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Sample records for multiplex add-on assay

  1. Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Schneider, Uffe Vest; Scheel, Troels

    2006-01-01

    BACKGROUND: Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently...... triggering receptor expressed on myeloid cells-1 (sTREM-1), and macrophage migration inhibiting factor (MIF) was developed and validated in-house. This 3-plex assay was added to a commercially available interleukin-1beta (IL-1beta), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor, and tumor...

  2. Development and validation of a multiplex add-on assay of biomarkers related to sepsis using xMAP technology

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Vest Schneider, Uffe; Scheel, Troels

    2006-01-01

    BACKGROUND: Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently...... triggering receptor expressed on myeloid cells-1 (sTREM-1), and macrophage migration inhibiting factor (MIF) was developed and validated in-house. This 3-plex assay was added to a commercially available interleukin-1beta (IL-1beta), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor, and tumor...

  3. Multiplexing a high-throughput liability assay to leverage efficiencies.

    Science.gov (United States)

    Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

    2009-06-01

    In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

  4. Opportunities and Challenges of Multiplex Assays: A Machine Learning Perspective.

    Science.gov (United States)

    Chen, Junfang; Schwarz, Emanuel

    2017-01-01

    Multiplex assays that allow the simultaneous measurement of multiple analytes in small sample quantities have developed into a widely used technology. Their implementation spans across multiple assay systems and can provide readouts of similar quality as the respective single-plex measures, albeit at far higher throughput. Multiplex assay systems are therefore an important element for biomarker discovery and development strategies but analysis of the derived data can face substantial challenges that may limit the possibility of identifying meaningful biological markers. This chapter gives an overview of opportunities and challenges of multiplexed biomarker analysis, in particular from the perspective of machine learning aimed at identification of predictive biological signatures.

  5. Multiplexed Dosing Assays by Digitally Definable Hydrogel Volumes

    DEFF Research Database (Denmark)

    Faralli, Adele; Melander, Fredrik; Larsen, Esben Kjær Unmack

    2016-01-01

    Stable and low-cost multiplexed drug sensitivity assays using small volumes of cells or tissue are in demand for personalized medicine, including patientspecific combination chemotherapy. Spatially defined projected light photopolymerization of hydrogels with embedded active compounds is introduc...

  6. Data transformation methods for multiplexed assays

    Science.gov (United States)

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  7. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Chinen, Isabel; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  8. Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Schneider, Uffe Vest; Scheel, Troels

    2006-01-01

    Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently used markers....

  9. Multiplex real-time PCR assay for Legionella species.

    Science.gov (United States)

    Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

    2015-12-01

    Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. A Novel Multiplex HRM Assay to Detect Clopidogrel Resistance.

    Science.gov (United States)

    Zhang, Lichen; Ma, Xiaowei; You, Guoling; Zhang, Xiaoqing; Fu, Qihua

    2017-11-22

    Clopidogrel is an antiplatelet medicine used to prevent blood clots in patients who have had a heart attack, stroke, or other symptoms. Variability in the clinical response to clopidogrel treatment has been attributed to genetic factors. In particular, five SNPs of rs4244285, rs4986893, rs12248560, rs662 and rs1045642 have been associated with resistance to clopidogrel therapy in Chinese population. This work involves the development of a multiplex high-resolution melting (HRM) assay to genotype all five of these loci in 2 tubes. Amplicons corresponding to distinct SNPs in a common tube were designed with the aid of uMelt prediction software to have different melting temperatures Tm by addition of a GC-rich tail to the 5' end of the certain primers. Two kinds of commercial methods, Digital Fluorescence Molecular Hybridization (DFMH) and Sanger sequencing, were used as a control. Three hundred sixteen DFMH pretested samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM. The sensitivity of HRM was 100% and the specificity was 99.93% reflecting detection of variants other than the known resistance SNPs. Multiplex HRM is an effective closed-tube, highly accurate, fast, and inexpensive method for genotyping the 5 clopidogrel resistance associated SNPs.

  11. Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

    Directory of Open Access Journals (Sweden)

    Angela Filomena

    2017-10-01

    Full Text Available Infection with Helicobacter pylori (H. pylori occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231 and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99% and specificity (100%. Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2% demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

  12. Design of a multiplex PCR assay for the simultaneous detection and confirmation of Neisseria gonorrhoeae.

    LENUS (Irish Health Repository)

    O'Callaghan, Isabelle

    2010-05-01

    To improve the detection of Neisseria gonorrhoeae by designing a multiplex PCR assay using two N gonorrhoeae-specific genes as targets, thereby providing detection and confirmation of a positive result simultaneously.

  13. Development of a multiplex PCR assay detecting 52 autosomal SNPs

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Phillips, C.; Børsting, Claus

    2006-01-01

    for amplifying 52 genomic DNA fragments, each containing one SNP, in a single tube, and accurately genotyping the PCR product mixture using two single base extension reactions. This multiplex approach reduces the cost of SNP genotyping and requires as little as 0.5 ng of genomic DNA to detect 52 SNPs. We used...

  14. A multiplex ligation detection assay for the characterization of Salmonella enterica strains

    DEFF Research Database (Denmark)

    Aarts, Henk J.M.; Vos, Pieter; Larsson, Jonas T.

    2011-01-01

    A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube® microarray detection. The fea...... assessors that support bio-traceability models....

  15. A multiplex ligation detection assay for the characterization of Salmonella enterica strains

    NARCIS (Netherlands)

    Aarts, H.J.M.; Vos, P.; Larsson, J.T.; Hoek, van A.H.A.M.; Huehn, S.; Weijers, T.; Gronlund, H.A.; Malorny, B.

    2011-01-01

    A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube (R) microarray detection. The

  16. Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

    Science.gov (United States)

    Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

    2017-09-01

    The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

  17. Recombinant Helicobacter bilis Protein P167 for Mouse Serodiagnosis in a Multiplex Microbead Assay

    OpenAIRE

    Feng, Sunlian; Kendall, Lon V.; Hodzic, Emir; Wong, Scott; Lorenzana, Edward; Freet, Kimberly; Ku, Karin S.; Luciw, Paul A.; Barthold, Stephen W.; Khan, Imran H.

    2004-01-01

    Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated t...

  18. Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

    DEFF Research Database (Denmark)

    Lundberg, Martin; Thorsen, Stine Buch; Assarsson, Erika

    2011-01-01

    A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...... sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex PLA thereby converts multiple target analytes into real-time PCR amplicons that are individually quantificatied using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent...

  19. A 50 SNP-multiplex mass spectrometry assay for human identification

    DEFF Research Database (Denmark)

    Wächter, Andrea; Mengel-From, Jonas; Børsting, Claus

    2008-01-01

    We developed a 50 SNP-multiplex assay for detection on a MALDI-TOF MS platform based on the SNPs in the 52 SNP-multiplex assay recently developed by the SNPforID Consortium. After PCR amplification, the products were purified on Qiagen columns and used as templates in one single base extension (SBE...... primers were extended with biotin labelled ddNTPs and purified on avidin beads ensuring that only the extended SBE primers were isolated and spotted on the MALDI-TOF anchor target. Detection of the 50 extended primers from the SBE reaction was performed in a mass range between 3000 and 10,000 m/z...

  20. 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

  1. Utility of a Multiplex PCR Assay for Detecting Herpesvirus DNA in Clinical Samples

    Science.gov (United States)

    Druce, Julian; Catton, Mike; Chibo, Doris; Minerds, Kirsty; Tyssen, David; Kostecki, Renata; Maskill, Bill; Leong-Shaw, Wendy; Gerrard, Marie; Birch, Chris

    2002-01-01

    A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods. PMID:11980951

  2. A multiplex real-time PCR assay for routine diagnosis of bacterial vaginosis

    NARCIS (Netherlands)

    Kusters, J. G.; Reuland, E. A.; Bouter, S.; Koenig, P.; Dorigo-Zetsma, J. W.

    2015-01-01

    A semi-quantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (BV) was evaluated in a prospective study in a population of Dutch women with complaints of abnormal vaginal discharge. The PCR targets Gardnerella vaginalis, Atopobium vaginae, Megasphaera phylotype 1, Lactobacillus

  3. Evaluation of multiplex assay platforms for detection of influenza hemagglutinin subtype specific antibody responses.

    Science.gov (United States)

    Li, Zhu-Nan; Weber, Kimberly M; Limmer, Rebecca A; Horne, Bobbi J; Stevens, James; Schwerzmann, Joy; Wrammert, Jens; McCausland, Megan; Phipps, Andrew J; Hancock, Kathy; Jernigan, Daniel B; Levine, Min; Katz, Jacqueline M; Miller, Joseph D

    2017-05-01

    Influenza hemagglutination inhibition (HI) and virus microneutralization assays (MN) are widely used for seroprevalence studies. However, these assays have limited field portability and are difficult to fully automate for high throughput laboratory testing. To address these issues, three multiplex influenza subtype-specific antibody detection assays were developed using recombinant hemagglutinin antigens in combination with Chembio, Luminex ® , and ForteBio ® platforms. Assay sensitivity, specificity, and subtype cross-reactivity were evaluated using a panel of well characterized human sera. Compared to the traditional HI, assay sensitivity ranged from 87% to 92% and assay specificity in sera collected from unexposed persons ranged from 65% to 100% across the platforms. High assay specificity (86-100%) for A(H5N1) rHA was achieved for sera from exposed or unexposed to hetorosubtype influenza HAs. In contrast, assay specificity for A(H1N1)pdm09 rHA using sera collected from A/Vietnam/1204/2004 (H5N1) vaccinees in 2008 was low (22-30%) in all platforms. Although cross-reactivity against rHA subtype proteins was observed in each assay platform, the correct subtype specific responses were identified 78%-94% of the time when paired samples were available for analysis. These results show that high throughput and portable multiplex assays that incorporate rHA can be used to identify influenza subtype specific infections. Published by Elsevier B.V.

