achieved with a threshold of only 10 μJ/mm2. Additionally, we investigated how tissue structure/geometry, tissue thickness, and staining dye concentration affect the tissue laser. It is further found that, despite large fluorescence spectral overlap between FITC and BODIPY in tissues, their lasing emissions could be clearly distinguished and controlled due to their narrow lasing bands and different lasing thresholds, thus enabling highly multiplexed detection. Our tissue laser platform can be broadly applicable to various types of tissues/diseases. It provides a new tool for a wide range of biological and biomedical applications, such as diagnostics/screening of tissues and identification/monitoring of biological transformations in tissue engineering.

  10. A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method.

    Science.gov (United States)

    Shiramaru, Sachi; Asakura, Masahiro; Inoue, Haruna; Nagita, Akira; Matsuhisa, Akio; Yamasaki, Shinji

    2012-07-01

    In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).

  11. The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform.

    Science.gov (United States)

    Škereňová, Markéta; Mikulová, Veronika; Čapoun, Otakar; Zima, Tomáš

    2016-01-01

    Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. The characteristics established in our study are in concordance with the manufacturer's specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.

  12. Rapid thyroid dysfunction screening based on serum surface-enhanced Raman scattering and multivariate statistical analysis

    Science.gov (United States)

    Tian, Dayong; Lü, Guodong; Zhai, Zhengang; Du, Guoli; Mo, Jiaqing; Lü, Xiaoyi

    2018-01-01

    In this paper, serum surface-enhanced Raman scattering and multivariate statistical analysis are used to investigate a rapid screening technique for thyroid function diseases. At present, the detection of thyroid function has become increasingly important, and it is urgently necessary to develop a rapid and portable method for the detection of thyroid function. Our experimental results show that, by using the Silmeco-based enhanced Raman signal, the signal strength greatly increases and the characteristic peak appears obviously. It is also observed that the Raman spectra of normal and anomalous thyroid function human serum are significantly different. Principal component analysis (PCA) combined with linear discriminant analysis (LDA) was used to diagnose thyroid dysfunction, and the diagnostic accuracy was 87.4%. The use of serum surface-enhanced Raman scattering technology combined with PCA–LDA shows good diagnostic performance for the rapid detection of thyroid function. By means of Raman technology, it is expected that a portable device for the rapid detection of thyroid function will be developed.

  13. Efficient exploration of multiplex networks

    Science.gov (United States)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2016-04-01

    Efficient techniques to navigate networks with local information are fundamental to sample large-scale online social systems and to retrieve resources in peer-to-peer systems. Biased random walks, i.e. walks whose motion is biased on properties of neighbouring nodes, have been largely exploited to design smart local strategies to explore a network, for instance by constructing maximally mixing trajectories or by allowing an almost uniform sampling of the nodes. Here we introduce and study biased random walks on multiplex networks, graphs where the nodes are related through different types of links organised in distinct and interacting layers, and we provide analytical solutions for their long-time properties, including the stationary occupation probability distribution and the entropy rate. We focus on degree-biased random walks and distinguish between two classes of walks, namely those whose transition probability depends on a number of parameters which is extensive in the number of layers, and those whose motion depends on intrinsically multiplex properties of the neighbouring nodes. We analyse the effect of the structure of the multiplex network on the steady-state behaviour of the walkers, and we find that heterogeneous degree distributions as well as the presence of inter-layer degree correlations and edge overlap determine the extent to which a multiplex can be efficiently explored by a biased walk. Finally we show that, in real-world multiplex transportation networks, the trade-off between efficient navigation and resilience to link failure has resulted into systems whose diffusion properties are qualitatively different from those of appropriately randomised multiplex graphs. This fact suggests that multiplexity is an important ingredient to include in the modelling of real-world systems.

  14. Multiplex real-time PCR assays for detection of eight Shiga toxin-producing Escherichia coli in food samples by melting curve analysis.

    Science.gov (United States)

    Singh, Prashant; Mustapha, Azlin

    2015-12-23

    Shiga toxin-producing Escherichia coli (STEC) are pathogenic strains of E. coli that can cause bloody diarrhea and kidney failure. Seven STEC serogroups, O157, O26, O45, O103, O111, O121 and O145 are responsible for more than 71% of the total infections caused by this group of pathogens. All seven serogroups are currently considered as adulterants in non-intact beef products in the U.S. In this study, two multiplex melt curve real-time PCR assays with internal amplification controls (IACs) were standardized for the detection of eight STEC serogroups. The first multiplex assay targeted E. coli serogroups O145, O121, O104, and O157; while the second set detected E. coli serogroups O26, O45, O103 and O111. The applicability of the assays was tested using 11 different meat and produce samples. For food samples spiked with a cocktail of four STEC serogroups with a combined count of 10 CFU/25 g food, all targets of the multiplex assays were detected after an enrichment period of 6h. The assays also worked efficiently when 325 g of food samples were spiked with 10 CFU of STECs. The assays are not dependent on fluorescent-labeled probes or immunomagnetic beads, and can be used for the detection of eight STEC serogroups in less than 11h. Routine preliminary screening of STECs in food samples is performed by testing for the presence of STEC virulence genes. The assays developed in this study can be useful as a first- or second-tier test for the identification of the eight O serogroup-specific genes in suspected food samples. Copyright © 2015. Published by Elsevier B.V.

  15. Rapid elemental analysis and provenance study of Blumea balsamifera DC using laser-induced breakdown spectroscopy.

    Science.gov (United States)

    Liu, Xiaona; Zhang, Qiao; Wu, Zhisheng; Shi, Xinyuan; Zhao, Na; Qiao, Yanjiang

    2014-12-31

    Laser-induced breakdown spectroscopy (LIBS) was applied to perform a rapid elemental analysis and provenance study of Blumea balsamifera DC. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were implemented to exploit the multivariate nature of the LIBS data. Scores and loadings of computed principal components visually illustrated the differing spectral data. The PLS-DA algorithm showed good classification performance. The PLS-DA model using complete spectra as input variables had similar discrimination performance to using selected spectral lines as input variables. The down-selection of spectral lines was specifically focused on the major elements of B. balsamifera samples. Results indicated that LIBS could be used to rapidly analyze elements and to perform provenance study of B. balsamifera.

  16. Simulation Framework for Rapid Entry, Descent, and Landing (EDL) Analysis. Volume 2; Appendices

    Science.gov (United States)

    Murri, Daniel G.

    2010-01-01

    The NASA Engineering and Safety Center (NESC) was requested to establish the Simulation Framework for Rapid Entry, Descent, and Landing (EDL) Analysis assessment, which involved development of an enhanced simulation architecture using the Program to Optimize Simulated Trajectories II (POST2) simulation tool. The assessment was requested to enhance the capability of the Agency to provide rapid evaluation of EDL characteristics in systems analysis studies, preliminary design, mission development and execution, and time-critical assessments. Many of the new simulation framework capabilities were developed to support the Agency EDL Systems Analysis (EDL-SA) team, that is conducting studies of the technologies and architectures that are required to enable higher mass robotic and human mission to Mars. The appendices to the original report are contained in this document.

  17. Simulation Framework for Rapid Entry, Descent, and Landing (EDL) Analysis, Phase 2 Results

    Science.gov (United States)

    Murri, Daniel G.

    2011-01-01

    The NASA Engineering and Safety Center (NESC) was requested to establish the Simulation Framework for Rapid Entry, Descent, and Landing (EDL) Analysis assessment, which involved development of an enhanced simulation architecture using the Program to Optimize Simulated Trajectories II simulation tool. The assessment was requested to enhance the capability of the Agency to provide rapid evaluation of EDL characteristics in systems analysis studies, preliminary design, mission development and execution, and time-critical assessments. Many of the new simulation framework capabilities were developed to support the Agency EDL-Systems Analysis (SA) team that is conducting studies of the technologies and architectures that are required to enable human and higher mass robotic missions to Mars. The findings, observations, and recommendations from the NESC are provided in this report.

  18. Simulation Framework for Rapid Entry, Descent, and Landing (EDL) Analysis. Volume 1

    Science.gov (United States)

    Murri, Daniel G.

    2010-01-01

    The NASA Engineering and Safety Center (NESC) was requested to establish the Simulation Framework for Rapid Entry, Descent, and Landing (EDL) Analysis assessment, which involved development of an enhanced simulation architecture using the Program to Optimize Simulated Trajectories II (POST2) simulation tool. The assessment was requested to enhance the capability of the Agency to provide rapid evaluation of EDL characteristics in systems analysis studies, preliminary design, mission development and execution, and time-critical assessments. Many of the new simulation framework capabilities were developed to support the Agency EDL Systems Analysis (EDL-SA) team, that is conducting studies of the technologies and architectures that are required to enable higher mass robotic and human mission to Mars. The findings of the assessment are contained in this report.

  19. Rapid Elemental Analysis and Provenance Study of Blumea balsamifera DC Using Laser-Induced Breakdown Spectroscopy

    Directory of Open Access Journals (Sweden)

    Xiaona Liu

    2014-12-01

    Full Text Available Laser-induced breakdown spectroscopy (LIBS was applied to perform a rapid elemental analysis and provenance study of Blumea balsamifera DC. Principal component analysis (PCA and partial least squares discriminant analysis (PLS-DA were implemented to exploit the multivariate nature of the LIBS data. Scores and loadings of computed principal components visually illustrated the differing spectral data. The PLS-DA algorithm showed good classification performance. The PLS-DA model using complete spectra as input variables had similar discrimination performance to using selected spectral lines as input variables. The down-selection of spectral lines was specifically focused on the major elements of B. balsamifera samples. Results indicated that LIBS could be used to rapidly analyze elements and to perform provenance study of B. balsamifera.

  20. Using latent class analysis to estimate the test characteristics of the γ-interferon test, the single intradermal comparative tuberculin test and a multiplex immunoassay under Irish conditions

    DEFF Research Database (Denmark)

    Clegg, Tracy A.; Duignan, Anthony; Whelan, Clare

    2011-01-01

    estimated in populations with defined infection status. However, these approaches can be problematic as there may be few herds in Ireland where freedom from infection is guaranteed. We used latent class models to estimate the diagnostic characteristics of existing (SICTT and ¿-IFN) and new (multiplex...... of sensitivity and specificity under field conditions in Ireland and suggest that the Enferplex-TB test has the potential to improve on current diagnostics for TB infection in cattle. The extent of that potential will be assessed in further studies....

  1. Antigen-induced cytokine and chemokine release test for tuberculosis infection using adsorption of stimulated whole blood on filter paper and multiplex analysis

    DEFF Research Database (Denmark)

    Skogstrand, Kristin; Thysen, Anna H; Jørgensen, Charlotte S

    2012-01-01

    (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. Results: Our multiplex measurements...... of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1β were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. Conclusion: Applying blood on filter paper after incubation...

  2. Advancing the High Throughput Identification of Liver Fibrosis Protein Signatures Using Multiplexed Ion Mobility Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Erin Shammel; Burnum-Johnson, Kristin E.; Jacobs, Jon M.; Diamond, Deborah L.; Brown, Roslyn N.; Ibrahim, Yehia M.; Orton, Daniel J.; Piehowski, Paul D.; Purdy, David E.; Moore, Ronald J.; Danielson, William F.; Monroe, Matthew E.; Crowell, Kevin L.; Slysz, Gordon W.; Gritsenko, Marina A.; Sandoval, John D.; Lamarche, Brian L.; Matzke, Melissa M.; Webb-Robertson, Bobbie-Jo M.; Simons, Brenna C.; McMahon, Brian J.; Bhattacharya, Renuka; Perkins, James D.; Carithers, Robert L.; Strom, Susan; Self, Steven; Katze, Michael G.; Anderson, Gordon A.; Smith, Richard D.

    2014-04-01

    Rapid diagnosis of disease states using less invasive, safer, and more clinically acceptable approaches than presently employed is an imperative goal for the field of medicine. While mass spectrometry (MS)-based proteomics approaches have attempted to meet these objectives, challenges such as the enormous dynamic range of protein concentrations in clinically relevant biofluid samples coupled with the need to address human biodiversity have slowed their employment. Herein, we report on the use of a new platform that addresses these challenges by coupling technical advances in rapid gas phase multiplexed ion mobility spectrometry (IMS) separations [1, 2] with liquid chromatography (LC) and MS to dramatically increase measurement sensitivity and throughput, further enabling future MS-based clinical applications. An initial application of the LC-IMS-MS platform for the analysis of blood serum samples from stratified post-liver transplant patients with recurrent fibrosis progression illustrates its potential utility for disease characterization and use in personalized medicine [3, 4].

  3. Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis.

    Science.gov (United States)

    Jiang, Lu Xi; Ren, Hong Yu; Zhou, Hai Jian; Zhao, Si Hong; Hou, Bo Yan; Yan, Jian Ping; Qin, Tian; Chen, Yu

    2017-08-01

    Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  4. Bond Percolation on Multiplex Networks

    Science.gov (United States)

    Hackett, A.; Cellai, D.; Gómez, S.; Arenas, A.; Gleeson, J. P.

    2016-04-01

    We present an analytical approach for bond percolation on multiplex networks and use it to determine the expected size of the giant connected component and the value of the critical bond occupation probability in these networks. We advocate the relevance of these tools to the modeling of multilayer robustness and contribute to the debate on whether any benefit is to be yielded from studying a full multiplex structure as opposed to its monoplex projection, especially in the seemingly irrelevant case of a bond occupation probability that does not depend on the layer. Although we find that in many cases the predictions of our theory for multiplex networks coincide with previously derived results for monoplex networks, we also uncover the remarkable result that for a certain class of multiplex networks, well described by our theory, new critical phenomena occur as multiple percolation phase transitions are present. We provide an instance of this phenomenon in a multiplex network constructed from London rail and European air transportation data sets.

  5. Bond Percolation on Multiplex Networks

    Directory of Open Access Journals (Sweden)

    A. Hackett

    2016-04-01

    Full Text Available We present an analytical approach for bond percolation on multiplex networks and use it to determine the expected size of the giant connected component and the value of the critical bond occupation probability in these networks. We advocate the relevance of these tools to the modeling of multilayer robustness and contribute to the debate on whether any benefit is to be yielded from studying a full multiplex structure as opposed to its monoplex projection, especially in the seemingly irrelevant case of a bond occupation probability that does not depend on the layer. Although we find that in many cases the predictions of our theory for multiplex networks coincide with previously derived results for monoplex networks, we also uncover the remarkable result that for a certain class of multiplex networks, well described by our theory, new critical phenomena occur as multiple percolation phase transitions are present. We provide an instance of this phenomenon in a multiplex network constructed from London rail and European air transportation data sets.

  6. Structural measures for multiplex networks.

    Science.gov (United States)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2014-03-01

    Many real-world complex systems consist of a set of elementary units connected by relationships of different kinds. All such systems are better described in terms of multiplex networks, where the links at each layer represent a different type of interaction between the same set of nodes rather than in terms of (single-layer) networks. In this paper we present a general framework to describe and study multiplex networks, whose links are either unweighted or weighted. In particular, we propose a series of measures to characterize the multiplexicity of the systems in terms of (i) basic node and link properties such as the node degree, and the edge overlap and reinforcement, (ii) local properties such as the clustering coefficient and the transitivity, and (iii) global properties related to the navigability of the multiplex across the different layers. The measures we introduce are validated on a genuinely multiplex data set of Indonesian terrorists, where information among 78 individuals are recorded with respect to mutual trust, common operations, exchanged communications, and business relationships.

  7. High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

    DEFF Research Database (Denmark)

    Hjelmsø, Mathis Hjort; Hansen, Lars Hestbjerg; Bælum, Jacob

    2014-01-01

    -resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized...... that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide...

  8. [Detection of Staphylococcus aureus, Shigella spp., Salmonella spp. in food by multiplex PCR].