  4. A multiplex branched DNA assay for parallel quantitative gene expression profiling.

    Science.gov (United States)

    Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

    2006-05-01

    We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

  5. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  6. Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

    Science.gov (United States)

    Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

    2013-01-01

    Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

  7. A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection

    Directory of Open Access Journals (Sweden)

    Van Neste Leander

    2012-06-01

    Full Text Available Abstract Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP. The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

  8. Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens.

    Science.gov (United States)

    Wadowsky, R M; Michaels, R H; Libert, T; Kingsley, L A; Ehrlich, G D

    1996-11-01

    A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.

  9. 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.

  10. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset

    Directory of Open Access Journals (Sweden)

    Nayereh Nouri

    2014-01-01

    Full Text Available Background: The Duchenne muscular dystrophy (DMD gene is located in the short arm of the X chromosome (Xp21. It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA over multiplex polymerase chain reaction (PCR assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

  11. Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

    Science.gov (United States)

    Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael

    2009-01-01

    A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

  12. Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

    2017-02-21

    Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

  13. Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

    Science.gov (United States)

    Williamson, Charles H D; Vazquez, Adam J; Hill, Karen; Smith, Theresa J; Nottingham, Roxanne; Stone, Nathan E; Sobek, Colin J; Cocking, Jill H; Fernández, Rafael A; Caballero, Patricia A; Leiser, Owen P; Keim, Paul; Sahl, Jason W

    2017-09-15

    Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present ( ha positive [ ha + ] or orfX + ). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene ( bont ) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Since Bo

  14. Microsatellite multiplex assay for the coral-eating crown-of-thorns starfish, Acanthaster cf. planci

    KAUST Repository

    Harrison, Hugo B.

    2015-03-20

    Population outbreaks of crown-of-thorns starfish (Acanthaster spp.) represent one of the most significant biological disturbances on Indo-Pacific coral reefs. Here, we combine 15 published and 11 newly isolated polymorphic microsatellite markers from the coral-eating starfish, A. cf. planci and describe their integration into four multiplex PCRs. All markers were polymorphic with a mean of 11.7 ± 1.9 SE alleles per locus and an average observed heterozygosity of 0.619 ± 0.049 SE across 195 genotyped individuals from the Great Barrier Reef. This multiplex assay provides an effective means of investigating the population dynamics of crown-of-thorns starfish and the initiation and spread of population outbreaks.

  15. Microsatellite multiplex assay for the coral-eating crown-of-thorns starfish, Acanthaster cf. planci

    KAUST Repository

    Harrison, Hugo B.; Saenz Agudelo, Pablo; Al-Salamah, Manalle; Messmer, Vanessa; Pratchett, Morgan S.; Berumen, Michael L.

    2015-01-01

    Population outbreaks of crown-of-thorns starfish (Acanthaster spp.) represent one of the most significant biological disturbances on Indo-Pacific coral reefs. Here, we combine 15 published and 11 newly isolated polymorphic microsatellite markers from the coral-eating starfish, A. cf. planci and describe their integration into four multiplex PCRs. All markers were polymorphic with a mean of 11.7 ± 1.9 SE alleles per locus and an average observed heterozygosity of 0.619 ± 0.049 SE across 195 genotyped individuals from the Great Barrier Reef. This multiplex assay provides an effective means of investigating the population dynamics of crown-of-thorns starfish and the initiation and spread of population outbreaks.

  16. Detection of four important Eimeria species by multiplex PCR in a single assay.

    Science.gov (United States)

    You, Myung-Jo

    2014-06-01

    The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

    Science.gov (United States)

    Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

    2015-02-06

    Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

  18. Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

    Science.gov (United States)

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Leitolis, Amanda; Crestani, Sandra; Foti, Leonardo; de Souza, Wayner Vieira; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2017-10-01

    Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD. Copyright © 2017 American Society for Microbiology.

  19. Screening for Proteolytic Activities in Snake Venom by Means of a Multiplexing ESI-MS Assay Scheme

    NARCIS (Netherlands)

    Liesener, A.; Perchuc, Anna-Maria; Schöni, Reto; Wilmer, Marianne; Karst, U.

    2005-01-01

    A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional

  20. Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

    Science.gov (United States)

    Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

    2017-01-01

    Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

  1. Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay

    Science.gov (United States)

    Li, Li; Yan, Hong-Bin; Blair, David; Lei, Meng-Tong; Cai, Jin-Zhong; Fan, Yan-Lei; Li, Jian-Qiu; Fu, Bao-Quan; Yang, Yu-Rong; McManus, Donald P.; Jia, Wan-Zhong

    2015-01-01

    Background Infections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool is needed to facilitate the detection of infected hosts and for species identification. Methodology/Principal Findings A single-tube multiplex PCR assay was established to differentiate the Echinococcus species responsible for infections in intermediate and definitive hosts. Primers specific for E. granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR accurately detected Echinococcus DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp. Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the multiplex PCR was 100% when evaluated using DNA isolated from other cestodes. Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E. multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with larval E. multilocularis and E. granulosus, respectively. Conclusions/Significance The multiplex PCR developed in this study is an efficient tool for discriminating E. granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis using feces collected from these definitive hosts. It can also be used for the identification

  2. A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

    Science.gov (United States)

    Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

    2017-06-01

    For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

    Science.gov (United States)

    Petryayeva, Eleonora; Algar, W Russ

    2014-03-18

    Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.

  4. Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts.

    Science.gov (United States)

    Galinski, Sabrina; Wichert, Sven P; Rossner, Moritz J; Wehr, Michael C

    2018-05-25

    G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are implicated in the physiological regulation of many biological processes. The high diversity of GPCRs and their physiological functions make them primary targets for therapeutic drugs. For the generation of novel compounds, however, selectivity towards a given target is a critical issue in drug development as structural similarities between members of GPCR subfamilies exist. Therefore, the activities of multiple GPCRs that are both closely and distantly related to assess compound selectivity need to be tested simultaneously. Here, we present a cell-based multiplexed GPCR activity assay, termed GPCRprofiler, which uses a β-arrestin recruitment strategy and combines split TEV protein-protein interaction and EXT-based barcode technologies. This approach enables simultaneous measurements of receptor activities of multiple GPCR-ligand combinations by applying massively parallelized reporter assays. In proof-of-principle experiments covering 19 different GPCRs, both the specificity of endogenous agonists and the polypharmacological effects of two known antipsychotics on GPCR activities were demonstrated. Technically, normalization of barcode reporters across individual assays allows quantitative pharmacological assays in a parallelized manner. In summary, the GPCRprofiler technique constitutes a flexible and scalable approach, which enables simultaneous profiling of compound actions on multiple receptor activities in living cells.

  5. Multiplex PCR assay for simultaneous detection of six major bacterial pathogens of rice.

    Science.gov (United States)

    Cui, Z; Ojaghian, M R; Tao, Z; Kakar, K U; Zeng, J; Zhao, W; Duan, Y; Vera Cruz, C M; Li, B; Zhu, B; Xie, G

    2016-05-01

    The aim of this study was to develop a multiplex PCR (mPCR) assay for rapid, sensitive and simultaneous detection of six important rice pathogens: Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Pseudomonas fuscovaginae, Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae subsp. avenae. Specific primers were designed through a bioinformatics pipeline. Sensitivity of detection was established using both traditional PCR and quantitative real-time PCR on isolated DNA and on bacterial cells both in vitro and in simulated diseased seeds and the parameters were optimized for an mPCR assay. A total of 150 bacterial strains were tested for specificity. The mPCR assay accurately predicted the presence of pathogens among 44 symptomatic and asymptomatic rice seed, sheath and leaf samples. This study confirmed that this mPCR assay is a rapid, reliable and simple tool for the simultaneous detection of six important rice bacterial pathogens. This study is the first report of a method allowing simultaneous detection of six major rice pathogens. The ability to use crude extracts from plants without bacterial isolation or DNA extraction enhances the value of this mPCR technology for rapid detection and aetiological/epidemiological studies. © 2016 The Society for Applied Microbiology.

  6. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

    International Nuclear Information System (INIS)

    Tanner, Scott D.; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I.

    2007-01-01

    Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration

  7. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

    Energy Technology Data Exchange (ETDEWEB)

    Tanner, Scott D. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)], E-mail: sd.tanner@utoronto.ca; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)

    2007-03-15

    Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration.

  8. Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with INNO-LiPA HPV Genotyping Extra Assay▿

    OpenAIRE

    Else, Elizabeth A.; Swoyer, Ryan; Zhang, Yuhua; Taddeo, Frank J.; Bryan, Janine T.; Lawson, John; Van Hyfte, Inez; Roberts, Christine C.

    2011-01-01

    Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and H...

  9. 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.

  10. Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

    Energy Technology Data Exchange (ETDEWEB)

    Cary,; Bruce, R [Santa Fe, NM; Stubben, Christopher J [Los Alamos, NM

    2011-03-22

    The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

  11. Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

    Science.gov (United States)

    Inderbitzin, Patrik; Davis, R. Michael; Bostock, Richard M.; Subbarao, Krishna V.

    2013-01-01

    Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers. PMID:23823707

  12. Rapid detection and strain typing of Chlamydia trachomatis using a highly multiplexed microfluidic PCR assay.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available Nucleic acid amplification tests (NAATs are recommended by the CDC for detection of Chlamydia trachomatis (Ct urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types. Based on amplified fragment length polymorphisms, the assay differentiates LGV, ocular, urogenital, and proctocolitis clades, and also serovars L1, L2, and L3 within the LGV group. The assay was evaluated in a blinded fashion using 95 clinical swabs, with 76 previously reported as urogenital Ct-positive samples and typed by ompA genotyping and/or Multi-Locus Sequence Typing. Results of the 13-plex assay showed that 51 samples fell within urogenital clade 2 or 4, 24 samples showed both clade 2 and 4 signatures, indicating possible mixed infection, gene rearrangement, or inter-clade recombination, and one sample was a noninvasive trachoma biovar (either a clade 3 or 4. The remaining 19 blinded samples were correctly identified as LGV clade 1 (3, ocular clade 3 (4, or as negatives (12. To date, no NAAT assay can provide a point-of-care applicable turnaround time for Ct detection while identifying clinically significant Ct strain types to inform appropriate treatment. Coupled with rapid DNA processing of clinical swabs (approximately 60 minutes from swab-in to result-out, the assay has significant potential as a rapid POC diagnostic for Ct infections.

  13. Development of a multiplex polymerase chain reaction assay for simultaneous identification of human enterovirus 71 and coxsackievirus A16

    Science.gov (United States)

    Thao, Nguyen Thi Thanh; Ngoc, Nguyen Thi Kim; Tú, Phan Văn; Thúy, Trần Thi; Cardosa, Mary Jane; McMinn, Peter Charles; Phuektes, Patchara

    2010-01-01

    Human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are two major aetiological agents of hand, foot and mouth disease (HFMD) in children. Recently there have been several large outbreaks of HFMD in Vietnam and the Asia-Pacific region. In this study, a multiplex RT-PCR assay was developed in order to detect simultaneously HEV71, CVA16 and other human enteroviruses. Enterovirus detection was performed with a mixture of three pairs of oligonucleotide primers: one pair of published primers for amplifying all known enterovirus genomes and two new primer pairs specific for detection of the VP1 genes of HEV71 and CVA16. Enterovirus isolates, CVA16 and HEV71 strains identified previously from patients with HFMD were examined to evaluate the sensitivity and specificity of the multiplex RT-PCR assay. The assay was then applied to the direct detection of these viruses in clinical specimens obtained from HFMD cases identified at Children's Hospital Number 2, Ho Chi Minh City, Vietnam. The multiplex RT-PCR assay showed 100% specificity in screening for enteroviruses and in identifying HEV71 and CVA16. Similar results were obtained when using the multiplex RT-PCR assay to screen for enteroviruses and to identify HEV71 and CVA16 in clinical specimens obtained from HFMD cases identified at the hospital. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of HEV71 or CVA16 infection in cases of HFMD and is also potentially useful for molecular epidemiological investigations. PMID:20863857

  14. Evaluation of a multiplex real-time PCR assay for the detection of respiratory viruses in clinical specimens.

    Science.gov (United States)

    Rheem, Insoo; Park, Joowon; Kim, Tae-Hyun; Kim, Jong Wan

    2012-11-01

    In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea). Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses. The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%. The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR.

  15. A new multiplex assay of 17 autosomal STRs and Amelogenin for forensic application.

    Directory of Open Access Journals (Sweden)

    Suhua Zhang

    Full Text Available This paper describes a newly devised autosomal short tandem repeat (STR multiplex polymerase chain reaction (PCR systems for 17 autosomal loci (D1S1656, D2S441, D3S1358, D3S3045, D6S477, D7S3048, D8S1132, D10S1435, D10S1248, D11S2368, D13S325, D14S608, D15S659, D17S1290, D18S535, D19S253 and D22-GATA198B05 and Amelogenin. Primers for the loci were designed and optimized so that all of the amplicons were distributed from 50 base pairs (bp to less than 500 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. Strategies were developed to overcome challenges that encountered in creating the final assay. The limits of the multiplex were tested, resulting in the successful amplification of genomic DNA range from 0.25-4 ng with 30 PCR cycles. A total of 681 individuals from the Chinese Han population were studied and forensic genetic data were present. No significant deviations from Hardy-Weinberg equilibrium were observed. A total of 180 alleles were detected for the 17 autosomal STRs. The cumulative mean exclusion chance in duos (CMECD was 0.999967, and cumulative mean exclusion chance in trios (CMECT was 0.99999995. We conclude that the present 17plex autosomal STRs assay provides an additional powerful tool for forensic applications.

  16. A multiplex PCR mini-barcode assay to identify processed shark products in the global trade.

    Directory of Open Access Journals (Sweden)

    Diego Cardeñosa

    Full Text Available Protecting sharks from overexploitation has become global priority after widespread population declines have occurred. Tracking catches and trade on a species-specific basis has proven challenging, in part due to difficulties in identifying processed shark products such as fins, meat, and liver oil. This has hindered efforts to implement regulations aimed at promoting sustainable use of commercially important species and protection of imperiled species. Genetic approaches to identify shark products exist but are typically based on sequencing or amplifying large DNA regions and may fail to work on heavily processed products in which DNA is degraded. Here, we describe a novel multiplex PCR mini-barcode assay based on two short fragments of the cytochrome oxidase I (COI gene. This assay can identify to species all sharks currently listed on the Convention of International Trade of Endangered Species (CITES and most shark species present in the international trade. It achieves species diagnosis based on a single PCR and one to two downstream DNA sequencing reactions. The assay is capable of identifying highly processed shark products including fins, cooked shark fin soup, and skin-care products containing liver oil. This is a straightforward and reliable identification method for data collection and enforcement of regulations implemented for certain species at all governance levels.

  17. Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods.

    Science.gov (United States)

    Ali, Md Eaqub; Razzak, Md Abdur; Hamid, Sharifah Bee Abd; Rahman, Md Mahfujur; Amin, Md Al; Rashid, Nur Raifana Abd; Asing

    2015-06-15

    Food falsification has direct impact on public health, religious faith, fair-trades and wildlife. For the first time, here we described a multiplex polymerase chain reaction assay for the accurate identification of five meat species forbidden in Islamic foods in a single assay platform. Five pairs of species-specific primers were designed targeting mitochondrial ND5, ATPase 6, and cytochrome b genes to amplify 172, 163, 141, 129 and 108 bp DNA fragments from cat, dog, pig, monkey and rat meats, respectively. All PCR products were identified in gel-images and electrochromatograms obtained from Experion Bioanalyzer. Species-specificity checking against 15 important meat and fish and 5 plant species detected no cross-species amplification. Screening of target species in model and commercial meatballs reflected its application to detect target species in process foods. The assay was tested to detect 0.01-0.02 ng DNA under raw states and 1% suspected meats in meatball formulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. 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

  19. 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

  20. Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples

    Directory of Open Access Journals (Sweden)

    Whyte Paul

    2008-09-01

    Full Text Available Abstract Background A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS for 6 hours and subsequent DNA extraction. Results The real-time multiplex PCR assay and traditional cultural method showed 100% inclusivity and 100% exclusivity on all strains tested. The real-time multiplex PCR assay was as sensitive as the traditional cultural method in detecting Salmonella in artificially contaminated chicken samples and correctly identified the serotype. Artificially contaminated chicken samples resulted in a detection limit of between 1 and 10 CFU per 25 g sample for both methods. A total of sixty-three naturally contaminated chicken samples were investigated by both methods and relative accuracy, relative sensitivity and relative specificity of the real-time PCR method were determined to be 89, 94 and 87%, respectively. Thirty cultures blind tested were correctly identified by the real-time multiplex PCR method. Conclusion Real-time PCR methodology can contribute to meet the need for rapid identification and detection methods in food testing laboratories.

  1. High throughput multiplex real time PCR assay for the simultaneous quantification of DNA and RNA viruses infecting cassava plants

    OpenAIRE

    Otti, Gerald; Bouvaine, Sophie; Kimata, Bernadetha; Mkamillo, Geoffrey; Kumar, Lava; Tomlins, Keith; Maruthi, M.N.