    Science.gov (United States)

    Li, Bo; Chen, Fusheng; Wang, Xiaohong; Shao, Yanchun

    2008-07-01

    To establish a multiplex PCR method for simultaneous detection of Staphylococcus aureus, Shigella spp., Salmonella spp. in food. Staphylococcus aureus was enriched by 7.5% NaCl broth while Shigella spp. and Salmonella spp. were enriched by GN medium . The primers were designed according to the gene nuc of Staphylococcus aureus, the gene ipaH of Shigella spp. and the gene invA of Salmonella spp. The target genes of these pathogens in food were amplified by multiplex PCR, which reaction conditions were optimized specifically. The multiplex PCR method established in this experment was of high specificity, which detection limit was 1 cfu/ml of Staphylococcus aureus, Shigella spp. and Salmonella spp. when the milk samples contaminated with these pathogens. The multiplex PCR method, which was rapid, convenient, and with high sensitivity, could be suitable for rapid detection of Staphylococcus aureus, Shigella spp., Salmonella spp. in food, and could have a great prospect.

  9. drLumi: An open-source package to manage data, calibrate, and conduct quality control of multiplex bead-based immunoassays data analysis.

    Directory of Open Access Journals (Sweden)

    Hector Sanz

    Full Text Available Multiplex bead-based immunoassays are used to measure concentrations of several analytes simultaneously. These assays include control standard curves (SC to reduce between-plate variability and normalize quantitation of analytes of biological samples. Suboptimal calibration might result in large random error and decreased number of samples with analyte concentrations within the limits of quantification. Suboptimal calibration may be a consequence of poor fitness of the functions used for the SC, the treatment of the background noise and the method used to estimate the limits of quantification. Currently assessment of fitness of curves is largely dependent on operator and that may add additional error. Moreover, there is no software to automate data managing and quality control. In this article we present a R package, drLumi, with functions for managing data, calibrating assays and performing quality control. To optimize the assay the package implements: i three dose-response functions, ii four approaches for treating background noise and iii three methods for estimating limits of quantifications. Other implemented functions are focused on the quality control of the fitted standard curve: detection of outliers, estimation of the confidence or prediction interval, and estimation of summary statistics. With demonstration purpose, we apply the software to 30 cytokines, chemokines and growth factors measured in a multiplex bead-based immunoassay in a study aiming to measure correlates of risk or protection from malaria of the RTS,S malaria vaccine nested in the Phase 3 randomized controlled trial of this vaccine.

  10. The new challenges of multiplex networks: Measures and models

    Science.gov (United States)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2017-02-01

    What do societies, the Internet, and the human brain have in common? They are all examples of complex relational systems, whose emerging behaviours are largely determined by the non-trivial networks of interactions among their constituents, namely individuals, computers, or neurons, rather than only by the properties of the units themselves. In the last two decades, network scientists have proposed models of increasing complexity to better understand real-world systems. Only recently we have realised that multiplexity, i.e. the coexistence of several types of interactions among the constituents of a complex system, is responsible for substantial qualitative and quantitative differences in the type and variety of behaviours that a complex system can exhibit. As a consequence, multilayer and multiplex networks have become a hot topic in complexity science. Here we provide an overview of some of the measures proposed so far to characterise the structure of multiplex networks, and a selection of models aiming at reproducing those structural properties and quantifying their statistical significance. Focusing on a subset of relevant topics, this brief review is a quite comprehensive introduction to the most basic tools for the analysis of multiplex networks observed in the real-world. The wide applicability of multiplex networks as a framework to model complex systems in different fields, from biology to social sciences, and the colloquial tone of the paper will make it an interesting read for researchers working on both theoretical and experimental analysis of networked systems.

  11. Helicity multiplexed broadband metasurface holograms

    Science.gov (United States)

    Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Yue Bun Pun, Edwin; Zhang, Shuang; Chen, Xianzhong

    2015-09-01

    Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices.

  12. Helicity multiplexed broadband metasurface holograms.

    Science.gov (United States)

    Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Pun, Edwin Yue Bun; Zhang, Shuang; Chen, Xianzhong

    2015-09-10

    Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices.

  13. Rapid analysis & design methodologies of High-Frequency LCLC Resonant Inverter as Electrodeless Fluorescent Lamp Ballast

    OpenAIRE

    Ang, Y A; Stone, D A; Bingham, Chris; Foster, M

    2007-01-01

    The papers presents methodologies for the analysis of 4th-order LCLC resonant power converters operating at 2.63 MHz as fluorescent lamp ballasts, where high frequency operation facilitates capacitive discharge into the tube, with near resonance operation at high load quality factor enabling high efficiency. State-variable dynamic descriptions of the converter are employed to rapidly determine the steady-state cyclic behaviour of the ballast during nominal operation. Simulation and experiment...

  14. The rapid analysis of fungal growth in the presence of inhibitory effects

    OpenAIRE

    Williams, Tyson

    2011-01-01

    For fungal contamination of foodstuffs, there are no fast, reliable, automated techniques to examine growth, nor have any predictive models been developed to describe the growth in the same way as for bacteria. Traditional plating methods can take 3 to 7 days to get adequate results depending on the fungal species utilised and well over a month for challenge testing, an unacceptable delay especially for the food industry. In this study two rapid analysis techniques were investi...

  15. Performance modeling, stochastic networks, and statistical multiplexing

    CERN Document Server

    Mazumdar, Ravi R

    2013-01-01

    This monograph presents a concise mathematical approach for modeling and analyzing the performance of communication networks with the aim of introducing an appropriate mathematical framework for modeling and analysis as well as understanding the phenomenon of statistical multiplexing. The models, techniques, and results presented form the core of traffic engineering methods used to design, control and allocate resources in communication networks.The novelty of the monograph is the fresh approach and insights provided by a sample-path methodology for queueing models that highlights the importan

  16. Rapid and Concomitant Analysis of Pharmaceuticals in Treated Wastewater by Coated Blade Spray Mass Spectrometry.

    Science.gov (United States)

    Poole, Justen J; Gómez-Ríos, German A; Boyaci, Ezel; Reyes-Garcés, Nathaly; Pawliszyn, Janusz

    2017-11-07

    The widespread use of pharmaceuticals in both human and animal populations, and the resultant contamination of surface waters from the outflow of water treatment facilities is an issue of growing concern. This has raised the need for analytical methods that can both perform rapid sample analysis and overcome the limitations of conventional analysis procedures, such as multistep workflows and tedious procedures. Coated blade spray (CBS) is a solid-phase microextraction based technique that enables the direct-to-mass-spectrometry analysis of extracted compounds via the use of limited organic solvent to desorb analytes and perform electrospray ionization. This paper documents how CBS can be applied for the concomitant tandem mass spectrometric (MS/MS) analysis of nine pharmaceuticals in treated wastewater. The total analysis times of less than 11 min provided limits of detection lower than 50 ng L-1 for all target compounds in river water. The CBS methodology was then compared to a conventional solid-phase extraction technique for the analysis of the final effluent of six wastewater treatment facilities. The experimental results strongly suggest that CBS offers scientists a viable alternative method for analyzing water samples that is both rapid and relatively solvent-free.

  17. Rapid Automated Dissolution and Analysis Techniques for Radionuclides in Recycle Process Streams

    Energy Technology Data Exchange (ETDEWEB)

    Sudowe, Ralf [Univ. of Nevada, Las Vegas, NV (United States). Radiochemistry Program and Health Physics Dept.; Roman, Audrey [Univ. of Nevada, Las Vegas, NV (United States). Radiochemistry Program; Dailey, Ashlee [Univ. of Nevada, Las Vegas, NV (United States). Radiochemistry Program; Go, Elaine [Univ. of Nevada, Las Vegas, NV (United States). Radiochemistry Program

    2013-07-18

    The analysis of process samples for radionuclide content is an important part of current procedures for material balance and accountancy in the different process streams of a recycling plant. The destructive sample analysis techniques currently available necessitate a significant amount of time. It is therefore desirable to develop new sample analysis procedures that allow for a quick turnaround time and increased sample throughput with a minimum of deviation between samples. In particular, new capabilities for rapid sample dissolution and radiochemical separation are required. Most of the radioanalytical techniques currently employed for sample analysis are based on manual laboratory procedures. Such procedures are time- and labor-intensive, and not well suited for situations in which a rapid sample analysis is required and/or large number of samples need to be analyzed. To address this issue we are currently investigating radiochemical separation methods based on extraction chromatography that have been specifically optimized for the analysis of process stream samples. The influence of potential interferences present in the process samples as well as mass loading, flow rate and resin performance is being studied. In addition, the potential to automate these procedures utilizing a robotic platform is evaluated. Initial studies have been carried out using the commercially available DGA resin. This resin shows an affinity for Am, Pu, U, and Th and is also exhibiting signs of a possible synergistic effects in the presence of iron.

  18. ImmunoCAP® ISAC and Microtest for multiplex allergen testing in people with difficult to manage allergic disease: a systematic review and cost analysis.

    Science.gov (United States)

    Westwood, Marie; Ramaekers, Bram; Lang, Shona; Armstrong, Nigel; Noake, Caro; de Kock, Shelley; Joore, Manuela; Severens, Johan; Kleijnen, Jos

    2016-09-01

    Allergy is a form of immune-mediated exaggerated sensitivity (hypersensitivity) to a substance that is either inhaled, swallowed, injected or comes into contact with the skin. Foreign substances that provoke allergies are called allergens. It has been claimed that multiplex allergen testing may help in diagnosing the cause of symptoms in patients with an unclear cause of allergy or who are allergic to more than one substance. To evaluate multiplex allergen testing [devices that can measure the presence of multiple immunoglobulin E (IgE) antibodies in a patient's blood at the same time], by assessing (1) clinical effectiveness (allergy symptoms, incidence of acute exacerbations, mortality, adverse events of testing and treatment, health-care presentations or admissions, health-related quality of life); (2) effects on treatment (diet, immunotherapy medications, other potential testing); (3) any additional diagnostic information provided by multiplex allergen testing; and (4) cost-effectiveness (cost of different assessment strategies). Fifteen databases were searched from 2005 to April 2015, including MEDLINE (via OvidSp), MEDLINE In-Process Citations, MEDLINE Daily Update, PubMed (National Library of Medicine), EMBASE, Cochrane Database of Systematic Reviews (CDSR), Cochrane Central Register of Controlled Trials (CENTRAL), Database of Abstracts of Reviews of Effects (DARE), Health Technology Assessment (HTA) database, Science Citation Index (SCI), Conference Proceedings Citation Index-Science (CPCI-S), BIOSIS Previews, Latin American and Caribbean Health Sciences Literature (LILACS), National Institute for Health Research (NIHR) HTA programme, and the US Food and Drug Administration (FDA); supplementary searches of conference proceedings and trials registries were performed. Review methods followed published guidance from the Cochrane Collaboration and the Centre for Reviews and Dissemination, University of York, UK. The methodological quality of included studies

  19. [Evaluation of a new blood gas analysis system: RapidPoint 500(®)].

    Science.gov (United States)

    Nicolas, Thierry; Cabrolier, Nadège; Bardonnet, Karine; Davani, Siamak

    2013-01-01

    We present here evaluation of a new blood gas analysis system, RapidPoint 500(®) (Siemens Healthcare Diagnostics). The aim of this research was to compare the ergonomics and analytical performances of this analyser with those of the RapidLab 1265 for the following parameters: pH, partial oxygen pressure, partial carbon dioxide pressure, sodium, potassium, ionized calcium, lactate and the CO-oximetry parameters: hemoglobin, oxyhemoglobin, carboxyhemoglobin, methemoglobin, reduced hemoglobin, neonatal bilirubin; as well as with the Dimension Vista 500 results for chloride and glucose. The Valtec protocol, recommended by the French Society of Clinical Biology (SFBC), was used to analyze the study results. The experiment was carried out over a period of one month in the Department of medical biochemistry. One hundred sixty five samples from adult patients admitted to the ER or hospitalized in intensive care were tested. The RapidPoint 500(®) was highly satisfactory from an ergonomic point of view. Intra-and inter- assay coefficients of variation (CV) with the three control levels were below those recommended by the SFBC for all parameters, and the comparative study gave coefficients of determination higher than 0.91. Taken together, the RapidPoint 500(®) appears fully satisfactory in terms of ergonomics and analytical performance.

  20. Super-multiplex vibrational imaging

    Science.gov (United States)

    Wei, Lu; Chen, Zhixing; Shi, Lixue; Long, Rong; Anzalone, Andrew V.; Zhang, Luyuan; Hu, Fanghao; Yuste, Rafael; Cornish, Virginia W.; Min, Wei

    2017-04-01

    The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a ‘colour barrier’, owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the

  1. An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture.

    Science.gov (United States)

    Doherty, Margaret; Bones, Jonathan; McLoughlin, Niaobh; Telford, Jayne E; Harmon, Bryan; DeFelippis, Michael R; Rudd, Pauline M

    2013-11-01

    Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Redundant Interdependencies Boost the Robustness of Multiplex Networks

    Directory of Open Access Journals (Sweden)

    Filippo Radicchi

    2017-01-01

    Full Text Available In the analysis of the robustness of multiplex networks, it is commonly assumed that a node is functioning only if its interdependent nodes are simultaneously functioning. According to this model, a multiplex network becomes more and more fragile as the number of layers increases. In this respect, the addition of a new layer of interdependent nodes to a preexisting multiplex network will never improve its robustness. Whereas such a model seems appropriate to understand the effect of interdependencies in the simplest scenario of a network composed of only two layers, it may seem unsuitable to characterize the robustness of real systems formed by multiple network layers. In fact, it seems unrealistic that a real system evolved, through the development of multiple layers of interactions, towards a fragile structure. In this paper, we introduce a model of percolation where the condition that makes a node functional is that the node is functioning in at least two of the layers of the network. The model reduces to the commonly adopted percolation model for multiplex networks when the number of layers equals two. For larger numbers of layers, however, the model describes a scenario where the addition of new layers boosts the robustness of the system by creating redundant interdependencies among layers. We prove this fact thanks to the development of a message-passing theory that is able to characterize the model in both synthetic and real-world multiplex graphs.

  3. Novel multiplex technology for diagnostic characterization of rheumatoid arthritis

    Science.gov (United States)

    2011-01-01

    Introduction The aim of this study was to develop a clinical-grade, automated, multiplex system for the differential diagnosis and molecular stratification of rheumatoid arthritis (RA). Methods We profiled autoantibodies, cytokines, and bone-turnover products in sera from 120 patients with a diagnosis of RA of arthritis, and 25 healthy individuals. We used a commercial bead assay to measure cytokine levels and developed an array assay based on novel multiplex technology (Immunological Multi-Parameter Chip Technology) to evaluate autoantibody reactivities and bone-turnover markers. Data were analyzed by Significance Analysis of Microarrays and hierarchical clustering software. Results We developed a highly reproducible, automated, multiplex biomarker assay that can reliably distinguish between RA patients and healthy individuals or patients with other inflammatory arthritides. Identification of distinct biomarker signatures enabled molecular stratification of early-stage RA into clinically relevant subtypes. In this initial study, multiplex measurement of a subset of the differentiating biomarkers provided high sensitivity and specificity in the diagnostic discrimination of RA: Use of 3 biomarkers yielded a sensitivity of 84.2% and a specificity of 93.8%, and use of 4 biomarkers a sensitivity of 59.2% and a specificity of 96.3%. Conclusions The multiplex biomarker assay described herein has the potential to diagnose RA with greater sensitivity and specificity than do current clinical tests. Its ability to stratify RA patients in an automated and reproducible manner paves the way for the development of assays that can guide RA therapy. PMID:21702928

  4. High-throughput droplet analysis and multiplex DNA detection in the microfluidic platform equipped with a robust sample-introduction technique

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jinyang; Ji, Xinghu [Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 (China); He, Zhike, E-mail: zhkhe@whu.edu.cn [Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 (China); Suzhou Institute of Wuhan University, Suzhou 215123 (China)

    2015-08-12

    In this work, a simple, flexible and low-cost sample-introduction technique was developed and integrated with droplet platform. The sample-introduction strategy was realized based on connecting the components of positive pressure input device, sample container and microfluidic chip through the tygon tubing with homemade polydimethylsiloxane (PDMS) adaptor, so the sample was delivered into the microchip from the sample container under the driving of positive pressure. This sample-introduction technique is so robust and compatible that could be integrated with T-junction, flow-focus or valve-assisted droplet microchips. By choosing the PDMS adaptor with proper dimension, the microchip could be flexibly equipped with various types of familiar sample containers, makes the sampling more straightforward without trivial sample transfer or loading. And the convenient sample changing was easily achieved by positioning the adaptor from one sample container to another. Benefiting from the proposed technique, the time-dependent concentration gradient was generated and applied for quantum dot (QD)-based fluorescence barcoding within droplet chip. High-throughput droplet screening was preliminarily demonstrated through the investigation of the quenching efficiency of ruthenium complex to the fluorescence of QD. More importantly, multiplex DNA assay was successfully carried out in the integrated system, which shows the practicability and potentials in high-throughput biosensing. - Highlights: • A simple, robust and low-cost sample-introduction technique was developed. • Convenient and flexible sample changing was achieved in microfluidic system. • Novel strategy of concentration gradient generation was presented for barcoding. • High-throughput droplet screening could be realized in the integrated platform. • Multiplex DNA assay was successfully carried out in the droplet platform.