    2016-01-01

    Aims: To develop a multiplex TaqMan-based real-time PCR assay (qPCR) for the simultaneous detection and quantification of both RNA and DNA viruses affecting cassava (Manihot esculenta) in eastern Africa.\\ud \\ud Methods and Results: The diagnostic assay was developed for two RNA viruses; Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) and two predominant DNA viruses; African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV), which cause t...

  2. OzPythonPlex: An optimised forensic STR multiplex assay set for the Australasian carpet python (Morelia spilota).

    Science.gov (United States)

    Ciavaglia, Sherryn; Linacre, Adrian

    2018-05-01

    Reptile species, and in particular snakes, are protected by national and international agreements yet are commonly handled illegally. To aid in the enforcement of such legislation, we report on the development of three 11-plex assays from the genome of the carpet python to type 24 loci of tetra-nucleotide and penta-nucleotide repeat motifs (pure, compound and complex included). The loci range in size between 70 and 550 bp. Seventeen of the loci are newly characterised with the inclusion of seven previously developed loci to facilitate cross-comparison with previous carpet python genotyping studies. Assays were optimised in accordance with human forensic profiling kits using one nanogram template DNA. Three loci are included in all three of the multiplex reactions as quality assurance markers, to ensure sample identity and genotyping accuracy is maintained across the three profiling assays. Allelic ladders have been developed for the three assays to ensure consistent and precise allele designation. A DNA reference database of allele frequencies is presented based on 249 samples collected from throughout the species native range. A small number of validation tests are conducted to demonstrate the utility of these multiplex assays. We suggest further appropriate validation tests that should be conducted prior to the application of the multiplex assays in criminal investigations involving carpet pythons. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Multiplex PCR assay discriminates rabbit, rat and squirrel meat in food chain.

    Science.gov (United States)

    Ahamad, Mohammad Nasir Uddin; Ali, Md Eaqub; Hossain, M A Motalib; Asing, Asing; Sultana, Sharmin; Jahurul, M H A

    2017-12-01

    Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.

  4. Multiplex polymerase chain reaction assay for the detection of minute virus of mice and mouse parvovirus infections in laboratory mice.

    Science.gov (United States)

    Wang, K W; Chueh, L L; Wang, M H; Huang, Y T; Fang, B H; Chang, C Y; Fang, M C; Chou, J Y; Hsieh, S C; Wan, C H

    2013-04-01

    Mouse parvoviruses are among the most prevalent infectious pathogens in contemporary mouse colonies. To improve the efficiency of routine screening for mouse parvovirus infections, a multiplex polymerase chain reaction (PCR) assay targeting the VP gene was developed. The assay detected minute virus of mice (MVM), mouse parvovirus (MPV) and a mouse housekeeping gene (α-actin) and was able to specifically detect MVM and MPV at levels as low as 50 copies. Co-infection with the two viruses with up to 200-fold differences in viral concentrations can easily be detected. The multiplex PCR assay developed here could be a useful tool for monitoring mouse health and the viral contamination of biological materials.

  5. 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.

  6. Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer

    Science.gov (United States)

    Jalal Kiani, Seyed; Shatizadeh Malekshahi, Somayeh; Yousefi Ghalejoogh, Zohreh; Ghavvami, Nastaran; Shafiei Jandaghi, Nazanin Zahra; Shahsiah, Reza; Jahanzad, Isa; Yavarian, Jila

    2015-01-01

    Background: Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs. Objectives: The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay. Patients and Methods: Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails. Results: Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%. Conclusions: The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner. PMID:26865940

  7. A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus

    NARCIS (Netherlands)

    E. Ghaznavi Rad (Ehsanollah); N.S. Mariana (Nor Shamsudin); Z. Sekawi (Zamberi); A.F. van Belkum (Alex); V. Neela (Vasanthakumari)

    2010-01-01

    textabstractA multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus

  8. 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.

  9. A Multiplexed Assay That Monitors Effects of Multiple Compound Treatment Times Reveals Candidate Immune-Enhancing Compounds.

    Science.gov (United States)

    Zhao, Ziyan; Henowitz, Liza; Zweifach, Adam

    2018-05-01

    We previously developed a flow cytometry assay that monitored lytic granule exocytosis in cytotoxic T lymphocytes stimulated by contacting beads coated with activating anti-CD3 antibodies. That assay was multiplexed in that responses of cells that did or did not receive the activating stimulus were distinguished via changes in light scatter accompanying binding of cells to beads, allowing us to discriminate compounds that activate responses on their own from compounds that enhance responses in cells that received the activating stimulus, all within a single sample. Here we add a second dimension of multiplexing by developing means to assess in a single sample the effects of treating cells with test compounds for different times. Bar-coding cells before adding them to test wells lets us determine compound treatment time while also monitoring activation status and response amplitude at the point of interrogation. This multiplexed assay is suitable for screening 96-well plates. We used it to screen compounds from the National Cancer Institute, identifying several compounds that enhance anti-LAMP1 responses. Multiple-treatment-time (MTT) screening enabled by bar-coding and read via high-throughput flow cytometry may be a generally useful method for facilitating the discovery of compounds of interest.

  10. Development of silicon photonic microring resonator biosensors for multiplexed cytokine assays and in vitro diagnostics

    Science.gov (United States)

    Luchansky, Matthew Sam

    In order to guide critical care therapies that are personalized to a patient's unique disease state, a diagnostic or theranostic medical device must quickly provide a detailed biomolecular understanding of disease onset and progression. This detailed molecular understanding of cellular processes and pathways requires the ability to measure multiple analytes in parallel. Though many traditional sensing technologies for biomarker analysis and fundamental biological studies (i.e. enzyme-linked immunosorbent assays, real-time polymerase chain reaction, etc.) rely on single-parameter measurements, it has become increasingly clear that the inherent complexity of many human illnesses and pathways necessitates quantitative and multiparameter analysis of biological samples. Currently used analytical methods are deficient in that they often provide either highly quantitative data for a single biomarker or qualitative data for many targets, but methods that simultaneously provide highly quantitative analysis of many targets have yet to be adequately developed. Fields such as medical diagnostics and cellular biology would benefit greatly from a technology that enables rapid, quantitative and reproducible assays for many targets within a single sample. In an effort to fill this unmet need, this doctoral dissertation describes the development of a clinically translational biosensing technology based on silicon photonics and developed in the chemistry research laboratory of Ryan C. Bailey. Silicon photonic microring resonators, a class of high-Q optical sensors, represent a promising platform for rapid, multiparameter in vitro measurements. The original device design utilizes 32-ring arrays for real-time biomolecular sensing without fluorescent labels, and these optical biosensors display great potential for more highly multiplexed (100s-1000s) measurements based on the impressive scalability of silicon device fabrication. Though this technology can be used to detect a variety of

  11. Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

    International Nuclear Information System (INIS)

    Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.; Selvapandiyan, Angamuthu; Hewlett, Indira; Duncan, Robert; Puri, Raj K.; Nakhasi, Hira L.; Kaplan, Gerardo G.

    2007-01-01

    Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients

  12. Clinical utility of an optimised multiplex real-time PCR assay for the identification of pathogens causing sepsis in Vietnamese patients

    Directory of Open Access Journals (Sweden)

    Ngo Tat Trung

    2018-02-01

    Conclusion: The combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients.

  13. Virus reactivations after autologous hematopoietic stem cell transplantation detected by multiplex PCR assay.

    Science.gov (United States)

    Inazawa, Natsuko; Hori, Tsukasa; Nojima, Masanori; Saito, Makoto; Igarashi, Keita; Yamamoto, Masaki; Shimizu, Norio; Yoto, Yuko; Tsutsumi, Hiroyuki

    2017-02-01

    Several studies have indicated that viral reactivations following allogeneic hematopoietic stem cell transplantation (allo-HSCT) are frequent, but viral reactivations after autologous HSCT (auto-HSCT) have not been investigated in detail. We performed multiplex polymerase chain reaction (PCR) assay to examine multiple viral reactivations simultaneously in 24 patients undergoing auto-HSCT between September 2010 and December 2012. Weekly whole blood samples were collected from pre- to 42 days post-HSCT, and tested for the following 13 viruses; herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), HHV-7, HHV-8, adeno virus (ADV), BK virus (BKV), JC virus (JCV), parvovirus B19 (B19V), and hepatitis B virus (HBV).  Fifteen (63%) patients had at least one type of viral reactivation. HHV6 (n = 10; 41.7%) was most frequently detected followed by EBV (n = 7; 29.2%). HHV-6 peaked on day 21 after HSCT and promptly declined. In addition, HBV, CMV, HHV7, and B19V were each detected in one patient. HHV6 reactivation was detected in almost half the auto-HSCT patients, which was similar to the incidence in allo-HSCT patients. The incidence of EBV was unexpectedly high. Viral infections in patients undergoing auto-HSCT were higher than previously reported in other studies. Although there were no particular complications of viral infection, we should pay attention to possible viral reactivations in auto-HSCT patients. J. Med. Virol. 89:358-362, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Comparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay

    Directory of Open Access Journals (Sweden)

    Dabisch-Ruthe Mareike

    2012-07-01

    Full Text Available Abstract Background A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. Methods The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP, were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens. Results To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml and RSV (103 copies/ml. The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found. The incidence of respiratory viruses was compared in tracheal secretion (TS samples (n = 100 of mechanically ventilated patients in winter (n = 50 and summer (n = 50. In winter, respiratory viruses were detected in 32 TS samples (64% by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32% and PIV-2 (20%. Multiple infections were detected

  15. Southeast Economic Add-on 1997

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — To collect data on an angler's last trip for revealed preference models and economic valuation purposes. Typically done as an add-on to the MRIP intercept survey and...

  16. Southeast Economic Add-on 2004

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — To collect data on an angler's last trip for revealed preference models and economic valuation purposes. Typically done as an add-on to the MRIP intercept survey and...

  17. Firefox add-ons for medical reference.

    Science.gov (United States)

    Hoy, Matthew B

    2010-07-01

    Firefox is a Web browser created by the Mozilla project, an open-source software group. Features of the browser include automated updates, advanced security and standards compliance, and the ability to add functionality through add-ons and extensions. First introduced in 2004, Firefox now accounts for roughly 30% of the browser market. This article will focus primarily on add-ons and extensions available for the browser that are useful to medical researchers.

  18. Detection of selected antibiotic resistance genes using multiplex PCR assay in mastitis pathogens in the Czech Republic

    Directory of Open Access Journals (Sweden)

    Vladimir Pyatov

    2017-01-01

    Full Text Available The aim of this research was to develop multiplex polymerase chain reaction assays for the detection of aminoglycoside (strA, strB, sulphonamide (sulI, sulII, tetracycline (tetA, tetB, tetK, tetM, tetO, macrolide and lincosamide (msrA, ermA, ermB, ermC, mefA/E genes of resistance in mastitis pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae. Applying the established assays, we investigated the distribution of antibiotic resistance genes in the above mentioned species isolated from milk samples in the Czech Republic. Each assay consisted of seven pairs of primers. Six of them amplified fragments of antibiotic resistance genes and one pair a fragment of a species specific gene. Polymerase chain reaction conditions were optimized to amplify seven gene fragments simultaneously in one reaction. In total, 249 isolates were used, among which 111 were positive for E. coli, 52 for S. aureus and 86 for Streptococcus spp. The majority (60.2% of bacteria carried at least one antibiotic resistance gene and 44.6% were multidrug-resistant. The designed multiplex polymerase chain reaction assays may be applied as diagnostic method to replace or complement standard techniques of antibiotic susceptibility testing in the mentioned pathogens.

  19. 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.

  20. Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

    Science.gov (United States)

    Yazdi, Soroush H; Giles, Kristen L; White, Ian M

    2013-11-05

    We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive

  1. 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

  2. Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines.

    Science.gov (United States)

    Košir, Alexandra Bogožalec; Spilsberg, Bjørn; Holst-Jensen, Arne; Žel, Jana; Dobnik, David

    2017-08-17

    Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.

  3. A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment

    Directory of Open Access Journals (Sweden)

    Tian Ding

    2017-05-01

    Full Text Available This study firstly developed a multiplex real-time PCR (RT-PCR technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus, Listeria monocytogenes (L. monocytogenes and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water in one reaction. Brain heart infusion (BHI broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

  4. Validation of the performance of a GMO multiplex screening assay based on microarray detection

    NARCIS (Netherlands)

    Leimanis, S.; Hamels, S.; Naze, F.; Mbongolo, G.; Sneyers, M.; Hochegger, R.; Broll, H.; Roth, L.; Dallmann, K.; Micsinai, A.; Dijk, van J.P.; Kok, E.J.

    2008-01-01

    A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct

  5. Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in Hypervariable Regions.

    Science.gov (United States)

    Aoki, Kimiko; Tanaka, Hiroyuki; Kawahara, Takashi

    2018-07-01

    The standard method for personal identification and verification of urine samples in doping control is short tandem repeat (STR) analysis using nuclear DNA (nDNA). The DNA concentration of urine is very low and decreases under most conditions used for sample storage; therefore, the amount of DNA from cryopreserved urine samples may be insufficient for STR analysis. We aimed to establish a multiplexed assay for urine mitochondrial DNA typing containing only trace amounts of DNA, particularly for Japanese populations. A multiplexed suspension-array assay using oligo-tagged microspheres (Luminex MagPlex-TAG) was developed to measure C-stretch length in hypervariable region 1 (HV1) and 2 (HV2), five single nucleotide polymorphisms (SNPs), and one polymorphic indel. Based on these SNPs and the indel, the Japanese population can be classified into five major haplogroups (D4, B, M7a, A, D5). The assay was applied to DNA samples from urine cryopreserved for 1 - 1.5 years (n = 63) and fresh blood (n = 150). The assay with blood DNA enabled Japanese subjects to be categorized into 62 types, exhibiting a discriminatory power of 0.960. The detection limit for cryopreserved urine was 0.005 ng of nDNA. Profiling of blood and urine pairs revealed that 5 of 63 pairs showed different C-stretch patterns in HV1 or HV2. The assay described here yields valuable information in terms of the verification of urine sample sources employing only trace amounts of recovered DNA. However, blood cannot be used as a reference sample.

  6. Clinical Application of a Multiplex Real-Time PCR Assay for Simultaneous Detection of Legionella Species, Legionella pneumophila, and Legionella pneumophila Serogroup 1

    OpenAIRE

    Benitez, Alvaro J.; Winchell, Jonas M.

    2014-01-01

    We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.

  7. A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections.

    Science.gov (United States)

    Díaz de Arce, Heidy; Pérez, Lester J; Frías, Maria T; Rosell, Rosa; Tarradas, Joan; Núñez, José I; Ganges, Llilianne

    2009-11-18

    Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5' non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89TCID(50). The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.

  8. Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay.

    Science.gov (United States)

    Das, S; Pingle, M R; Muñoz-Jordán, J; Rundell, M S; Rondini, S; Granger, K; Chang, G-J J; Kelly, E; Spier, E G; Larone, D; Spitzer, E; Barany, F; Golightly, L M

    2008-10-01

    The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

  9. Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays

    DEFF Research Database (Denmark)

    Iftner, Thomas; Germ, Liesje; Swoyer, Ryan

    2009-01-01

    methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay...... (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons...... were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example...

  10. The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay.

    Science.gov (United States)

    Hilton, Hugo G; Moesta, Achim K; Guethlein, Lisbeth A; Blokhuis, Jeroen; Parham, Peter; Norman, Paul J

    2015-10-01

    Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR-Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR-Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Development of a multiplex PCR assay for rapid and simultaneous detection of four genera of fish pathogenic bacteria.

    Science.gov (United States)

    Zhang, D F; Zhang, Q Q; Li, A H

    2014-11-01

    Species of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus are the most common fish pathogenic bacteria that cause economically devastating losses in aquaculture. A multiplex polymerase chain reaction (mPCR) was developed for the simultaneous detection and differentiation of the four genera of fish pathogenic bacteria. Through the use of genus-specific primers instead of species-specific ones, the current mPCR covered much more target bacterial species compared with previously reported species-specific mPCR methods. The specificity of the four putative genus-specific primers was validated experimentally while used exclusively (uniplex PCR) or combined (mPCR) against bacterial genomic DNA templates of the target bacteria and nontarget bacteria. The PCR amplicons for the following genera were obtained as expected: Aeromonas (875 bp), Vibrio (524 bp), Edwardsiella (302 bp) and Streptococcus (197 bp), and the fragments could be separated clearly on the agarose gel electrophoresis. The mPCR did not produce nonspecific amplification products when used to amplify 21 nontarget species of bacteria. The mPCR detection limits for each target bacterial genera were 50 colony-forming units (CFU) in pure culture and 100 CFU in fish tissue samples. In conclusion, the mPCR assay was proven to be a powerful alternative to the conventional culture-based method, given its rapid, specific, sensitive and reliable detection of target pathogens. The fish pathogenic bacteria of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus-specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The mPCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species-specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This m

  12. 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.

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

    Science.gov (United States)

    Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

    2018-01-01

    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. PMID:29425124

  14. Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal.

    Science.gov (United States)

    Bluhm, B H; Flaherty, J E; Cousin, M A; Woloshuk, C P

    2002-12-01

    The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6 gene involved in trichothecene biosynthesis and the FUM5 gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearum and at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearum and its differentiation from F. verticillioides were accomplished prior to visible fungal growth at cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industry's effort to monitor quality and safety.