  5. Performance analysis of an OAM multiplexing-based MIMO FSO system over atmospheric turbulence using space-time coding with channel estimation.

    Science.gov (United States)

    Zhang, Yan; Wang, Ping; Guo, Lixin; Wang, Wei; Tian, Hongxin

    2017-08-21

    The average bit error rate (ABER) performance of an orbital angular momentum (OAM) multiplexing-based free-space optical (FSO) system with multiple-input multiple-output (MIMO) architecture has been investigated over atmospheric turbulence considering channel estimation and space-time coding. The impact of different types of space-time coding, modulation orders, turbulence strengths, receive antenna numbers on the transmission performance of this OAM-FSO system is also taken into account. On the basis of the proposed system model, the analytical expressions of the received signals carried by the k-th OAM mode of the n-th receive antenna for the vertical bell labs layered space-time (V-Blast) and space-time block codes (STBC) are derived, respectively. With the help of channel estimator carrying out with least square (LS) algorithm, the zero-forcing criterion with ordered successive interference cancellation criterion (ZF-OSIC) equalizer of V-Blast scheme and Alamouti decoder of STBC scheme are adopted to mitigate the performance degradation induced by the atmospheric turbulence. The results show that the ABERs obtained by channel estimation have excellent agreement with those of turbulence phase screen simulations. The ABERs of this OAM multiplexing-based MIMO system deteriorate with the increase of turbulence strengths. And both V-Blast and STBC schemes can significantly improve the system performance by mitigating the distortions of atmospheric turbulence as well as additive white Gaussian noise (AWGN). In addition, the ABER performances of both space-time coding schemes can be further enhanced by increasing the number of receive antennas for the diversity gain and STBC outperforms V-Blast in this system for data recovery. This work is beneficial to the OAM FSO system design.

  6. Child impact on family functioning: a multivariate analysis in multiplex families with children and mothers both affected by attention-deficit/hyperactivity disorder (ADHD).

    Science.gov (United States)

    Fleck, Katja; Jacob, Christian; Philipsen, Alexandra; Matthies, Swantje; Graf, Erika; Hennighausen, Klaus; Haack-Dees, Barbara; Weyers, Peter; Warnke, Andreas; Rösler, Michael; Retz, Wolfgang; von Gontard, Alexander; Hänig, Susan; Freitag, Christine; Sobanski, Esther; Schumacher-Stien, Martina; Poustka, Luise; Bliznak, Lucia; Becker, Katja; Holtmann, Martin; Colla, Michael; Gentschow, Laura; Kappel, Viola; Jaite, Charlotte; Jans, Thomas

    2015-09-01

    ADHD seriously impacts family functioning, even the more in families with simultaneous parental and child ADHD. The aim of the study was to examine associations between family impact of child ADHD and child, mother and family characteristics in multiplex families with children and mothers both affected by ADHD. One hundred and forty-four mother-child pairs were assessed (children: mean age 9.4 ± 1.7 years, 73.6 % male). Family impact of child ADHD was rated by mothers using the Family Impact Questionnaire (FIQ). Multiple linear regression analyses were performed with child and maternal psychopathology and basic family characteristics such as employment, partnership status and number of children as predictors and FIQ subscores as criteria. Rates of variance explained by family variables were 49 % for negative feelings towards the child, 37 % for impact on partnership, 31 % for impact on social life and 27 % for impact on finances (p ADHD and co-morbid symptoms of the mother. The strongest independent predictor was oppositional defiant child behaviour. In ADHD multiplex families, mothers' perception of the impact of an ADHD child on its family can be explained to a substantial degree by child psychopathology, maternal psychopathology and basic family characteristics. Although a cross-sectional design does not allow for causal interpretations, the findings of this study offer important targets for the treatment of ADHD in a family context pointing to the need for assessing and treating parental mental health and co-morbid symptoms besides ADHD core symptoms.

  7. Quantitative and ultrasensitive detection of multiplex cardiac biomarkers in lateral flow assay with core-shell SERS nanotags.

    Science.gov (United States)

    Zhang, Di; Huang, Li; Liu, Bing; Ni, Haibin; Sun, Liangdong; Su, Enben; Chen, Hongyuan; Gu, Zhongze; Zhao, Xiangwei

    2018-05-30

    Rapid and sensitive quantification of multiplex proteins in a wide concentration range is challenging in high throughput analysis. Herein, we proposed a lateral flow assay (LFA) based on core-shell surface enhanced Raman scattering (SERS) nanotags for multiplex and quantitative detection of cardiac biomarkers for the early diagnosis of acute myocardial infarction (AMI). In practice, Raman dyes (RDs) were embedded into the interior-gap of silver core and gold shell nanoparticles (NPs) to form SERS nanotags as labels instead of gold colloids and three test lines were employed in the strip for the detection of three cardiac biomarkers, Myo, cTnI, and CK-MB, respectively. Due to the amplified signal of the SERS nanotags and the high surface area to volume ratio (SVR) of porous nitrocellulose (NC) membrane, ultrasensitive quantification of protein markers with wide linear dynamic range (LDR) was realized, which is crucial for the quick detection of multiplex biomarkers in the same sample without pretreatments at bedsides. This method makes it possible for LFA in point of care testing (POCT) to be comparable with chemiluminescence immunoassay (CLIA) used in labs. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Finite element analysis (FEA) of dental implant fixture for mechanical stability and rapid osseointegration

    Science.gov (United States)

    Tabassum, Shafia; Murtaza, Ahmar; Ali, Hasan; Uddin, Zia Mohy; Zehra, Syedah Sadaf

    2017-10-01

    For rapid osseointegration of dental implant fixtures, various surface treatments including plasma spraying, hydroxyapatite coating, acid-etching, and surface grooving are used. However undesirable effects such as chemical modifications, loss of mechanical properties, prolonged processing times and post production treatment steps are often associated with these techniques. The osseointegration rate of the dental implants can be promoted by increasing the surface area of the dental implant, thus increasing the bone cells - implant material contact and allow bone tissues to grow rapidly. Additive Manufacturing (AM) techniques can be used to fabricate dental implant fixtures with desirable surface area in a single step manufacturing process. AM allows the use of Computer Aided Designing (CAD) for customised rapid prototyping of components with precise control over geometry. In this study, the dental implant fixture that replaces the tooth root was designed on commercially available software COMSOL. Nickel - titanium alloy was selected as build materials for dental implant. The geometry of the dental fixture was varied by changing the interspacing distance (thread pitch) and number of threads to increase the total surface area. Three different microstructures were introduced on the surface of dental implant. The designed models were used to examine the effect of changing geometries on the total surface area. Finite Element Analysis (FEA) was performed to investigate the effect of changing geometries on the mechanical properties of the dental implant fixtures using stress analysis.

  9. Time-varying multiplex network: Intralayer and interlayer synchronization

    Science.gov (United States)

    Rakshit, Sarbendu; Majhi, Soumen; Bera, Bidesh K.; Sinha, Sudeshna; Ghosh, Dibakar

    2017-12-01

    A large class of engineered and natural systems, ranging from transportation networks to neuronal networks, are best represented by multiplex network architectures, namely a network composed of two or more different layers where the mutual interaction in each layer may differ from other layers. Here we consider a multiplex network where the intralayer coupling interactions are switched stochastically with a characteristic frequency. We explore the intralayer and interlayer synchronization of such a time-varying multiplex network. We find that the analytically derived necessary condition for intralayer and interlayer synchronization, obtained by the master stability function approach, is in excellent agreement with our numerical results. Interestingly, we clearly find that the higher frequency of switching links in the layers enhances both intralayer and interlayer synchrony, yielding larger windows of synchronization. Further, we quantify the resilience of synchronous states against random perturbations, using a global stability measure based on the concept of basin stability, and this reveals that intralayer coupling strength is most crucial for determining both intralayer and interlayer synchrony. Lastly, we investigate the robustness of interlayer synchronization against a progressive demultiplexing of the multiplex structure, and we find that for rapid switching of intralayer links, the interlayer synchronization persists even when a large number of interlayer nodes are disconnected.

  10. A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis

    Science.gov (United States)

    Gering, E.; Atkinson, C.T.

    2004-01-01

    Measures of parasitemia by intraerythrocytic hematozoan parasites are normally expressed as the number of infected erythrocytes per n erythrocytes and are notoriously tedious and time consuming to measure. We describe a protocol for generating rapid counts of nucleated erythrocytes from digital micrographs of thin blood smears that can be used to estimate intensity of hematozoan infections in nonmammalian vertebrate hosts. This method takes advantage of the bold contrast and relatively uniform size and morphology of erythrocyte nuclei on Giemsa-stained blood smears and uses ImageJ, a java-based image analysis program developed at the U.S. National Institutes of Health and available on the internet, to recognize and count these nuclei. This technique makes feasible rapid and accurate counts of total erythrocytes in large numbers of microscope fields, which can be used in the calculation of peripheral parasitemias in low-intensity infections.

  11. The acoustic sensor for rapid analysis of bacterial cells in the conductive suspensions.

    Science.gov (United States)

    Borodina, I A; Zaitsev, B D; Guliy, O; Teplykh, A A; Shikhabudinov, A M

    2017-11-01

    The possibility of using the acoustic sensor on the basis of a two-channel delay line for rapid analysis of bacterial cells in the conductive suspensions was investigated. The dependencies of change in phase and insertion loss of output signal of the sensor on conductivity of buffer solution with various concentrations of cells due to a specific interaction "bacterial cells - mini-antibodies" for electrically open and electrically shorted channels of delay line were measured. It has been found that these changes have the most values for the electrically open channel. It has been also shown that the sensor rapidly responds to the specific interaction and the time stabilization of the phase and insertion loss of output signal is less than 10min. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Laser induced breakdown spectroscopy (LIBS) as a rapid tool for material analysis

    Science.gov (United States)

    Hussain, T.; Gondal, M. A.

    2013-06-01

    Laser induced breakdown spectroscopy (LIBS) is a novel technique for elemental analysis based on laser-generated plasma. In this technique, laser pulses are applied for ablation of the sample, resulting in the vaporization and ionization of sample in hot plasma which is finally analyzed by the spectrometer. The elements are identified by their unique spectral signatures. LIBS system was developed for elemental analysis of solid and liquid samples. The developed system was applied for qualitative as well as quantitative measurement of elemental concentration present in iron slag and open pit ore samples. The plasma was generated by focusing a pulsed Nd:YAG laser at 1064 nm on test samples to study the capabilities of LIBS as a rapid tool for material analysis. The concentrations of various elements of environmental significance such as cadmium, calcium, magnesium, chromium, manganese, titanium, barium, phosphorus, copper, iron, zinc etc., in these samples were determined. Optimal experimental conditions were evaluated for improving the sensitivity of developed LIBS system through parametric dependence study. The laser-induced breakdown spectroscopy (LIBS) results were compared with the results obtained using standard analytical technique such as inductively couple plasma emission spectroscopy (ICP). Limit of detection (LOD) of our LIBS system were also estimated for the above mentioned elements. This study demonstrates that LIBS could be highly appropriate for rapid online analysis of iron slag and open pit waste.

  13. Electroanalytical Sensors and Devices for Multiplexed Detection of Foodborne Pathogen Microorganisms

    Directory of Open Access Journals (Sweden)

    Susana Campuzano

    2009-07-01

    Full Text Available The detection and identification of pathogen microorganisms still rely on conventional culturing techniques, which are not suitable for on-site monitoring. Therefore, a great research challenge in this field is focused on the need to develop rapid, reliable, specific, and sensitive methods to detect these bacteria at low cost. Moreover, the growing interest in biochip development for large scale screening analysis implies improved miniaturization, reduction of analysis time and cost, and multi-analyte detection, which has nowadays become a crucial challenge. This paper reviews multiplexed foodborne pathogen microorganisms detection methods based on electrochemical sensors incorporating microarrays and other platforms. These devices usually involve antibody-antigen and DNA hybridization specific interactions, although other approaches such as the monitoring of oxygen consumption are also considered.

  14. Multiplexed microsatellite recovery using massively parallel sequencing

    Science.gov (United States)

    Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

    2011-01-01

    Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

  15. Towards effective visual analytics on multiplex and multilayer networks

    DEFF Research Database (Denmark)

    Rossi, Luca; Magnani, Matteo

    2015-01-01

    In this article we discuss visualisation strategies for multiplex networks. Since Moreno’s early works on network analysis, visualisation has been one of the main ways to understand networks thanks to its ability to summarise a complex structure into a single representation highlighting multiple ...

  16. Multiplexed noise shaping structures for Delta-Sigma modulators

    Science.gov (United States)

    Czarnul, Z.; Yesilyurt, A. G.; Temes, G. C.

    1991-01-01

    A new structure for realizing a multiplexed noise-shaping A/D convertor is proposed. It improves the performance of first- or second-order Delta-Sigma modulators by employing additional feedback paths for suppressing the quantizer error. The structure, its theoretical analysis, and simulations confirming the improved resolution are presented.

  17. Preparation of highly multiplexed small RNA sequencing libraries.

    Science.gov (United States)

    Persson, Helena; Søkilde, Rolf; Pirona, Anna Chiara; Rovira, Carlos

    2017-08-01

    MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.

  18. The Rapid Analysis of Scattering from Periodic Dielectric Structures Using Accelerated Cartesian Expansions (ACE)

    CERN Document Server

    Baczewski, Andrew D; Shanker, Balasubramaniam

    2011-01-01

    The analysis of fields in periodic dielectric structures arise in numerous applications of recent interest, ranging from photonic bandgap (PBG) structures and plasmonically active nanostructures to metamaterials. To achieve an accurate representation of the fields in these structures using numerical methods, dense spatial discretization is required. This, in turn, affects the cost of analysis, particularly for integral equation based methods, for which traditional iterative methods require O(N^2) operations, N being the number of spatial degrees of freedom. In this paper, we introduce a method for the rapid solution of volumetric electric field integral equations used in the analysis of doubly periodic dielectric structures. The crux of our method is the ACE algorithm, which is used to evaluate the requisite potentials in O(N) cost. Results are provided that corroborate our claims of acceleration without compromising accuracy, as well as the application of our method to a number of compelling photonics applic...