  15. A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

    Science.gov (United States)

    Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

    2011-12-01

    Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Results of Multiplex Polymerase Chain Reaction Assay to Identify Urethritis Pathogens

    Directory of Open Access Journals (Sweden)

    Mehmet Sarıer

    2017-03-01

    Full Text Available Objective: The purpose of this study was to evaluate the results of multiplex polymerase chain reaction (PCR test applied to identify the pathogens in male patients who attended our urology clinic with a pre-diagnosis of urethritis related with sexual intercourse. Materials and Methods: In this study, we included a total of 91 male patients, who sought medical advice in our clinic between August 2015 and October 2016 due to complaints of urethral discharge, dysuria and urethral itching, having a visible urethral discharge during the physical examination or a positive leukocyte esterase test (Combur-Test®-Roche in the first urine sample. In the urethral swab samples of these patients, urethritis pathogens were searched with a multiplex PCR test. The multiplex PCR kit, which is able to identify nine pathogens and produced by PathoFinder® (Holland, was used in the process. The pathogens that could be detected by the kit were Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Gardnerella vaginalis, Trichomonas vaginalis, Treponema pallidum, and Candida albicans. Results: The average age of the subjects was 35.1 (19-57 years. Sixty one out of 91 patients (67% were found to have a pathogen in the urethral swab sample. In 45 patients (49.4%, only one pathogen, in 12 (13.1% - two different pathogens and in 4 (4.3% patients, 3 different pathogens were detected. The pathogens found were as follows: Ureaplasma urealyticum in 22 patients (27.1%, Gardnerella vaginalis in 15 (18.6%, Neisseria gonorrhoeae in 13 (16.1%, Mycoplasma genitalium (10 patients; 12.3%, Mycoplasma hominis (8 patients; 9.9%, Chlamydia trachomatis (8 patients; 9.9%, Trichomonas vaginalis (3 patients; 3.8%, and Candida albicans (2 patients; 2.4%. None of the patients were identified with Treponema pallidum. None of the pathogens were identified in 30 patients (32.9% whose samples were examined by PCR method. Conclusion

  17. A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Ghaznavi-Rad, Ehsanollah; Nor Shamsudin, Mariana; Sekawi, Zamberi; van Belkum, Alex; Neela, Vasanthakumari

    2010-10-01

    A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.

  18. Fuzzy-logic based strategy for validation of multiplex methods: example with qualitative GMO assays.

    Science.gov (United States)

    Bellocchi, Gianni; Bertholet, Vincent; Hamels, Sandrine; Moens, W; Remacle, José; Van den Eede, Guy

    2010-02-01

    This paper illustrates the advantages that a fuzzy-based aggregation method could bring into the validation of a multiplex method for GMO detection (DualChip GMO kit, Eppendorf). Guidelines for validation of chemical, bio-chemical, pharmaceutical and genetic methods have been developed and ad hoc validation statistics are available and routinely used, for in-house and inter-laboratory testing, and decision-making. Fuzzy logic allows summarising the information obtained by independent validation statistics into one synthetic indicator of overall method performance. The microarray technology, introduced for simultaneous identification of multiple GMOs, poses specific validation issues (patterns of performance for a variety of GMOs at different concentrations). A fuzzy-based indicator for overall evaluation is illustrated in this paper, and applied to validation data for different genetically modified elements. Remarks were drawn on the analytical results. The fuzzy-logic based rules were shown to be applicable to improve interpretation of results and facilitate overall evaluation of the multiplex method.

  19. Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

    Science.gov (United States)

    Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

    2014-01-01

    A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

  20. Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

    Directory of Open Access Journals (Sweden)

    Maria Frølund

    Full Text Available A novel multiplex quantitative real-time polymerase chain reaction (qPCR for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

  1. Use of multiplex polymerase chain reaction-based assay to conduct epidemiological studies on bovine hemoparasites in Mexico.

    Science.gov (United States)

    Figueroa, J V; Alvarez, J A; Ramos, J A; Vega, C A; Buening, G M

    1993-01-01

    A study was conducted to test the applicability of a Polymerase Chain Reaction (PCR)-based approach for the simultaneous detection of the bovine hemoparasites Babesia bigemina, B. bovis and Anaplasma marginale. Bovine blood samples from cattle ranches of a previously determined enzootic zone in the Yucatan Peninsula of Mexico, were collected from peripheral blood and processed for PCR analysis. Blood samples were subjected to DNA amplification by placing an aliquot in a reaction tube containing oligonucleotide primers specific for DNA of each hemoparasite species. The PCR products were detected by Dot-Blot nucleic acid hybridization utilizing nonradioactive, species-specific, digoxigenin PCR-labeled DNA probes. Four hundred twenty one field samples analyzed by the multiplex PCR-DNA probe assay showed 66.7%, 60.1% and 59.6% prevalence rates for B. bigemina, B. bovis and A. marginale, respectively. The multiplex PCR analysis showed that animals with single, double or triple infection could be detected with the parasite specific DNA probes. The procedure is proposed as a valuable tool for the epidemiological analysis in regions where the hemoparasite species are concurrently infecting cattle.

  2. 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.

  3. A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine.

    Science.gov (United States)

    Erickson, A; Fisher, M; Furukawa-Stoffer, T; Ambagala, A; Hodko, D; Pasick, J; King, D P; Nfon, C; Ortega Polo, R; Lung, O

    2018-04-01

    Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional

  4. Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Fernandez, Jovita

    2005-01-01

    In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed......-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating...... probes. The limits of detection ware found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid...

  5. An analytical approach to reduce between-plate variation in multiplex assays that measure antibodies to Plasmodium falciparum antigens.

    Science.gov (United States)

    Fang, Rui; Wey, Andrew; Bobbili, Naveen K; Leke, Rose F G; Taylor, Diane Wallace; Chen, John J

    2017-07-17

    Antibodies play an important role in immunity to malaria. Recent studies show that antibodies to multiple antigens, as well as, the overall breadth of the response are associated with protection from malaria. Yet, the variability and reliability of antibody measurements against a combination of malarial antigens using multiplex assays have not been well characterized. A normalization procedure for reducing between-plate variation using replicates of pooled positive and negative controls was investigated. Sixty test samples (30 from malaria-positive and 30 malaria-negative individuals), together with five pooled positive-controls and two pooled negative-controls, were screened for antibody levels to 9 malarial antigens, including merozoite antigens (AMA1, EBA175, MSP1, MSP2, MSP3, MSP11, Pf41), sporozoite CSP, and pregnancy-associated VAR2CSA. The antibody levels were measured in triplicate on each of 3 plates, and the experiments were replicated on two different days by the same technician. The performance of the proposed normalization procedure was evaluated with the pooled controls for the test samples on both the linear and natural-log scales. Compared with data on the linear scale, the natural-log transformed data were less skewed and reduced the mean-variance relationship. The proposed normalization procedure using pooled controls on the natural-log scale significantly reduced between-plate variation. For malaria-related research that measure antibodies to multiple antigens with multiplex assays, the natural-log transformation is recommended for data analysis and use of the normalization procedure with multiple pooled controls can improve the precision of antibody measurements.

  6. A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

    Science.gov (United States)

    Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

    2017-03-01

    Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with type 2 (as of April 2016), laboratories will need to conform to much more stringent laboratory biosafety regulations when handling live poliovirus strains. In this study, a poliovirus binding inhibition multiplex immunoassay (polio MIA) using inactivated poliovirus vaccine (IPV-Salk) was developed for simultaneous quantification of serum antibodies directed to all three poliovirus types. Our assay shows a good correlation with the NT and an excellent correlation with the ELISA-based binding inhibition assay (POBI). The assay is highly type-specific and reproducible. Additionally, serum sample throughput increases about fivefold relative to NT and POBI and the amount of serum needed is reduced by more than 90%. In conclusion, the polio MIA can be used as a safe and high throughput application, especially for large-scale surveillance and vaccine studies, reducing laboratory time and serum amounts needed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach.

    Science.gov (United States)

    Yu, Y; Zhao, Z; Jiang, D; Wu, Z; Li, S

    2013-10-01

    A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282 bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously. The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach. © 2013 The Society for Applied Microbiology.

  8. Real-Time, Fast Neutron Coincidence Assay of Plutonium With a 4-Channel Multiplexed Analyzer and Organic Scintillators

    Science.gov (United States)

    Joyce, Malcolm J.; Gamage, Kelum A. A.; Aspinall, M. D.; Cave, F. D.; Lavietes, A.

    2014-06-01

    The design, principle of operation and the results of measurements made with a four-channel organic scintillator system are described. The system comprises four detectors and a multiplexed analyzer for the real-time parallel processing of fast neutron events. The function of the real-time, digital multiple-channel pulse-shape discrimination analyzer is described together with the results of laboratory-based measurements with 252Cf, 241Am-Li and plutonium. The analyzer is based on a single-board solution with integrated high-voltage supplies and graphical user interface. It has been developed to meet the requirements of nuclear materials assay of relevance to safeguards and security. Data are presented for the real-time coincidence assay of plutonium in terms of doubles count rate versus mass. This includes an assessment of the limiting mass uncertainty for coincidence assay based on a 100 s measurement period and samples in the range 0-50 g. Measurements of count rate versus order of multiplicity for 252Cf and 241Am-Li and combinations of both are also presented.

  9. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

    Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

  10. On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

    Science.gov (United States)

    Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

    2013-07-25

    A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples

    Directory of Open Access Journals (Sweden)

    Jiyun Li

    2017-10-01

    Full Text Available The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 102 cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry.

  12. Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order

    Science.gov (United States)

    G. J. Bilodeau; F. N. Martin; M. D. Coffey; C. L. Blomquist

    2014-01-01

    A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed...

  13. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.

    NARCIS (Netherlands)

    Quint, K.D.; Doorn, L.J. van; Kleter, B.; Koning, M.N. de; Munckhof, H.A. van den; Morre, S.A.; Harmsel, B. ter; Weiderpass, E.; Harbers, G.; Melchers, W.J.G.; Quint, W.G.V.

    2007-01-01

    Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT

  14. RHD genotype and zygosity analysis in the Chinese Southern Han D plus , D- and D variant donors using the multiplex ligation-dependent probe amplification assay

    NARCIS (Netherlands)

    Ji, Y. L.; Luo, H.; Wen, J. Z.; Haer-Wigman, L.; Veldhuisen, B.; Wei, L.; Wang, Z.; Ligthart, P.; Lodén-van Straaten, M.; Fu, Y. S.; van der Schoot, C. E.; Luo, G. P.

    2017-01-01

    Background and ObjectivesSeveral comprehensive genotyping platforms for determining red blood cell (RBC) antigens have been established and validated for use in the Caucasian and Black populations, but not for the Chinese. The multiplex ligation-dependent probe amplification (MLPA) assay was

  15. Comprehensive genotyping for 18 blood group systems using a multiplex ligation-dependent probe amplification assay shows a high degree of accuracy

    NARCIS (Netherlands)

    Haer-Wigman, Lonneke; Ji, Yanli; Lodén, Martin; de Haas, Masja; van der Schoot, C. Ellen; Veldhuisen, Barbera

    2013-01-01

    In recent years genotyping methods have been implemented in blood banks as alternative to comprehensive serologic typing. We evaluated a newly developed assay for convenient and comprehensive genotyping of blood group alleles based on multiplex ligation-dependent probe amplification (MLPA)

  16. Clinical utility of an optimised multiplex real-time PCR assay for the identification of pathogens causing sepsis in Vietnamese patients.

    Science.gov (United States)

    Tat Trung, Ngo; Van Tong, Hoang; Lien, Tran Thi; Van Son, Trinh; Thanh Huyen, Tran Thi; Quyen, Dao Thanh; Hoan, Phan Quoc; Meyer, Christian G; Song, Le Huu

    2018-02-01

    For the identification of bacterial pathogens, blood culture is still the gold standard diagnostic method. However, several disadvantages apply to blood cultures, such as time and rather large volumes of blood sample required. We have previously established an optimised multiplex real-time PCR method in order to diagnose bloodstream infections. In the present study, we evaluated the diagnostic performance of this optimised multiplex RT-PCR in blood samples collected from 110 septicaemia patients enrolled at the 108 Military Central Hospital, Hanoi, Vietnam. Positive results were obtained by blood culture, the Light Cylcler-based SeptiFast ® assay and our multiplex RT-PCR in 35 (32%), 31 (28%), and 31 (28%) samples, respectively. Combined use of the three methods confirmed 50 (45.5%) positive cases of bloodstream infection, a rate significantly higher compared to the exclusive use of one of the three methods (P=0.052, 0.012 and 0.012, respectively). The sensitivity, specificity and area under the curve (AUC) of our assay were higher compared to that of the SeptiFast ® assay (77.4%, 86.1% and 0.8 vs. 67.7%, 82.3% and 0.73, respectively). Combined use of blood culture and multiplex RT-PCR assay showed a superior diagnostic performance, as the sensitivity, specificity, and AUC reached 83.3%, 100%, and 0.95, respectively. The concordance between blood culture and the multiplex RT-PCR assay was highest for Klebsiella pneumonia (100%), followed by Streptococcus spp. (77.8%), Escherichia coli (66.7%), Staphylococcus spp. (50%) and Salmonella spp. (50%). In addition, the use of the newly established multiplex RT-PCR assay increased the spectrum of identifiable agents (Acintobacter baumannii, 1/32; Proteus mirabilis, 1/32). The combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients. Copyright © 2017 The

  17. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    Energy Technology Data Exchange (ETDEWEB)

    Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  18. Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

    Science.gov (United States)

    Baker, Harold N.; Murphy, Robin; Lopez, Erica; Garcia, Carlos

    2012-01-01

    The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results. In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample. Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and

  19. Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs.

    Science.gov (United States)

    Ambroise, Jérôme; Butoescu, Valentina; Robert, Annie; Tombal, Bertrand; Gala, Jean-Luc

    2015-06-25

    Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex

  20. Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip.

    Science.gov (United States)

    Smoldovskaya, Olga; Feyzkhanova, Guzel; Arefieva, Alla; Voloshin, Sergei; Ivashkina, Olga; Reznikov, Yuriy; Rubina, Alla

    2016-01-01

    Immunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay. Multiplex fluorescent immunoassay of 139 patients' blood serum samples was carried out using biological microchips (biochips). sIgE concentrations for the chosen allergens and their recombinant components were measured. ROC analysis was used for comparison of the results and determination of diagnostic accuracy. The results for the birch pollen extract and its recombinant allergens have shown that the diagnostic accuracy of the methods utilizing the whole allergen extract, its major component Bet v 1 and the combination of major and minor components (Bet v 1 and Bet v 2) was the same. Values for diagnostic accuracy for the cat dander extract and its major recombinant component Fel d 1 were equal. In contrast with birch pollen and cat dander allergens, using of recombinant components of timothy grass pollen (Phl p 1, Phl p 5, Phl p 7 and Phl p 12) did not allow reaching the diagnostic accuracy of using natural extract. Multiplex analysis of samples obtained from patients with allergy to birch pollen and cat dander using biological microchips has shown that comparable accuracy was observed for the assay with natural extracts and recombinant allergens. In the case of timothy grass allergen, using the recombinant components may be insufficient.

  1. A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella enterica serotype Typhimurium

    Directory of Open Access Journals (Sweden)

    Brisabois Anne

    2011-06-01

    Full Text Available Abstract Background Typhimurium is the main serotype of Salmonella enterica subsp. enterica implicated in food-borne diseases worldwide. This study aimed to detect the prevalence of ten markers combined in a macro-array based on multiplex real-time PCR. We targeted characteristic determinants located on pathogenicity islands (SPI-2 to -5, virulence plasmid pSLT and Salmonella genomic island 1 (SGI1 as well as a specific 16S-23S rRNA intergenic spacer sequence of definitive type 104 (DT104. To investigate antimicrobial resistance, the study also targeted the presence of genes involved in sulfonamide (sul1 and beta-lactam (blaTEM resistance. Finally, the intI1 determinant encoding integrase from class 1 integron was also investigated. Results A total of 538 unrelated S. Typhimurium strains isolated between 1999 and 2009 from various sources, including food animals, food products, human and environmental samples were studied. Based on the combined presence or absence of these markers, we distinguished 34 different genotypes, including three major genotypes encountered in 75% of the studied strains, Although SPI determinants were almost always detected, SGI1, intI1, sul1 and blaTEM determinants were found 47%, 52%, 54% and 12% of the time respectively, varying according to isolation source. Low-marker patterns were most often detected in poultry sources whereas full-marker patterns were observed in pig, cattle and human sources. Conclusion The GeneDisc® assay developed in this study madeit easier to explore variability within serotype Typhimurium by analyzing ten relevant gene determinants in a large collection of strains. This real-time multiplex method constitutes a valuable tool for strains characterization on epidemiological purposes.

  2. Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens.

    Science.gov (United States)

    Qu, X S; Wanner, L A; Christ, B J

    2011-03-01

    To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Real-time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real-time PCR assay. The multiplex real-time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C(t) values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. This multiplex real-time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  3. A high throughput single nucleotide polymorphism multiplex assay for parentage assignment in New Zealand sheep.

    Directory of Open Access Journals (Sweden)

    Shannon M Clarke

    Full Text Available Accurate pedigree information is critical to animal breeding systems to ensure the highest rate of genetic gain and management of inbreeding. The abundance of available genomic data, together with development of high throughput genotyping platforms, means that single nucleotide polymorphisms (SNPs are now the DNA marker of choice for genomic selection studies. Furthermore the superior qualities of SNPs compared to microsatellite markers allows for standardization between laboratories; a property that is crucial for developing an international set of markers for traceability studies. The objective of this study was to develop a high throughput SNP assay for use in the New Zealand sheep industry that gives accurate pedigree assignment and will allow a reduction in breeder input over lambing. This required two phases of development--firstly, a method of extracting quality DNA from ear-punch tissue performed in a high throughput cost efficient manner and secondly a SNP assay that has the ability to assign paternity to progeny resulting from mob mating. A likelihood based approach to infer paternity was used where sires with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non-parentage are assigned. An 84 "parentage SNP panel" was developed that assigned, on average, 99% of progeny to a sire in a problem where there were 3,000 progeny from 120 mob mated sires that included numerous half sib sires. In only 6% of those cases was there another sire with at least a 0.02 probability of paternity. Furthermore dam information (either recorded, or by genotyping possible dams was absent, highlighting the SNP test's suitability for paternity testing. Utilization of this parentage SNP assay will allow implementation of progeny testing into large commercial farms where the improved accuracy of sire assignment and genetic evaluations will increase genetic gain in the sheep industry.