  19. GSMA: Gene Set Matrix Analysis, An Automated Method for Rapid Hypothesis Testing of Gene Expression Data

    Directory of Open Access Journals (Sweden)

    Chris Cheadle

    2007-01-01

    Full Text Available Background: Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The assignment of functional information to these complex patterns remains a challenging task in effectively interpreting data and correlating results from across experiments, projects and laboratories. Methods which allow the rapid and robust evaluation of multiple functional hypotheses increase the power of individual researchers to data mine gene expression data more efficiently.Results: We have developed (gene set matrix analysis GSMA as a useful method for the rapid testing of group-wise up- or downregulation of gene expression simultaneously for multiple lists of genes (gene sets against entire distributions of gene expression changes (datasets for single or multiple experiments. The utility of GSMA lies in its flexibility to rapidly poll gene sets related by known biological function or as designated solely by the end-user against large numbers of datasets simultaneously.Conclusions: GSMA provides a simple and straightforward method for hypothesis testing in which genes are tested by groups across multiple datasets for patterns of expression enrichment.

  20. Using mind mapping techniques for rapid qualitative data analysis in public participation processes.

    Science.gov (United States)

    Burgess-Allen, Jilla; Owen-Smith, Vicci

    2010-12-01

    In a health service environment where timescales for patient participation in service design are short and resources scarce, a balance needs to be achieved between research rigour and the timeliness and utility of the findings of patient participation processes. To develop a pragmatic mind mapping approach to managing the qualitative data from patient participation processes. While this article draws on experience of using mind maps in a variety of participation processes, a single example is used to illustrate the approach. In this example mind maps were created during the course of patient participation focus groups. Two group discussions were also transcribed verbatim to allow comparison of the rapid mind mapping approach with traditional thematic analysis of qualitative data. The illustrative example formed part of a local alcohol service review which included consultation with local alcohol service users, their families and staff groups. The mind mapping approach provided a pleasing graphical format for representing the key themes raised during the focus groups. It helped stimulate and galvanize discussion and keep it on track, enhanced transparency and group ownership of the data analysis process, allowed a rapid dynamic between data collection and feedback, and was considerably faster than traditional methods for the analysis of focus groups, while resulting in similar broad themes. This study suggests that the use of a mind mapping approach to managing qualitative data can provide a pragmatic resolution of the tension between limited resources and quality in patient participation processes. © 2010 The Authors. Health Expectations © 2010 Blackwell Publishing Ltd.

  1. Rapid assessment of large scale vegetation change based on multi-temporal phenological analysis

    Science.gov (United States)

    Cai, Danlu; Guan, Yanning; Guo, Shan; Yan, Baoping; Xing, Zhi; Zhang, Chunyan; Piao, Yingchao; An, Xudong; Kang, Lihua

    2011-11-01

    Detecting vegetation change is critical for earth system and sustainability science. The existing methods, however, show several limitations, including inevitable selection of imagery acquisition dates, affection from vegetation related noise on temporal trajectory analysis, and assumptions due to vegetation classification model. This paper presents a multitemporal phenological frequency analysis over a relatively short period (MTPFA-SP) methodology to detect vegetation changes. This MTPFA-SP methodology bases on the amplitude components of fast Fourier transforming (FFT) and is implemented with two steps. First, NDVI time series over two periods are transformed with FFT into frequency domain, separately. Second, amplitude components with phenological information from Step 1 are selected for further change comparison. In this methodology, component selection shows physical meanings of natural vegetation process in frequency domain. Comparisons among those selected components help enhance the ability to rapidly detect vegetation changes. To validate this MTPFA-SP methodology, we detect changes between two periods (2001-2005 and 2006-2010) in the eastern Tibet Plateau area and make two kinds of assessments. The first is for a larger scale, including statistic analysis of altitudinal zonality and latitudinal zonality. The second assessment is for rapid detection of vegetation change location. Landsat TM image were employed to validate the result.

  2. VAPI: low-cost, rapid automated visual inspection system for Petri plate analysis

    Science.gov (United States)

    Chatburn, L. T.; Kirkup, B. C.; Polz, M. F.

    2007-09-01

    Most culture-based microbiology tasks utilize a petri plate during processing, but rarely do the scientists capture the full information available from the plate. In particular, visual analysis of plates is an under-developed rich source of data that can be rapid and non-invasive. However, collecting this data has been limited by the difficulties of standardizing and quantifying human observations, by the limits of a scientists' fatigue, and by the cost of automating the process. The availability of specialized counting equipment and intelligent camera systems has not changed this - they are prohibitively expensive for many laboratories, only process a limited number of plate types, are often destructive to the sample, and have limited accuracy. This paper describes an automated visual inspection solution, VAPI, that employs inexpensive consumer computing hardware and digital cameras along with custom cross-platform open-source software written in C++, combining Trolltech's Qt GUI toolkit with Intel's OpenCV computer vision library. The system is more accurate than common commercial systems costing many times as much, while being flexible in use and offering comparable responsiveness. VAPI not only counts colonies but also sorts and enumerates colonies by morphology, tracks colony growth by time series analysis, and provides other analytical resources. Output to XML files or directly to a database provides data that can be easily maintained and manipulated by the end user, offering ready access for system enhancement, interaction with other software systems, and rapid development of advanced analysis applications.

  3. The flood event explorer - a web based framework for rapid flood event analysis

    Science.gov (United States)

    Schröter, Kai; Lüdtke, Stefan; Kreibich, Heidi; Merz, Bruno

    2015-04-01

    Flood disaster management, recovery and reconstruction planning benefit from rapid evaluations of flood events and expected impacts. The near real time in-depth analysis of flood causes and key drivers for flood impacts requires a close monitoring and documentation of hydro-meteorological and socio-economic factors. Within the CEDIM's Rapid Flood Event Analysis project a flood event analysis system is developed which enables the near real-time evaluation of large scale floods in Germany. The analysis system includes functionalities to compile event related hydro-meteorological data, to evaluate the current flood situation, to assess hazard intensity and to estimate flood damage to residential buildings. A German flood event database is under development, which contains various hydro-meteorological information - in the future also impact information -for all large-scale floods since 1950. This data base comprises data on historic flood events which allow the classification of ongoing floods in terms of triggering processes and pre-conditions, critical controls and drivers for flood losses. The flood event analysis system has been implemented in a database system which automatically retrieves and stores data from more than 100 online discharge gauges on a daily basis. The current discharge observations are evaluated in a long term context in terms of flood frequency analysis. The web-based frontend visualizes the current flood situation in comparison to any past flood from the flood catalogue. The regional flood data base for Germany contains hydro-meteorological data and aggregated severity indices for a set of 76 historic large-scale flood events in Germany. This data base has been used to evaluate the key drivers for the flood in June 2013.

  4. Rapid analysis of vessel elements (RAVE): a tool for studying physiologic, pathologic and tumor angiogenesis.

    Science.gov (United States)

    Seaman, Marc E; Peirce, Shayn M; Kelly, Kimberly

    2011-01-01

    Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI) that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity), and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening.

  5. Rapid analysis of vessel elements (RAVE: a tool for studying physiologic, pathologic and tumor angiogenesis.

    Directory of Open Access Journals (Sweden)

    Marc E Seaman

    Full Text Available Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity, and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening.

  6. Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

    Science.gov (United States)

    Turingan, Rosemary S; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F

    2016-01-01

    Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time. A Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1-15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis. The LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample

  7. A novel method for perceptual assessment of small room acoustics using rapid sensory analysis

    DEFF Research Database (Denmark)

    Kaplanis, Neofytos; Bech, Søren; Lokki, Tapio

    2016-01-01

    small rooms and car cabins. In-situ measurements were performed to obtain a range of possible acoustical settings, by varying physically the spaces under investigation. The measured responses were spatiallyanalyzed and synthesized to reproduce the observed fields in the laboratory. Expert listeners were...... presented with auralized sound over a loudspeaker array and followed a rapid sensory analysis protocol. The elicited attributes and ratings are analyzed and possible links to the acoustical properties of these spaces are discussed. [This study is a part of Marie Curie Network on Dereverberation...

  8. Arsenic analysis II: rapid separation and quantification of inorganic arsenic plus metabolites and arsenobetaine from urine.

    Science.gov (United States)

    Nixon, D E; Moyer, T P

    1992-12-01

    We describe the rapid separation of inorganic arsenic plus metabolites from arsenobetaine or seafood arsenic in urine. Traditional, high-pressure liquid chromatography is replaced by disposable silica-based cation-exchange cartridges for this separation. Both fractions are quickly separated and collected for analysis by atomic absorption spectrophotometry. Analytical recovery of both fractions is > or = 95%, with an overall precision (CV) ranging from 1.6% to 6.4%. Using this method, we correctly identified the sources of arsenic exposure, whether of inorganic or seafood origin, in 11 urine specimens supplied by the Centre de Toxicologie du Quebec.

  9. Rapid quality assessment of Radix Aconiti Preparata using direct analysis in real time mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Zhu Hongbin; Wang Chunyan; Qi Yao [Changchun Center of Mass Spectrometry and Chemical Biology Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); University of Chinese Academy of Sciences, Beijing 100039 (China); Song Fengrui, E-mail: songfr@ciac.jl.cn [Changchun Center of Mass Spectrometry and Chemical Biology Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Liu Zhiqiang; Liu Shuying [Changchun Center of Mass Spectrometry and Chemical Biology Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China)

    2012-11-08

    Highlights: Black-Right-Pointing-Pointer DART MS combined with PCA and HCA was used to rapidly identify markers of Radix Aconiti. Black-Right-Pointing-Pointer The DART MS behavior of six aconitine-type alkaloids was investigated. Black-Right-Pointing-Pointer Chemical markers were recognized between the qualified and unqualified samples. Black-Right-Pointing-Pointer DART MS was shown to be an effective tool for quality control of Radix Aconiti Preparata. - Abstract: This study presents a novel and rapid method to identify chemical markers for the quality control of Radix Aconiti Preparata, a world widely used traditional herbal medicine. In the method, the samples with a fast extraction procedure were analyzed using direct analysis in real time mass spectrometry (DART MS) combined with multivariate data analysis. At present, the quality assessment approach of Radix Aconiti Preparata was based on the two processing methods recorded in Chinese Pharmacopoeia for the purpose of reducing the toxicity of Radix Aconiti and ensuring its clinical therapeutic efficacy. In order to ensure the safety and effectivity in clinical use, the processing degree of Radix Aconiti should be well controlled and assessed. In the paper, hierarchical cluster analysis and principal component analysis were performed to evaluate the DART MS data of Radix Aconiti Preparata samples in different processing times. The results showed that the well processed Radix Aconiti Preparata, unqualified processed and the raw Radix Aconiti could be clustered reasonably corresponding to their constituents. The loading plot shows that the main chemical markers having the most influence on the discrimination amongst the qualified and unqualified samples were mainly some monoester diterpenoid aconitines and diester diterpenoid aconitines, i.e. benzoylmesaconine, hypaconitine, mesaconitine, neoline, benzoylhypaconine, benzoylaconine, fuziline, aconitine and 10-OH-mesaconitine. The established DART MS approach in

  10. Environmental science applications with Rapid Integrated Mapping and analysis System (RIMS)

    Science.gov (United States)

    Shiklomanov, A.; Prusevich, A.; Gordov, E.; Okladnikov, I.; Titov, A.

    2016-11-01

    The Rapid Integrated Mapping and analysis System (RIMS) has been developed at the University of New Hampshire as an online instrument for multidisciplinary data visualization, analysis and manipulation with a focus on hydrological applications. Recently it was enriched with data and tools to allow more sophisticated analysis of interdisciplinary data. Three different examples of specific scientific applications with RIMS are demonstrated and discussed. Analysis of historical changes in major components of the Eurasian pan-Arctic water budget is based on historical discharge data, gridded observational meteorological fields, and remote sensing data for sea ice area. Express analysis of the extremely hot and dry summer of 2010 across European Russia is performed using a combination of near-real time and historical data to evaluate the intensity and spatial distribution of this event and its socioeconomic impacts. Integrative analysis of hydrological, water management, and population data for Central Asia over the last 30 years provides an assessment of regional water security due to changes in climate, water use and demography. The presented case studies demonstrate the capabilities of RIMS as a powerful instrument for hydrological and coupled human-natural systems research.

  11. Evaluation of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the identification of Campylobacter strains isolated from diarrheal patients in Japan.

    Science.gov (United States)

    Kabir, S M Lutful; Kikuchi, Ken; Asakura, Masahiro; Shiramaru, Sachi; Tsuruoka, Naoki; Goto, Aeko; Hinenoya, Atsushi; Yamasaki, Shinji

    2011-01-01

    We have developed a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection and identification of Campylobacter jejuni, C. coli, and C. fetus. The applicability of this assay was evaluated with 325 Campylobacter strains isolated from diarrheal patients in Japan and the results were compared with those obtained by other genetic methods, including hipO gene detection and 16S rRNA gene sequencing. Of the 325 strains analyzed, 314 and 11 were identified as C. jejuni and C. coli, respectively, by combination of hipO gene detection and 16S rRNA gene sequencing. When the multiplex PCR assay was employed, 309, 310, and 314 strains were identified as C. jejuni on the basis of cdtA, cdtB, and cdtC gene-specific primers, respectively. Similarly, 11, 11, and 10 strains were identified as C. coli on the basis of cdtA, cdtB, and cdtC gene-specific primers, respectively. Sequence analysis of the cdt gene region of 6 strains (5 C. jejuni and 1 C. coli) which did not yield specific PCR products in any of the cdt gene-based multiplex PCR assays revealed deletions or mutations of the cdt genes. Pulsed-field gel electrophoresis indicated that C. jejuni and C. coli strains were genetically diverse. Taken together, these findings suggest that the cdtC gene-based multiplex PCR seems to be a particularly simple and rapid method for differentiating between species of Campylobacter strains, such as C. jejuni and C. coli. However, combination of these multiplex PCR assays will allow more accurate identification.

  12. Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A/B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin.

    Science.gov (United States)

    Beck, Eric T; Jurgens, Lisa A; Kehl, Sue C; Bose, Michael E; Patitucci, Teresa; LaGue, Elizabeth; Darga, Patrick; Wilkinson, Kimberly; Witt, Lorraine M; Fan, Jiang; He, Jie; Kumar, Swati; Henrickson, Kelly J

    2010-01-01

    Rapid, semiautomated, and fully automated multiplex real-time RT-PCR assays were developed and validated for the detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV) from nasopharyngeal specimens. The assays can detect human H1N1, H3N2, and swine-origin (S-OIV) H1N1 Flu A viruses and were effectively used to distinguish Flu A infections (of all subtypes) from Flu B and RSV infections during the current S-OIV outbreak in Milwaukee, WI. The analytical limits of detection were 10(-2) to 10(1) TCID(50)/ml depending on the platform and analyte and showed only one minor cross-reaction among 23 common respiratory pathogens (intermittent cross-reaction to adenovirus at >10(7) TCID(50)/ml). A total of 100 clinical samples were tested by tissue culture, both automated assays, and the US Food and Drug Administration-approved ProFlu+ assay. Both the semiautomated and fully automated assays exhibited greater overall (Flu A, Flu B, and RSV combined) clinical sensitivities (93 and 96%, respectively) and individual Flu A sensitivities (100%) than the Food and Drug Administration-approved test (89% overall sensitivity and 93% Flu A sensitivity). All assays were 99% specific. During the S-OIV outbreak in Milwaukee, WI, the fully automated assay was used to test 1232 samples in 2 weeks. Flu A was detected in 134 clinical samples (126 H1N1 S-OIV, 5 H1N1 [human], and 1 untyped) with 100% positive agreement compared with other "in-house" validated molecular assays, with only 2 false-positive results. Such accurate testing using automated high-throughput molecule systems should allow clinicians and public health officials to react quickly and effectively during viral outbreaks.

  13. Qualitative and Quantitative Multiplexed Proteomic Analysis of Complex Yeast Protein Fractions That Modulate the Assembly of the Yeast Prion Sup35p

    Science.gov (United States)

    Redeker, Virginie; Hughes, Chris; Savistchenko, Jimmy; Vissers, Johannes P. C.; Melki, Ronald

    2011-01-01

    Background The aggregation of the baker's yeast prion Sup35p is at the origin of the transmissible [PSI+] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI+] formation. Results We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI+] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI+] and [psi−] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes. Conclusion Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI+] formation. Our results highlight the complexity of the cellular changes accompanying [PSI+] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly. PMID:21931608

  14. Analysis of Signal-to-Crosstalk Ratio Variations due to Four-Wave Mixing in Dense Wavelength Division Multiplexing Systems Implemented with Standard Single-Mode Fibers

    Directory of Open Access Journals (Sweden)

    Sait Eser KARLIK

    2016-10-01

    Full Text Available In this paper, variation of the signal-to-crosstalk ratio (SXR due to effects of four-wave mixing (FWM has been analyzed on center channels of 5-, 7-, 9-channel dense wavelength division multiplexing (DWDM systems implemented with G.652 standard single-mode fibers (SSMFs for 12.5 GHz, 25 GHz, 50 GHz and 100 GHz equal channel spacing values. Center channels on such systems are the most severely impacted channels by FWM. Therefore, results obtained are the worst-case values for the DWDM system performance and important for system design. Simulations have been performed for systems using three different commercially available SMFs having different design parameter values for chromatic dispersion, dispersion slope, nonlinearity coefficient and attenuation coefficient which are all in the scope of the G.652 Recommendation of Telecommunication Standardization Sector of International Telecommunication Union (ITU-T for SSMFs. In those simulations, under the impact of FWM, variation of SXR with variations in input powers, channel spacings and link lengths have been observed. Simulation results display the combined effect of the optical fiber and system design parameters on FWM performance of DWDM systems and give important clues for not only long-haul but also access network implementations of DWDM systems.

  15. Multiplexed Colorimetric Solid-Phase Extraction

    Science.gov (United States)

    Gazda, Daniel B.; Fritz, James S.; Porter, Marc D.

    2009-01-01

    Multiplexed colorimetric solid-phase extraction (MC-SPE) is an extension of colorimetric solid-phase extraction (C-SPE) an analytical platform that combines colorimetric reagents, solid phase extraction, and diffuse reflectance spectroscopy to quantify trace analytes in water. In CSPE, analytes are extracted and complexed on the surface of an extraction membrane impregnated with a colorimetric reagent. The analytes are then quantified directly on the membrane surface using a handheld diffuse reflectance spectrophotometer. Importantly, the use of solid-phase extraction membranes as the matrix for impregnation of the colorimetric reagents creates a concentration factor that enables the detection of low concentrations of analytes in small sample volumes. In extending C-SPE to a multiplexed format, a filter holder that incorporates discrete analysis channels and a jig that facilitates the concurrent operation of multiple sample syringes have been designed, enabling the simultaneous determination of multiple analytes. Separate, single analyte membranes, placed in a readout cartridge create unique, analyte-specific addresses at the exit of each channel. Following sample exposure, the diffuse reflectance spectrum of each address is collected serially and the Kubelka-Munk function is used to quantify each water quality parameter via calibration curves. In a demonstration, MC-SPE was used to measure the pH of a sample and quantitate Ag(I) and Ni(II).

  16. Rapid differentiation of mycobacteria by simplex real-time PCR with melting temperature calling analysis.

    Science.gov (United States)

    Lin, L; Yin, X; Wang, Q

    2015-09-01

    This study aimed to develop a rapid, simple and cost-effective method for the differentiation of Mycobacterium species. A total of 80 clinical mycobacterial isolates belonging to 12 different species and 16 reference strains of 16 different species were differentiated by the simplex real-time PCR coupled with melting temperature calling analysis. By comparing their melting profiles with those of the reference strains, all clinical mycobacterial isolates were differentiated as Mycobacterium tuberculosis complex or nontuberculous mycobacteria, and the latter were further divided into five groups. In comparison with 16S-23S internal transcribed spacer sequencing method as the gold standard method, both sensitivity and specificity of the assay were 100% when it was used for the differentiation between Myco. tuberculosis complex and nontuberculous mycobacteria. The simplex real-time PCR coupled with melting temperature calling analysis could be an alternative method for the differentiation between Myco. tuberculosis complex and nontuberculous mycobacteria. Rapid differentiation of mycobacteria could shorten the diagnostic time of mycobacterial diseases. It is also helpful for achieving optimal therapy and appropriate patient management. © 2015 The Society for Applied Microbiology.

  17. Rapid analysis of acetone in human plasma by derivatization desorption electrospray ionization.

    Science.gov (United States)

    Xu, Guangming; Chen, Bo; Liu, Guozhu; Yao, Shouzhuo

    2010-09-01

    Desorption electrospray ionization mass spectrometry (DESI MS) is rapidly becoming a powerful analysis tool for a wide variety of samples in the open air. By combining DESI MS and derivatization, acetone in human blood samples, one of the diabetes mellitus (DM) biomarkers, was rapidly analyzed. Prior to DESI, the plasma sample was first exposed to hydroxylamine hydrochloride and acetone in plasma was derived into fixed oxime. The reactive products were validated by DESI/MS/MS. Quantification was performed in the positive-ion electrospray ionization mode by monitoring of the product ions at m/z 74 for acetone oxime and m/z 116 for 4-methyl-2-pentanone oxime as the internal standard. The limit of detection was of the order of magnitude of 0.10 ppbv and the linear range comprised two decades of concentration, covering the relevant concentration range of acetone in plasma samples. The method has achieved fast, high-throughput analysis of acetone in human plasma with little beforehand preparation and an experiment time of only a few seconds.

  18. Absolute quantitation of proteins in human blood by multiplexed multiple reaction monitoring mass spectrometry.

    Science.gov (United States)

    Percy, Andrew J; Chambers, Andrew G; Parker, Carol E; Borchers, Christoph H

    2013-01-01

    Multiple reaction monitoring (MRM)-mass spectrometry (MS) with stable isotope-labeled standards (SIS) has proven adept in rapidly, precisely, and accurately quantifying proteins in complex biological samples. The impetus behind the early use of multiplexed MRM in proteomics was to expedite the verification and validation stages of the protein biomarker pipeline for clinical utility, which involves the analysis of hundreds or even thousands of samples. Moreover, once a multiplexed assay has been developed, however, it can be turned around and used for biomarker discovery, as has been demonstrated for cancer biomarkers by our laboratory and by others. Overall, these MRM-based methods compare favorably with antibody-based techniques, such as ELISAs or protein arrays, in that MRM-based methods are less expensive and can be developed more rapidly. There are two MRM-based platforms that are currently being developed: a standard-flow and a nano-flow LC/ESI-MRM-MS (liquid chromatography-electrospray ionization) platform. In this book chapter, we describe a recent study in which we evaluated these two platforms, both interfaced to the same mass spectrometer. This study demonstrated the enhanced performance metrics (in terms of sensitivity, dynamic range, and robustness) of the standard-flow ultra-high performance liquid chromatography (UHPLC) system compared to the nano-flow HPLC-Chip for the absolute quantitation of 48 plasma proteins. Using the standard-flow platform, we also developed two high-throughput assays for the analysis of a panel of 67 cardiovascular disease (CVD) biomarkers in non-depleted and non-enriched human plasma and a panel of 25 putative biomarkers in dried human blood spots (DBS). Since the nanoLC/MRM-MS platform has advantages under sample-limited conditions and for the analysis of certain specific peptides, the protocols for both systems are described here.

  19. Rapid, autonomous analysis of HPGe gamma-ray spectra III: Plutonium identification and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Gosnell, Thomas B. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Wong, James L. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2014-02-11

    RadID is a gamma-ray spectrum analysis program originally written to assist in the detection of the illicit movement of nuclear material. It is specific to the rapid analysis of HPGe gamma-ray data to reveal the radionuclide signatures of interest that may be present in the spectra. It is an autonomous, rule-based heuristic system that can identify well over 200 radioactive sources in about one second. RadID does not require knowledge of the detector efficiency, the source-todetector distance, or the geometry of the inspected radiation source—including any shielding. In this third of a three-document series we discuss how RadID detects the presence of plutonium isotopes and determines a number of its characteristics, most notably: the spectral characteristics of weapons-grade plutonium, reactor-grade plutonium, and heat-source plutonium used in radioisotope thermoelectric generators.

  20. A method for multiplex gene synthesis employing error correction based on expression.

    Directory of Open Access Journals (Sweden)

    Timothy H-C Hsiau

    Full Text Available Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.

  1. Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

    Directory of Open Access Journals (Sweden)

    Yellman Christopher M

    2009-01-01

    Full Text Available Abstract Background Short-read high-throughput DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as S. cerevisiae. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina's platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII and a reference sample (input DNA in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs. Results We developed a barcoding ChIP-Seq method for the concurrent analysis of transcription factor binding sites in yeast. Our multiplex strategy generated high quality data that was indistinguishable from data obtained with non-barcoded libraries. None of the barcoded adapters induced differences relative to a non-barcoded adapter when applied to the same DNA sample. We used this method to map the binding sites for Cse4, Ste12 and Pol II throughout the yeast genome and we found 148 binding targets for Cse4, 823 targets for Ste12 and 2508 targets for PolII. Cse4 was strongly bound to all yeast centromeres as expected and the remaining non-centromeric targets correspond to highly expressed genes in rich media. The presence of Cse4 non-centromeric binding sites was not reported previously. Conclusion We designed a multiplex short-read DNA sequencing method to perform efficient ChIP-Seq in yeast and other small genome model organisms. This method produces accurate results with higher throughput and reduced cost. Given constant improvements in high-throughput sequencing technologies, increasing

  2. Time-division-multiplexed 3D shearography

    Science.gov (United States)

    James, Stephen W.; Tatam, Ralph P.

    1999-08-01

    The development of a time-division-multiplexed 3D digital shearography instrument is described. The system provides simultaneous measurement of the in-plane and out-of-plane deformation gradients, allowing full surface strain analysis. The object under investigation is sequentially illuminated from three directions by three fiber coupled high power laser diode sources, and imaged onto a CCD camera through a single shearing interferometer. The pulsing of the sources is synchronized with the camera frame rate. Phase stepping is achieved using laser diode wavelength tuning combined with a path length imbalance in the shearing interferometer. The source pulsing schedule and image acquisition are controlled from a PC. An analysis of the optimum illumination geometry is presented. The performance of the system is evaluated on laboratory test samples.

  3. Multiplexed operation of a micromachined ultrasonic droplet ejector array.

    Science.gov (United States)

    Forbes, Thomas P; Degertekin, F Levent; Fedorov, Andrei G

    2007-10-01

    A dual-sample ultrasonic droplet ejector array is developed for use as a soft-ionization ion source for multiplexed mass spectrometry (MS). Such a multiplexed ion source aims to reduce MS analysis time for multiple analyte streams, as well as allow for the synchronized ejection of the sample(s) and an internal standard for quantitative results and mass calibration. Multiplexing is achieved at the device level by division of the fluid reservoir and separating the active electrodes of the piezoelectric transducer for isolated application of ultrasonic wave energy to each domain. The transducer is mechanically shaped to further reduce the acoustical crosstalk between the domains. Device design is performed using finite-element analysis simulations and supported by experimental characterization. Isolated ejection of approximately 5 microm diameter water droplets from individual domains in the micromachined droplet ejector array at around 1 MHz frequency is demonstrated by experiments. The proof-of-concept demonstration using a dual-sample device also shows potential for multiplexing with larger numbers of analytes.

  4. The Future of Multiplexed Eukaryotic Genome Engineering.

    Science.gov (United States)

    Thompson, David B; Aboulhouda, Soufiane; Hysolli, Eriona; Smith, Cory J; Wang, Stan; Castanon, Oscar; Church, George M

    2017-12-28

    Multiplex genome editing is the simultaneous introduction of multiple distinct modifications to a given genome. Though in its infancy, maturation of this field will facilitate powerful new biomedical research approaches and will enable a host of far-reaching biological engineering applications, including new therapeutic modalities and industrial applications, as well as "genome writing" and de-extinction efforts. In this Perspective, we focus on multiplex editing of large eukaryotic genomes. We describe the current state of multiplexed genome editing, the current limits of our ability to multiplex edits, and provide perspective on the many applications that fully realized multiplex editing technologies would enable in higher eukaryotic genomes. We offer a broad look at future directions, covering emergent CRISPR-based technologies, advances in intracellular delivery, and new DNA assembly approaches that may enable future genome editing on a massively multiplexed scale.

  5. Spatially Multiplexed Micro-Spectrophotometry in Bright Field Mode for Thin Film Characterization

    Science.gov (United States)

    Pini, Valerio; Kosaka, Priscila M.; Ruz, Jose J.; Malvar, Oscar; Encinar, Mario; Tamayo, Javier; Calleja, Montserrat

    2016-01-01

    Thickness characterization of thin films is of primary importance in a variety of nanotechnology applications, either in the semiconductor industry, quality control in nanofabrication processes or engineering of nanoelectromechanical systems (NEMS) because small thickness variability can strongly compromise the device performance. Here, we present an alternative optical method in bright field mode called Spatially Multiplexed Micro-Spectrophotometry that allows rapid and non-destructive characterization of thin films over areas of mm2 and with 1 μm of lateral resolution. We demonstrate an accuracy of 0.1% in the thickness characterization through measurements performed on four microcantilevers that expand an area of 1.8 mm2 in one minute of analysis time. The measured thickness variation in the range of few tens of nm translates into a mechanical variability that produces an error of up to 2% in the response of the studied devices when they are used to measure surface stress variations. PMID:27338398

  6. Spatially Multiplexed Micro-Spectrophotometry in Bright Field Mode for Thin Film Characterization

    Directory of Open Access Journals (Sweden)

    Valerio Pini

    2016-06-01

    Full Text Available Thickness characterization of thin films is of primary importance in a variety of nanotechnology applications, either in the semiconductor industry, quality control in nanofabrication processes or engineering of nanoelectromechanical systems (NEMS because small thickness variability can strongly compromise the device performance. Here, we present an alternative optical method in bright field mode called Spatially Multiplexed Micro-Spectrophotometry that allows rapid and non-destructive characterization of thin films over areas of mm2 and with 1 μm of lateral resolution. We demonstrate an accuracy of 0.1% in the thickness characterization through measurements performed on four microcantilevers that expand an area of 1.8 mm2 in one minute of analysis time. The measured thickness variation in the range of few tens of nm translates into a mechanical variability that produces an error of up to 2% in the response of the studied devices when they are used to measure surface stress variations.

  7. Multiplex PCR for Differential Identification of Broad Tapeworms (Cestoda: Diphyllobothrium) Infecting Humans▿

    Science.gov (United States)

    Wicht, Barbara; Yanagida, Tetsuya; Scholz, Tomáš; Ito, Akira; Jiménez, Juan A.; Brabec, Jan

    2010-01-01

    The specific identification of broad tapeworms (genus Diphyllobothrium) infecting humans is very difficult to perform by morphological observation. Molecular analysis by PCR and sequencing represents the only reliable tool to date to identify these parasites to the species level. Due to the recent spread of human diphyllobothriosis in several countries, a correct diagnosis has become crucial to better understand the distribution and the life cycle of human-infecting species as well as to prevent the introduction of parasites to disease-free water systems. Nevertheless, PCR and sequencing, although highly precise, are too complicated, long, and expensive to be employed in medical laboratories for routine diagnostics. In the present study we optimized a cheap and rapid molecular test for the differential identification of the most common Diphyllobothrium species infecting humans (D. latum, D. dendriticum, D. nihonkaiense, and D. pacificum), based on a multiplex PCR with the cytochrome c oxidase subunit 1 gene of mitochondrial DNA. PMID:20592146

  8. Rapid susceptibility testing and microcolony analysis of Candida spp. cultured and imaged on porous aluminum oxide.

    Science.gov (United States)

    Ingham, Colin J; Boonstra, Sjoukje; Levels, Suzanne; de Lange, Marit; Meis, Jacques F; Schneeberger, Peter M

    2012-01-01

    Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO) support combined with microscopy offers a route to more rapid results. Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture), and voriconazole and itraconazole (7 h culture). Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170) and E-test (85.9% agreement; n = 170). The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2%) and the least good for the triazoles (79.4 to 82.4%). Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications.

  9. Rapid susceptibility testing and microcolony analysis of Candida spp. cultured and imaged on porous aluminum oxide.

    Directory of Open Access Journals (Sweden)

    Colin J Ingham

    Full Text Available BACKGROUND: Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO support combined with microscopy offers a route to more rapid results. METHODS: Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture, and voriconazole and itraconazole (7 h culture. SIGNIFICANCE: Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170 and E-test (85.9% agreement; n = 170. The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2% and the least good for the triazoles (79.4 to 82.4%. Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications.

  10. A rapid analysis of plasma/serum ethylene and propylene glycol by headspace gas chromatography.

    Science.gov (United States)

    Ehlers, Alexandra; Morris, Cory; Krasowski, Matthew D

    2013-12-01

    A rapid headspace-gas chromatography (HS-GC) method was developed for the analysis of ethylene glycol and propylene glycol in plasma and serum specimens using 1,3-propanediol as the internal standard. The method employed a single-step derivitization using phenylboronic acid, was linear to 200 mg/dL and had a lower limit of quantitation of 1 mg/dL suitable for clinical analyses. The analytical method described allows for laboratories with HS-GC instrumentation to analyze ethanol, methanol, isopropanol, ethylene glycol, and propylene glycol on a single instrument with rapid switch-over from alcohols to glycols analysis. In addition to the novel HS-GC method, a retrospective analysis of patient specimens containing ethylene glycol and propylene glycol was also described. A total of 36 patients ingested ethylene glycol, including 3 patients who presented with two separate admissions for ethylene glycol toxicity. Laboratory studies on presentation to hospital for these patients showed both osmolal and anion gap in 13 patients, osmolal but not anion gap in 13 patients, anion but not osmolal gap in 8 patients, and 1 patient with neither an osmolal nor anion gap. Acidosis on arterial blood gas was present in 13 cases. Only one fatality was seen; this was a patient with initial serum ethylene glycol concentration of 1282 mg/dL who died on third day of hospitalization. Propylene glycol was common in patients being managed for toxic ingestions, and was often attributed to iatrogenic administration of propylene glycol-containing medications such as activated charcoal and intravenous lorazepam. In six patients, propylene glycol contributed to an abnormally high osmolal gap. The common presence of propylene glycol in hospitalized patients emphasizes the importance of being able to identify both ethylene glycol and propylene glycol by chromatographic methods.

  11. Rapid maxillary expansion and obstructive sleep apnea: A review and meta-analysis.

    Science.gov (United States)

    Machado-Júnior, A-J; Zancanella, E; Crespo, A-N

    2016-07-01

    OSAS during childhood leads to significant physical and neuropsychomotor impairment. Thus, it needs to be recognized and treated early in order to avoid or attenuate the chronic problems associated with OSAS, which are deleterious to a child's development. Adenotonsillectomy and, in select cases, continuous positive airwaypressure (CPAP) have been the preferred treatments for OSAS in children, and yet they are ineffective at fully ameliorating the disease. Minimally invasive treatments have recently been proposed, comprising intra-oral and extra-oral devices as well as speech therapy. Objetive: to conduct a meta-analysis on studies from around the world that used rapid maxillary expansion (RME) to treat OSAS in children. We performed a meta-analysis of studies using RME for OSA treatment in children. A literature survey was conductedusing PubMed and Medline for English articles published up to December 2014 with the following descriptors: SleepApnea, Obstructive, Children, Treatment, Orthodontic, Othopaedic, Maxillaryexpansion. Studies were included in the meta-analysisif they were case-controlled studies, randomized, and involved non-syndromic children aged 0 to 12years old diagnosed with OSA by the polysomnography apnea-hypopnea index (AHI) before and after the intervention, submitted RME only. In all, 10 articles conformed to the inclusion criteria and were included in this meta-analysis. The total sample size across all these articles was 215 children, having a mean age of 6.7 years,of whom58.6%were male. The mean AHI during the follow-up was -6.86 (p <0.0001). We concluded that rapid maxillary expansion (RME) in children with OSAS appears to be an effective treatment for this syndrome. Further randomized clinical studies are needed to determine the effectiveness of RME in adults.

  12. Sequential injection analysis with chemiluminescence detection for rapid monitoring of commercial Calendula officinalis extractions.

    Science.gov (United States)

    Hughes, Rachel R; Scown, David; Lenehan, Claire E

    2015-01-01

    Plant extracts containing high levels of antioxidants are desirable due to their reported health benefits. Most techniques capable of determining the antioxidant activity of plant extracts are unsuitable for rapid at-line analysis as they require extensive sample preparation and/or long analysis times. Therefore, analytical techniques capable of real-time or pseudo real-time at-line monitoring of plant extractions, and determination of extraction endpoints, would be useful to manufacturers of antioxidant-rich plant extracts. To develop a reliable method for the rapid at-line extraction monitoring of antioxidants in plant extracts. Calendula officinalis extracts were prepared from dried flowers and analysed for antioxidant activity using sequential injection analysis (SIA) with chemiluminescence (CL) detection. The intensity of CL emission from the reaction of acidic potassium permanganate with antioxidants within the extract was used as the analytical signal. The SIA-CL method was applied to monitor the extraction of C. officinalis over the course of a batch extraction to determine the extraction endpoint. Results were compared with those from ultra high performance liquid chromatography (UHPLC). Pseudo real-time, at-line monitoring showed the level of antioxidants in a batch extract of Calendula officinalis plateaued after 100 min of extraction. These results correlated well with those of an offline UHPLC study. SIA-CL was found to be a suitable method for pseudo real-time monitoring of plant extractions and determination of extraction endpoints with respect to antioxidant concentrations. The method was applied at-line in the manufacturing industry. Copyright © 2015 John Wiley & Sons, Ltd.

  13. Rapid detection of Listeria monocytogenes in milk using confocal micro-Raman spectroscopy and chemometric analysis.

    Science.gov (United States)

    Wang, Junping; Xie, Xinfang; Feng, Jinsong; Chen, Jessica C; Du, Xin-jun; Luo, Jiangzhao; Lu, Xiaonan; Wang, Shuo

    2015-07-02

    Listeria monocytogenes is a facultatively anaerobic, Gram-positive, rod-shape foodborne bacterium causing invasive infection, listeriosis, in susceptible populations. Rapid and high-throughput detection of this pathogen in dairy products is critical as milk and other dairy products have been implicated as food vehicles in several outbreaks. Here we evaluated confocal micro-Raman spectroscopy (785 nm laser) coupled with chemometric analysis to distinguish six closely related Listeria species, including L. monocytogenes, in both liquid media and milk. Raman spectra of different Listeria species and other bacteria (i.e., Staphylococcus aureus, Salmonella enterica and Escherichia coli) were collected to create two independent databases for detection in media and milk, respectively. Unsupervised chemometric models including principal component analysis and hierarchical cluster analysis were applied to differentiate L. monocytogenes from Listeria and other bacteria. To further evaluate the performance and reliability of unsupervised chemometric analyses, supervised chemometrics were performed, including two discriminant analyses (DA) and soft independent modeling of class analogies (SIMCA). By analyzing Raman spectra via two DA-based chemometric models, average identification accuracies of 97.78% and 98.33% for L. monocytogenes in media, and 95.28% and 96.11% in milk were obtained, respectively. SIMCA analysis also resulted in satisfied average classification accuracies (over 93% in both media and milk). This Raman spectroscopic-based detection of L. monocytogenes in media and milk can be finished within a few hours and requires no extensive sample preparation. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Rapid Holocene coastal change revealed by high-resolution micropaleontological analysis, Pamlico Sound, North Carolina, USA

    Science.gov (United States)

    Grand, Pre C.; Culver, S.J.; Mallinson, D.J.; Farrell, K.M.; Corbett, D.R.; Horton, B.P.; Hillier, C.; Riggs, S.R.; Snyder, S.W.; Buzas, M.A.

    2011-01-01

    Foraminiferal analyses of 404 contiguous samples, supported by diatom, lithologic, geochronologic and seismic data, reveal both rapid and gradual Holocene paleoenvironmental changes in an 8.21-m vibracore taken from southern Pamlico Sound, North Carolina. Data record initial flooding of a latest Pleistocene river drainage and the formation of an estuary 9000. yr ago. Estuarine conditions were punctuated by two intervals of marine influence from approximately 4100 to 3700 and 1150 to 500. cal. yr BP. Foraminiferal assemblages in the muddy sand facies that accumulated during these intervals contain many well-preserved benthic foraminiferal species, which occur today in open marine settings as deep as the mid shelf, and significant numbers of well-preserved planktonic foraminifera, some typical of Gulf Stream waters. We postulate that these marine-influenced units resulted from temporary destruction of the southern Outer Banks barrier islands by hurricanes. The second increase in marine influence is coeval with increased rate of sea-level rise and a peak in Atlantic tropical cyclone activity during the Medieval Climate Anomaly. This high-resolution analysis demonstrates the range of environmental variability and the rapidity of coastal change that can result from the interplay of changing climate, sea level and geomorphology in an estuarine setting. ?? 2011 University of Washington.

  15. Rapid quantitative and qualitative analysis of biofilm production by Staphylococcus epidermidis under static growth conditions.

    Science.gov (United States)

    Waters, Elaine M; McCarthy, Hannah; Hogan, Siobhan; Zapotoczna, Marta; O'Neill, Eoghan; O'Gara, James P

    2014-01-01

    Rapid screening of biofilm forming capacity by Staphylococcus epidermidis is possible using in vitro assays with 96-well plates. This method first developed by Christensen et al. in 1985 is fast and does not require specialized instruments. Thus, laboratories with standard microbiology infrastructure and a 96-well plate reader can easily use this technique to generate data on the biofilm phenotypes of multiple S. epidermidis strains and clinical isolates. Furthermore, this method can be adapted to gain insights into biofilm regulation and the characteristics of biofilms produced by different S. epidermidis isolates. Although this assay is extremely useful for showing whether individual strains are biofilm-positive or biofilm-negative and distinguishing between form weak, moderate or strong biofilm, it is important to acknowledge that the absolute levels of biofilm produced by an individual strain can vary significantly between experiments meaning that strict adherence to the protocol used is of paramount importance. Furthermore, measuring biofilm under static conditions does not generally reflect in vivo conditions in which bacteria are often subjected to shear stresses under flow conditions. Hence, the biofilm characteristics of some strains are dramatically different under flow and static conditions. Nevertheless, rapid measurement of biofilm production under static conditions is a useful tool in the analysis of the S. epidermidis biofilm phenotype.

  16. Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis.

    Science.gov (United States)

    Li, Juan; Zhao, Guang-Hui; Lin, RuiQing; Blair, David; Sugiyama, Hiromu; Zhu, Xing-Quan

    2015-11-01

    Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

  17. Rapid analysis of drug dissolution by paper spray ionization mass spectrometry.

    Science.gov (United States)

    Liu, Yang; Liu, Ning; Zhou, Ya-Nan; Lin, Lan; He, Lan

    2017-03-20

    With a great quantity of solid dosage tested by dissolution technology, developing a rapid and sensitive method to access the content of drug within dissolution media is highly desired by analysts and scientists. Traditionally, dissolution media is not compatible with mass spectrometry since the inorganic salts in the media might damage the mass spectrometer. Here, paper spray ionization mass spectrometry (PSI-MS), one of the ambient mass spectrometry technologies, is developed to characterize the content of drugs in dissolution media. The porous structure of paper can effectively retain salts from entering mass spectrometer. This makes the measurement of drug content within dissolution media by mass spectrometer possible. After the experimental parameters were optimized, calibration curves of model drugs - enalapril, quinapril and benazepril were established by using corresponding deuterated internal standards. PSI-MS was then deployed to characterize the content of enalapril from the dissolution testing of enalapril tablets. The results from PSI-MS are comparable to those from HPLC characterization. More importantly, the analysis time of 6 samples is shortened from 90min to 6min. Detection limit of enalapril maleate tablets by PSI-MS is 1/300 of LC. PSI-MS is rapid, sensitive and accurate in analyzing drug content from dissolution tests. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Quantitative analysis of lipid droplet fusion: inefficient steady state fusion but rapid stimulation by chemical fusogens.

    Directory of Open Access Journals (Sweden)

    Samantha Murphy

    Full Text Available Lipid droplets (LDs are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s, followed by the slower merging (100 s of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types.

  19. Quantitative analysis of lipid droplet fusion: inefficient steady state fusion but rapid stimulation by chemical fusogens.

    Science.gov (United States)

    Murphy, Samantha; Martin, Sally; Parton, Robert G

    2010-12-23

    Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types.

  20. Rapid point-of-care HIV testing in pregnant women: a systematic review and meta-analysis.

    Science.gov (United States)

    Pai, Nitika Pant; Tulsky, Jacqueline Peterson; Cohan, Deborah; Colford, John M; Reingold, Arthur L

    2007-02-01

    Rapid, point-of-care human immunodeficiency virus (HIV) testing has the potential to enhance strategies to prevent mother-to-child transmission (MTCT) of HIV infection. Rapid tests need minimal laboratory infrastructure and can be performed by health workers with minimal training. In our systematic review and meta-analysis, we aimed to summarize the overall diagnostic accuracy of rapid HIV tests in pregnancy, and outcomes such as acceptability, patient preference, feasibility and impact of rapid testing. We searched four major databases, identified and screened 1377 citations, and included 17 studies that met our eligibility criteria. Analyses of these studies suggested that the overall sensitivity and specificity of blood-based rapid tests was high compared with oral rapid tests. A two-step testing strategy, particularly parallel testing, was found to be superior to single-test strategy in labour and delivery settings. Acceptability of rapid tests and patient preference was variable across studies. Overall, rapid HIV testing was highly accurate compared with conventional tests and offer a clear advantage of enabling the implementation of timely interventions to reduce MTCT of HIV. To improve diagnostic accuracy and to reduce false-positive results, it may be necessary to use two rapid tests during labour and delivery.

  1. Multiplex PCR for rapid genotyping of Flavobacterium columnare

    Science.gov (United States)

    Columnaris disease, caused by the Gram-negative bacterium Flavobacterium columnare, is one of the leading causes of disease losses to the catfish industry in the Southeast USA. Recent research in our laboratory has deciphered the genetic diversity among F. columnare isolates through whole genome seq...

  2. Spatial-Temporal Feature Analysis on Single-Trial Event Related Potential for Rapid Face Identification.

    Science.gov (United States)

    Jiang, Lei; Wang, Yun; Cai, Bangyu; Wang, Yueming; Wang, Yiwen

    2017-01-01

    The event-related potential (ERP) is the brain response measured in electroencephalography (EEG), which reflects the process of human cognitive activity. ERP has been introduced into brain computer interfaces (BCIs) to communicate the computer with the subject's intention. Due to the low signal-to-noise ratio of EEG, most ERP studies are based on grand-averaging over many trials. Recently single-trial ERP detection attracts more attention, which enables real time processing tasks as rapid face identification. All the targets needed to be retrieved may appear only once, and there is no knowledge of target label for averaging. More interestingly, how the features contribute temporally and spatially to single-trial ERP detection has not been fully investigated. In this paper, we propose to implement a local-learning-based (LLB) feature extraction method to investigate the importance of spatial-temporal components of ERP in a task of rapid face identification using single-trial detection. Comparing to previous methods, LLB method preserves the nonlinear structure of EEG signal distribution, and analyze the importance of original spatial-temporal components via optimization in feature space. As a data-driven methods, the weighting of the spatial-temporal component does not depend on the ERP detection method. The importance weights are optimized by making the targets more different from non-targets in feature space, and regularization penalty is introduced in optimization for sparse weights. This spatial-temporal feature extraction method is evaluated on the EEG data of 15 participants in performing a face identification task using rapid serial visual presentation paradigm. Comparing with other methods, the proposed spatial-temporal analysis method uses sparser (only 10% of the total) features, and could achieve comparable performance (98%) of single-trial ERP detection as the whole features across different detection methods. The interesting finding is that the N250 is

  3. Cost-effective and rapid blood analysis on a cell-phone.

    Science.gov (United States)

    Zhu, Hongying; Sencan, Ikbal; Wong, Justin; Dimitrov, Stoyan; Tseng, Derek; Nagashima, Keita; Ozcan, Aydogan

    2013-04-07

    We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. We evaluated the performance of this cell-phone based blood analysis platform using anonymous human blood samples and achieved comparable results to a standard bench-top hematology analyser. Test results can either be stored on the cell-phone memory or be transmitted to a central server, providing remote diagnosis opportunities even in field settings.

  4. Finite element analysis of gradually and rapidly varied unsteady flow in open channel : II. Applications

    Energy Technology Data Exchange (ETDEWEB)

    Han, Kun Yeun; Park, Jae Hong; Lee, Eul Rae [Kyungpook National University, Taegu (Korea, Republic of)

    1997-02-28

    Petrov-Galerkin finite element model for analyzing dynamic wave equation is applied to gradually and rapidly varied unsteady flow. The model is verified by applying to hydraulic jump, nonlinear disturbance propagation in frictionless horizontal channel and dam-break analysis. It shows stable and accurate results compared with analytical solutions for various cases. The model is applied to a surge propagation in a frictionless horizontal channel. Three-dimensional water surface profiles show that the computed result converges to the analytical one with sharp discontinuity. The model is also applied to the Taehwa River to analyze unsteady flood wave propagation. The computed results have good agreements with those of DWOPER model in terms of discharge and stage hydrographs. (author). 19 refs., 22 figs.

  5. Bienzymatic Biosensor for Rapid Detection of Aspartame by Flow Injection Analysis

    Directory of Open Access Journals (Sweden)

    Maria-Cristina Radulescu

    2014-01-01

    Full Text Available A rapid, simple and stable biosensor for aspartame detection was developed. Alcohol oxidase (AOX, carboxyl esterase (CaE and bovine serum albumin (BSA were immobilised with glutaraldehyde (GA onto screen-printed electrodes modified with cobalt-phthalocyanine (CoPC. The biosensor response was fast. The sample throughput using a flow injection analysis (FIA system was 40 h−1 with an RSD of 2.7%. The detection limits for both batch and FIA measurements were 0.1 µM for methanol and 0.2 µM for aspartame, respectively. The enzymatic biosensor was successfully applied for aspartame determination in different sample matrices/commercial products (liquid and solid samples without any pre-treatment step prior to measurement.

  6. [Study on rapid determination and analysis of rocket kerosene by near infrared spectrum and chemometrics].

    Science.gov (United States)

    Xia, Ben-Li; Cong, Ji-Xin; Li, Xia; Wang, Xuan-Jun

    2011-06-01

    The rocket kerosene quality properties such as density, distillation range, viscosity and iodine value were successfully measured based on their near-infrared spectrum (NIRS) and chemometrics. In the present paper, more than 70 rocket kerosene samples were determined by near infrared spectrum, the models were built using the partial least squares method within the appropriate wavelength range. The correlation coefficients (R2) of every rocket kerosene's quality properties ranged from 0.862 to 0.999. Ten unknown samples were determined with the model, and the result showed that the prediction accuracy of near infrared spectrum method accords with standard analysis requirements. The new method is well suitable for replacing the traditional standard method to rapidly determine the properties of the rocket kerosene.

  7. Rapid confirmatory analysis of avermectin residues in milk by liquid chromatography tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Nurullah Ozdemir

    2016-01-01

    Full Text Available Our study developed a quick method for confirmatory analysis of avermectins (abamectin B1a, doramectin, ivermectin B1a, eprinomectin B1a, and moxidectin in bovine milk according to the European Commission Decision 2002/657/EC requirements. Avermectins were liquid–liquid extracted with acetonitrile, followed by an evaporation step, and then analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry in the negative ion mode. An in-house method validation was performed and the data reported on specificity, linearity, recovery, limit of detection, limit of quantitation, decision limit, and detection capability. The advantage of this method is that low levels of avermectins are detectable and quantitatively confirmed at a rapid rate in milk.

  8. Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix.

    Science.gov (United States)

    Barta, C; Ronai, Z; Sasvari-Szekely, M; Guttman, A

    2001-01-01

    Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the most common mutation in the 21-hydroxylase gene is presented using primer extension and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.

  9. Laser and LED phototherapy on midpalatal suture after rapid maxilla expansion: Raman and histological analysis.

    Science.gov (United States)

    Rosa, Cristiane Becher; Habib, Fernando Antonio Lima; de Araújo, Telma Martins; Dos Santos, Jean Nunes; Cangussu, Maria Cristina T; Barbosa, Artur Felipe Santos; de Castro, Isabele Cardoso Vieira; Pinheiro, Antônio Luiz Barbosa

    2017-02-01

    The aim of this study was to analyze the effect of laser or LED phototherapy on the acceleration of bone formation at the midpalatal suture after rapid maxilla expansion. Forty-five rats were divided into groups at 7 days (control, expansion, expansion and laser irradiation, and expansion and LED irradiation) and into 14 days (expansion, expansion and laser in the 1st week, expansion and LED in the 1st week, expansion and laser in the 1st and 2nd weeks, expansion and LED in the 1st and 2nd weeks). Laser/LED irradiation occurred every 48 h. Expansion was accomplished with a spatula and maintained with a triple helicoid of 0.020-in stainless steel orthodontic wire. A diode laser (λ780 nm, 70 mW, spot of 0.04 cm2, t = 257 s, SAEF of 18 J/cm2) or a LED (λ850 ± 10 nm, 150 ± 10 mW, spot of 0.5 cm2, t = 120 s, SAEF of 18 J/cm2) was applied in one point in the midpalatal suture immediately behind the upper incisors. Raman spectroscopy and histological analyses of the suture region were carried and data was submitted to statistical analyses (p ≤ 0.05). Raman spectrum analysis demonstrated that irradiation increases hydroxyapatite in the midpalatal suture after expansion. In the histological analysis of various inflammation, there was a higher production of collagen and osteoblastic activity and less osteoclastic activity. The results showed that LED irradiation associated to rapid maxillary expansion improves bone repair and could be an alternative to the use of laser in accelerating bone formation in the midpalatal suture.

  10. High resolution multiplexed functional imaging in live embryos (Conference Presentation)

    Science.gov (United States)

    Xu, Dongli; Zhou, Weibin; Peng, Leilei

    2017-02-01

    Fourier multiplexed fluorescence lifetime imaging (FmFLIM) scanning laser optical tomography (FmFLIM-SLOT) combines FmFLIM and Scanning laser optical tomography (SLOT) to perform multiplexed 3D FLIM imaging of live embryos. The system had demonstrate multiplexed functional imaging of zebrafish embryos genetically express Foster Resonant Energy Transfer (FRET) sensors. However, previous system has a 20 micron resolution because the focused Gaussian beam diverges quickly from the focused plane, makes it difficult to achieve high resolution imaging over a long projection depth. Here, we present a high-resolution FmFLIM-SLOT system with achromatic Bessel beam, which achieves 3 micron resolution in 3D deep tissue imaging. In Bessel-FmFLIM-SLOT, multiple laser excitation lines are firstly intensity modulated by a Michelson interferometer with a spinning polygon mirror optical delay line, which enables Fourier multiplexed multi-channel lifetime measurements. Then, a spatial light modulator and a prism are used to transform the modulated Gaussian laser beam to an achromatic Bessel beam. The achromatic Bessel beam scans across the whole specimen with equal angular intervals as sample rotated. After tomography reconstruction and the frequency domain lifetime analysis method, both the 3D intensity and lifetime image of multiple excitation-emission can be obtained. Using Bessel-FmFLIM-SLOT system, we performed cellular-resolution FLIM tomography imaging of live zebrafish embryo. Genetically expressed FRET sensors in these embryo will allow non-invasive observation of multiple biochemical processes in vivo.

  11. Direct object detection from field-of-view multiplexed measurements

    Science.gov (United States)

    Shilling, Richard Z.; Muise, Robert R.

    2017-04-01

    A compressive imaging model is proposed that multiplexes segments of the field of view (FOV) onto an infrared focal plane array (FPA). Similar to compound imaging, our model is based on combining pixels from a surface comprising of the different parts of the FOV. We formalize this superposition of pixels in a global multiplexing process reducing the number of detectors required for the FPA. We present an analysis of the signal-to-noise ratio for the full rank and compressive collection paradigms for a target detection and tracking scenario. We then apply automated target detection algorithms directly on the measurement sequence for this multiplexing model. We extend the target training and detection processes for the application directly on the encoded measurements. Optimal measurement codes for this application may imply abandoning the ability to reconstruct the actual scene in favor of reconstructing the locations of interesting objects. We present a simulated case study as well as real data results from a visible FOV multiplexing camera.

  12. Precoded spatial multiplexing MIMO system with spatial component interleaver.

    Science.gov (United States)

    Gao, Xiang; Wu, Zhanji

    In this paper, the performance of precoded bit-interleaved coded modulation (BICM) spatial multiplexing multiple-input multiple-output (MIMO) system with spatial component interleaver is investigated. For the ideal precoded spatial multiplexing MIMO system with spatial component interleaver based on singular value decomposition (SVD) of the MIMO channel, the average pairwise error probability (PEP) of coded bits is derived. Based on the PEP analysis, the optimum spatial Q-component interleaver design criterion is provided to achieve the minimum error probability. For the limited feedback precoded proposed scheme with linear zero forcing (ZF) receiver, in order to minimize a bound on the average probability of a symbol vector error, a novel effective signal-to-noise ratio (SNR)-based precoding matrix selection criterion and a simplified criterion are proposed. Based on the average mutual information (AMI)-maximization criterion, the optimal constellation rotation angles are investigated. Simulation results indicate that the optimized spatial multiplexing MIMO system with spatial component interleaver can achieve significant performance advantages compared to the conventional spatial multiplexing MIMO system.

  13. Rapid assessment of populations trends of invasive species: Singular Spectrum Analysis (SSA

    Directory of Open Access Journals (Sweden)

    DANA, ED

    2010-01-01

    Full Text Available Singular Spectrum Analysis (SSA is a powerful analytical approach for biodi-versity management. Its main advan-tages are due to its intuitive processing and visualization, since mathematical workflow is conceptually similar to the widely accepted Principal Components Analysis. Detailed analyses of popula-tion trends with mathematical tools are often difficult to achieve for managers by a number of reasons (large numbers or areas monitored, large number of species, insufficient statistics skills, strong knowledge level in demographic analyses, etc.. SSA has been used since the 1970’s in signal processing to clarify signal vs. noisy information, but it has also been used in climate change analy-sis and other developmental areas. Be-sides, SSA is a rapid-learning method for technicians and managers with medium level of mathematical knowledge. Free software in Unix environment is avail-able. Unfortunately, no free and friendly software is available for Win-dows SO. Although R package may offer solutions for really advanced users, it does not fit real work situations for managers of biological invasions. Cater-pillar (Gistat Group, Ltd is by now, the best option found by the author in terms of price, facility for results inter-pretation and time consumed in learn-ing. The main disadvantage is the poor content of tutorial files

  14. SASqPCR: robust and rapid analysis of RT-qPCR data in SAS.

    Directory of Open Access Journals (Sweden)

    Daijun Ling

    Full Text Available Reverse transcription quantitative real-time PCR (RT-qPCR is a key method for measurement of relative gene expression. Analysis of RT-qPCR data requires many iterative computations for data normalization and analytical optimization. Currently no computer program for RT-qPCR data analysis is suitable for analytical optimization and user-controllable customization based on data quality, experimental design as well as specific research aims. Here I introduce an all-in-one computer program, SASqPCR, for robust and rapid analysis of RT-qPCR data in SAS. This program has multiple macros for assessment of PCR efficiencies, validation of reference genes, optimization of data normalizers, normalization of confounding variations across samples, and statistical comparison of target gene expression in parallel samples. Users can simply change the macro variables to test various analytical strategies, optimize results and customize the analytical processes. In addition, it is highly automatic and functionally extendable. Thus users are the actual decision-makers controlling RT-qPCR data analyses. SASqPCR and its tutorial are freely available at http://code.google.com/p/sasqpcr/downloads/list.

  15. Evaluation of the multiplex PCR Allplex-GI assay in the detection of bacterial pathogens in diarrheic stool samples.

    Science.gov (United States)

    Martín, Ariadna; Pérez-Ayala, Ana; Chaves, Fernando; Lora, David; Orellana, M Ángeles

    2017-10-31

    Rapid and accurate detection of the pathogens that cause gastrointestinal infection is important for appropriate therapy and proper infection control. This study assesses the performance of a new molecular assay for simultaneous detection of 13 different gastrointestinal bacteria in stool specimens. Using the Allplex GI-Bacteria (AGI-BI/AGI-BII) assay, a total of 394 stool samples were tested and the results were compared with culturing on selective differential followed by identification by mass spectroscopy. Discordant results were analyzed by a different multiplex PCR method, the Fast-Track Diagnostics Bacterial gastroenteritis (FTD-BG). The routine method (RM) detected 109 (27.7%) positive samples and the Allplex-GI assay, 261 (66.2%). Analysis of discordant results revealed that the molecular assay detected 44 pathogens that were not detected by the RM, including 23 Campylobacter spp., 11 Salmonella spp, 3 Y. enterocolitica, 2 EIEC/Shigella spp, 2 E. coli 0157, 2 C. difficile and 1 Aeromonas spp. Five cases not detected by the molecular method were detected by the RM (3 Aeromonas spp, 1 Salmonella spp and 1 Y. enterocolitica). For all targets, the percentages of sensitivity and specificity were >95%, except for Aeromonas spp., which were 81% and 99% respectively. This study suggests that Allplex-GI multiplex PCR is a sensitive and specific assay that enables a rapid and accurate diagnosis of bacterial gastrointestinal infections. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Temporal correlations in social multiplex networks

    CERN Document Server

    Starnini, Michele; Pastor-Satorras, Romualdo

    2016-01-01

    Social interactions are composite, involve different communication layers and evolve in time. However, a rigorous analysis of the whole complexity of social networks has been hindered so far by lack of suitable data. Here we consider both the multi-layer and dynamic nature of social relations by analysing a diverse set of empirical temporal multiplex networks. We focus on the measurement and characterization of inter-layer correlations to investigate how activity in one layer affects social acts in another layer. We define observables able to detect when genuine correlations are present in empirical data, and single out spurious correlation induced by the bursty nature of human dynamics. We show that such temporal correlations do exist in social interactions where they act to depress the tendency to concentrate long stretches of activity on the same layer and imply some amount of potential predictability in the connection patterns between layers. Our work sets up a general framework to measure temporal correl...

  17. [The rapid analysis of fatty acids in vegetable oils by near infrared spectrum].

    Science.gov (United States)

    Yu, Yan-Bo; Zang, Peng; Fu, Yuan-Hua; Zhang, Lu-Da; Yan, Yan-Lu; Chen, Bin

    2008-07-01

    In this research, The functional components of vegetable oils were analyzed by near infrared (NIR) spectral technology. The optimum conditions of mathematics model of four components (C16 : 0, C18 : 0, C18 : 1, C18 : 2) were studied, including the sample set selection, chemical value analysis, the detection methods and condition. Chemical value was analyzed by HPLC. 52 samples were selected, 41 for modeling set and 11 for testing set. All samples were placed in 5mm thick sample pools and swept by near infrared (NIR) with discrimination factor 8 cm(-1) without any other disposal. Using PLS methods sated model. Data were processed by first derivative method and centering method. 5 000-9 000 cm(-1) spectral region was analyzed. Correlating index (r), RMSECV and RMSEP were chose as evaluation index. The result demonstrated that the correlation between the reference value of the modeling sample set and the near infrared predictive value were r(C16 : 0) = 0.891, r(C18 : 0) = 0.837, r(C18 : 1) = 0.982, r(C18 : 2) = 0.971, respectively. And the correlation between the reference value of the testing sample set and the near infrared predictive value were 0.921, 0.891, 0.946 and 0.949, respectively. It proved that the near infrared predictive value was linear with chemical value and the mathematical model established for components of vegetable oils was feasible. For validation, 8 unknown samples were selected to be analysis by infrared (NIR). The result demonstrated that error between predict value and chemical value was less than 10%. That was to say infrared (NIR) had a good veracity in analysis components of vegetable oil. Because infrared (NIR) spectral technology is convenient, rapid than HPLC in oil components analysis, moreover, infrared (NIR) can analyze many components at the same time. It must have great application prospect in vegetable oil components analysis.

  18. Multiplexed lateral flow biosensors: Technological advances for radically improving point-of-care diagnoses.

    Science.gov (United States)

    Li, Jia; Macdonald, Joanne

    2016-09-15

    Lateral flow biosensors are a leading technology in point-of-care diagnostics due to their simplicity, rapidness and low cost. Their primacy in this arena continues through technological breakthroughs such as multiplexing: the detection of more than one biomarker in a single assay. Multiplexing capacity is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases and reducing diagnostic cost. Here we review, for the first time, the various types and strategies employed for creating multiplexed lateral flow biosensors. These are classified into four main categories in terms of specific application or multiplexing level, namely linear, parameter, spatial and conceptual. We describe the practical applications and implications for each approach and compare their advantages and disadvantages. Importantly, multiplexing is still subject to limitations of the traditional lateral flow biosensor, such as sensitivity and specificity. However, by pushing the limitations of the traditional medium into the multiplex arena, several technological breakthroughs are emerging with novel solutions that further expand the utility of lateral flow biosensing for point-of-care applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Usefulness of capillary electrophoresis-based multiplex PCR assay for species-specific identification of Candida spp.

    Science.gov (United States)

    Mallus, F; Martis, S; Serra, C; Loi, G; Camboni, T; Manzin, A

    2013-02-15

    The study evaluated the performances of a commercial multiplex PCR assay, the Seegene Seeplex STI Master Panel 3, for Candida spp. identification. Eighty clinical strains of Candida spp. were identified with this system and a homemade multiplex PCR assay. The results were also compared with those obtained with two phenotypic methods. The study provided a preliminary evaluation of a multiplex assay from Seegene that uses capillary electrophoresis as the detection of amplified products. The Seeplex assay was found to be a rapid and useful method for identifying large numbers of yeast isolates in the clinical laboratory context. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Flexible Multiplexed Surface Temperature Sensor

    Science.gov (United States)

    Daryabeigi, Kamran; Dillon-Townes, L. A.; Johnson, Preston B.; Ash, Robert L.

    1995-01-01

    Unitary array of sensors measures temperatures at points distributed over designated area on surface. Useful in measuring surface temperatures of aerodynamic models and thermally controlled objects. Made of combination of integrated-circuit microchips and film circuitry. Temperature-sensing chips scanned at speeds approaching 10 kHz. Operating range minus 40 degrees C to 120 degrees C. Flexibility of array conforms to curved surfaces. Multiplexer eliminates numerous monitoring cables. Control of acquisition and recording of data effected by connecting array to microcomputers via suitable interface circuitry.

  1. Multiplex detection of respiratory pathogens

    Energy Technology Data Exchange (ETDEWEB)

    McBride, Mary [Brentwood, CA; Slezak, Thomas [Livermore, CA; Birch, James M [Albany, CA

    2012-07-31

    Described are kits and methods useful for detection of respiratory pathogens (influenza A (including subtyping capability for H1, H3, H5 and H7 subtypes) influenza B, parainfluenza (type 2), respiratory syncytial virus, and adenovirus) in a sample. Genomic sequence information from the respiratory pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  2. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

    Science.gov (United States)

    Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

    2016-01-01

    Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (PPCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods. PMID

  3. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

    Science.gov (United States)

    Dinoop, K P; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R P; Narayanan, P

    2016-01-01

    Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( PPCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

  4. Mesoscopic structures reveal the network between the layers of multiplex data sets

    Science.gov (United States)

    Iacovacci, Jacopo; Wu, Zhihao; Bianconi, Ginestra

    2015-10-01

    Multiplex networks describe a large variety of complex systems, whose elements (nodes) can be connected by different types of interactions forming different layers (networks) of the multiplex. Multiplex networks include social networks, transportation networks, or biological networks in the cell or in the brain. Extracting relevant information from these networks is of crucial importance for solving challenging inference problems and for characterizing the multiplex networks microscopic and mesoscopic structure. Here we propose an information theory method to extract the network between the layers of multiplex data sets, forming a "network of networks." We build an indicator function, based on the entropy of network ensembles, to characterize the mesoscopic similarities between the layers of a multiplex network, and we use clustering techniques to characterize the communities present in this network of networks. We apply the proposed method to study the Multiplex Collaboration Network formed by scientists collaborating on different subjects and publishing in the American Physical Society journals. The analysis of this data set reveals the interplay between the collaboration networks and the organization of knowledge in physics.

  5. Multiplex lexical networks reveal patterns in early word acquisition in children

    Science.gov (United States)

    Stella, Massimo; Beckage, Nicole M.; Brede, Markus

    2017-04-01

    Network models of language have provided a way of linking cognitive processes to language structure. However, current approaches focus only on one linguistic relationship at a time, missing the complex multi-relational nature of language. In this work, we overcome this limitation by modelling the mental lexicon of English-speaking toddlers as a multiplex lexical network, i.e. a multi-layered network where N = 529 words/nodes are connected according to four relationship: (i) free association, (ii) feature sharing, (iii) co-occurrence, and (iv) phonological similarity. We investigate the topology of the resulting multiplex and then proceed to evaluate single layers and the full multiplex structure on their ability to predict empirically observed age of acquisition data of English speaking toddlers. We find that the multiplex topology is an important proxy of the cognitive processes of acquisition, capable of capturing emergent lexicon structure. In fact, we show that the multiplex structure is fundamentally more powerful than individual layers in predicting the ordering with which words are acquired. Furthermore, multiplex analysis allows for a quantification of distinct phases of lexical acquisition in early learners: while initially all the multiplex layers contribute to word learning, after about month 23 free associations take the lead in driving word acquisition.

  6. A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis.

    Science.gov (United States)

    Lledías, Fernando; Hernández, Felipe; Rivas, Viridiana; García-Mendoza, Abisaí; Cassab, Gladys I; Nieto-Sotelo, Jorge

    2017-08-01

    Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.

  7. Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

    Science.gov (United States)

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

  8. Rapid enzymatic hydrolysis of masked deoxynivalenol and zearalenone prior to liquid chromatography mass spectrometry or immuniassay analysis

    NARCIS (Netherlands)

    Nielen, M.W.F.; Weijers, C.A.G.M.; Peters, J.; Weignerová, L.; Zuilhof, H.; Franssen, M.C.R.

    2014-01-01

    Recently it has been shown that conjugates (‘masked mycotoxins’) may contribute to the total daily intake of hazardous mycotoxins. Therefore, there is an urgent need for rapid analysis methods that assess the level of both free and masked mycotoxins in food and feed. However, the analysis of masked

  9. Rapid fluctuation of the vaginal microbiota measured by Gram stain analysis.

    Science.gov (United States)

    Brotman, Rebecca M; Ravel, Jacques; Cone, Richard A; Zenilman, Jonathan M

    2010-08-01

    The aetiology of bacterial vaginosis (BV) remains unknown. To describe longitudinal changes in vaginal microbiota. Thirty-nine women (mean age 36.8 years; 22 (56.4%) African-American) self-collected vaginal specimens twice weekly for 16 weeks as part of a vaginal douching cessation study. In an analysis where each woman serves as her own control, conditional logistic regression was used to evaluate daily, time-varying factors associated with a woman's incident BV episode(s) as compared with her persistently BV-negative sample(s). BV was defined by a Nugent's Gram stain score >or=7. 46.2% of participants had BV in the first 4 weeks of observation. Rapid fluctuation of vaginal microbiota was observed in 226 transitions to BV or spontaneous remission. Duration of BV was often short: 51% of the episodes lasted for only one sample interval (3 days). Among women who had at least one BV episode, the median number of episodes per woman was 8.7 (SD 7.4, range 1-22). Lubricant use 1 day before specimen collection (adjusted OR (aOR)=11.75, 95% CI 1.96 to 70.27) and rectal sex 2 days before (aOR=4.48, 95% CI 2.79 to 7.17) were associated with BV onset. Rapid fluctuation of the vaginal microbiota was seen. Longitudinal studies with long intervals between sampling are likely to miss episodes of BV. Recent report of lubricant use and rectal sex were associated with incident BV.

  10. Classification of Bacillus and Brevibacillus species using rapid analysis of lipids by mass spectrometry.

    Science.gov (United States)

    AlMasoud, Najla; Xu, Yun; Trivedi, Drupad K; Salivo, Simona; Abban, Tom; Rattray, Nicholas J W; Szula, Ewa; AlRabiah, Haitham; Sayqal, Ali; Goodacre, Royston

    2016-11-01

    Bacillus are aerobic spore-forming bacteria that are known to lead to specific diseases, such as anthrax and food poisoning. This study focuses on the characterization of these bacteria by the detection of lipids extracted from 33 well-characterized strains from the Bacillus and Brevibacillus genera, with the aim to discriminate between the different species. For the purpose of analysing the lipids extracted from these bacterial samples, two rapid physicochemical techniques were used: matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography in conjunction with mass spectrometry (LC-MS). The findings of this investigation confirmed that MALDI-TOF-MS could be used to identify different bacterial lipids and, in combination with appropriate chemometrics, allowed for the discrimination between these different bacterial species, which was supported by LC-MS. The average correct classification rates for the seven species of bacteria were 62.23 and 77.03 % based on MALDI-TOF-MS and LC-MS data, respectively. The Procrustes distance for the two datasets was 0.0699, indicating that the results from the two techniques were very similar. In addition, we also compared these bacterial lipid MALDI-TOF-MS profiles to protein profiles also collected by MALDI-TOF-MS on the same bacteria (Procrustes distance, 0.1006). The level of discrimination between lipids and proteins was equivalent, and this further indicated the potential of MALDI-TOF-MS analysis as a rapid, robust and reliable method for the classification of bacteria based on different bacterial chemical components. Graphical abstract MALDI-MS has been successfully developed for the characterization of bacteria at the subspecies level using lipids and benchmarked against HPLC.

  11. Finite element analysis of rapid canine retraction through reducing resistance and distraction

    Directory of Open Access Journals (Sweden)

    Junjie XUE

    2014-01-01

    Full Text Available Objective: The aims of this study were to compare different surgical approaches to rapid canine retraction by designing and selecting the most effective method of reducing resistance by a three-dimensional finite element analysis. Material and Methods: Three-dimensional finite element models of different approaches to rapid canine retraction by reducing resistance and distraction were established, including maxillary teeth, periodontal ligament, and alveolar. The models were designed to dissect the periodontal ligament, root, and alveolar separately. A 1.5 N force vector was loaded bilaterally to the center of the crown between first molar and canine, to retract the canine distally. The value of total deformation was used to assess the initial displacement of the canine and molar at the beginning of force loading. Stress intensity and force distribution were analyzed and evaluated by Ansys 13.0 through comparison of equivalent (von Mises stress and maximum shear stress. Results: The maximum value of total deformation with the three kinds of models occurred in the distal part of the canine crown and gradually reduced from the crown to the apex of the canine; compared with the canines in model 3 and model 1, the canine in model 2 had the maximum value of displacement, up to 1.9812 mm. The lowest equivalent (von Mises stress and the lowest maximum shear stress were concentrated mainly on the distal side of the canine root in model 2. The distribution of equivalent (von Mises stress and maximum shear stress on the PDL of the canine in the three models was highly concentrated on the distal edge of the canine cervix. . Conclusions: Removal of the bone in the pathway of canine retraction results in low stress intensity for canine movement. Periodontal distraction aided by surgical undermining of the interseptal bone would reduce resistance and effectively accelerate the speed of canine retraction.

  12. Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

    Science.gov (United States)

    Felker, Daniel L.; Burggraf, Larry W.

    2014-01-01

    Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating. PMID:24375142

  13. Rapid solid-phase extraction and analysis of resveratrol and other polyphenols in red wine.

    Science.gov (United States)

    Hashim, Shima N N S; Schwarz, Lachlan J; Boysen, Reinhard I; Yang, Yuanzhong; Danylec, Basil; Hearn, Milton T W

    2013-10-25

    Red wine has long been credited as a good source of health-beneficial antioxidants, including the bioactive polyphenols catechin, quercetin, and (E)-resveratrol. In this paper, we report the application of reusable molecularly imprinted polymers (MIPs) for the selective and robust solid-phase extraction (SPE) and rapid analysis of (E)-resveratrol (LOD=8.87×10(-3) mg/L, LOQ=2.94×10(-2) mg/L), along with a range of other polyphenols from an Australian Pinot noir red wine. Optimization of the molecularly imprinted solid-phase extraction (MISPE) protocol resulted in the significant enrichment of (E)-resveratrol and several structurally related polyphenols. These secondary metabolites were subsequently identified by RP-HPLC and μLC-ESI ion trap MS/MS methods. The developed MISPE protocol employed low volumes of environmentally benign solvents selected according to the Green Chemistry principles, and resulted in the recovery of 99% of the total (E)-resveratrol present. These results further demonstrate the potential of generic protocols for the analysis of target compound with health beneficial properties within the food and nutraceutical industries using tailor-made MIPs. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Flash Profile for rapid descriptive analysis in sensory characterization of passion fruit juice

    Directory of Open Access Journals (Sweden)

    Flávia Daiana Montanuci

    2015-07-01

    Full Text Available The Flash Profile is a descriptive analysis method derived from Free-Choice Profile, in which each taster chooses and uses his/her own words to evaluate the product while comparing several attributes. Four passion fruit juices were analyzed, two juices were produced with concentrated juice, one with pulp and one with reconstituted juice; all juices had different levels of sugar, some had gum and dyes. This study aimed to evaluate the physicochemical properties (color, titratable acidity and solid content as well as sensory analysis like Flash profile and affective test. In physicochemical characterization and in Flash Profile, the juice A (pulp had higher solid content and consistence, the juice B (concentrated juice was the least acidic and presented the lowest value of soluble solids and presented strong aroma and flavor of passion-fruit, the juice C (reconstituted juice was pale yellow and showed artificial flavor and the juice D (concentrated juice was the most acidic, consistent with the natural flavor. In the acceptance test, all the juices scored 5-6, indicating that panelists tasters neither liked nor disliked. Flash Profile proved to be an easy and rapid technique showing a good correlation between panelists and the attributes and confirmed the results of physicochemical characterization.

  15. Rapid Mapping and Deformation Analysis over Cultural Heritage and Rural Sites Based on Persistent Scatterer Interferometry