  4. One-Step Multiplex RT-qPCR Assay for the detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

    International Nuclear Information System (INIS)

    Settypalli, T.B.K.; Lamien, C.; Spergser, J.; Lelenta, M.; Wade, A.; Gelaye, E.; Loitsch, A.; Minoungou, G.; Thiaucourt, F.; Diallo, A.

    2016-01-01

    Full text: Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in

  5. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp. capripneumoniae.

    Directory of Open Access Journals (Sweden)

    Tirumala Bharani Kumar Settypalli

    Full Text Available Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV, Peste de petits ruminants virus (PPRV, Pasteurella multocida (PM and Mycoplasma capricolum ssp. capripneumonia (Mccp in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in

  6. Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.

    Science.gov (United States)

    Li, Baoguang; Liu, Huanli; Wang, Weimin

    2017-11-09

    Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency. The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s. The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella

  7. An automated multiplex specific IgE assay system using a photoimmobilized microarray.

    Science.gov (United States)

    Ito, Yoshihiro; Moritsugu, Nozomi; Matsue, Takahisa; Mitsukoshi, Kiyomi; Ayame, Hirohito; Okochi, Norihiko; Hattori, Hideshi; Tashiro, Hideo; Sato, Sakura; Ebisawa, Motohiro

    2012-11-15

    An automated microarray diagnostic system for specific IgE using photoimmobilized allergen has been developed. Photoimmobilization is useful for preparing microarrays, where various types of biological components are covalently immobilized on a plate. Because the immobilization is based on a photo-induced radical cross-linking reaction, it does not require specific functional groups on the immobilized components. Here, an aqueous solution of a photoreactive poly(ethylene glycol)-based polymer was spin-coated on a plate, and an aqueous solution of each allergen was microspotted on the coated plate and allowed to dry in air. Finally, the plate was irradiated with an ultraviolet lamp for covalent immobilization. An automated machine using these plates was developed for the assay of antigen-specific IgE. Initially, the patient serum was added to the microarray plate, and after reaction of the microspotted allergen with IgE, the adsorbed IgE was detected by a peroxidase-conjugated anti-IgE-antibody. The chemical luminescence intensity of the substrate decomposed by the peroxidase was automatically detected using a sensitive charge-coupled device camera. All the allergens were immobilized stably using this method, which was used to screen for allergen-specific IgE. The results were comparable with those using conventional specific IgE. Using this system, six different allergen-specific IgE were assayed using 10 μL of serum within a period of 20 min. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

    Science.gov (United States)

    Liu, Yichun; Lee, May-Ann; Ooi, Eng-Eong; Mavis, Yeo; Tan, Ai-Ling; Quek, Hung-Hiang

    2003-09-01

    A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

  9. Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

    Science.gov (United States)

    Bray, Mark-Anthony; Singh, Shantanu; Han, Han; Davis, Chadwick T.; Borgeson, Blake; Hartland, Cathy; Kost-Alimova, Maria; Gustafsdottir, Sigrun M.; Gibson, Christopher C.; Carpenter, Anne E.

    2016-01-01

    In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks. PMID:27560178

  10. Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

    Science.gov (United States)

    Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

    2010-07-01

    Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

  11. Systematic Dissection of Sequence Elements Controlling σ70 Promoters Using a Genomically-Encoded Multiplexed Reporter Assay in E. coli.

    Science.gov (United States)

    Urtecho, Guillaume; Tripp, Arielle D; Insigne, Kimberly; Kim, Hwangbeom; Kosuri, Sriram

    2018-02-01

    Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In E. coli , decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange (RMCE) system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations. We build and test a library of 10,898 σ70 promoter variants consisting of all combinations of a set of eight -35 elements, eight -10 elements, three UP elements, eight spacers, and eight backgrounds. We find that the -35 and -10 sequence elements can explain approximately 74% of the variance in promoter strength within our dataset using a simple log-linear statistical model. Neural network models can explain greater than 95% of the variance in our dataset, and show the increased power is due to nonlinear interactions of other elements such as the spacer, background, and UP elements.

  12. Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Feroze Ahmed Ganaie

    2015-01-01

    Full Text Available Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment] in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.

  13. Alteration in sample preparation to increase the yield of multiplex Polymerase Chain Reaction assay for diagnosis of genital ulcer disease

    Directory of Open Access Journals (Sweden)

    G Rao

    2013-01-01

    Full Text Available Purpose: Genital Ulcer Disease (GUD is common sexually transmitted infection (STI. Multiple studies have shown that GUDs are strongly associated with the transmission and the acquisition of HIV infection. An accurate diagnosis of common etiology of GUD namely Herpes, syphilis and Chancroid is possible using Multiplex PCR (M-PCR. However, frequent presence of Polymerase Chain Reaction inhibitors in the ulcer swab specimen limits the performance of the assay. In order to overcome this problem, alternative specimen preparation method was used. Materials and Methods: To determine the common etiology, GUD specimens obtained under an STI operations research study were tested with M-PCR after the samples were prepared using Roche Amplicor specimen preparation kit. PCR inhibiting samples were identified from that, which showed negative results. These samples were subjected to phenol-chloroform extraction and ethanol precipitation before the conduct of M-PCR on them. Results: Of the 237 GUD specimens tested, in 145 etiologies could be detected, whereas 92 samples were found negative. Further spiking with one of the target DNA, 128 of the negative samples were found to contain the inhibitors. These 126 samples were then subjected to phenol chloroform extraction and ethanol precipitation followed by M-PCR. Using this method for sample preparation, etiology could be determined in 46 (23% additional samples. This success rate of altered sample preparation method has been lower than that has reported. Conclusion: The results indicate that sample preparation using phenol chloroform extraction and ethanol precipitation, prior to M-PCR helps to eliminate the inhibitors and increase the yield of the assay. However, being a laborious procedure, it may be used for samples giving negative results after the screening by Roche Amplicor specimen preparation kit.

  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. Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India

    Directory of Open Access Journals (Sweden)

    Anita Chakravarti

    2016-01-01

    Full Text Available Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary-care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR. Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. Results: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.

  16. Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes

    Science.gov (United States)

    TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time PCR. One tube contained a primer-probe set specific for G. pallida (pale cyst nematode) multiplexed with another prim...

  17. Impact of radiotherapy on PBMCs DNA repair capacity - Use of a multiplexed functional repair assay

    International Nuclear Information System (INIS)

    Sauvaigo, S.; Sarrazy, F.; Breton, J.; Caillat, S.; Chapuis, V.

    2012-01-01

    Radiation therapy is an essential part of cancer treatment as about 50% of patients will receive radiations at least once. Significant broad variation in radiosensitivity has been demonstrated in patients. About 5-10% of patients develop acute toxicity after radiotherapy. Therefore there is a need for the identification of markers able to predict the occurrence of adverse effects and thus adapt the radiotherapy regimen for radiosensitive patients. As a first step toward this goal, and considering the DNA repair defects associated with hypersensitivity radiation syndromes, we investigated the DNA repair phenotype of patients receiving radiotherapy. More precisely, we used a functional repair assay on support to follow the evolution of the glycosylases/AP endonuclease activities of PBMCs extracts of a series of patients during the time course of radiotherapy. For each patient, we collected one PBMCs sample before the first radiotherapy application (S1) and three samples after (S2 to S4) (one day and one week after application 1, and one at the end of the radiotherapy protocol). These four samples have been analysed for 11 donors. Clustering analyses of the results demonstrated a great heterogeneity of responses among the patients. Interestingly, this heterogeneity decreased between S1 and S4 where only 2 classes of patients remained if we except one patient that exhibited an atypical DNA repair phenotype. Furthermore, we showed that repair of several oxidized bases significantly increased between S1 and S3 or S4 (8oxoG, thymine glycol, A paired with 8oxoG), suggesting an adaptation of patients repair systems to the oxidative stress generated by the ionising radiations. Our preliminary results provided evidence that the DNA repair phenotype was impacted by the radiotherapy regimen. Further characterization of patients with known repair defects are needed to determine if atypical repair phenotypes could be associated with radiotherapy complications. Finally

  18. Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

    Science.gov (United States)

    Park, Chul Min; Lee, Kyunghoon; Jun, Sun-Hee; Song, Sang Hoon; Song, Junghan

    2017-08-15

    Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R 2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting.

    Directory of Open Access Journals (Sweden)

    Jermaine Khumalo

    Full Text Available Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children.We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis.From 292 samples, bacterial DNA was detected in 12 (4.1% and viral nucleic acids in 94 (32%. Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10% of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR.In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation.

  20. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Fichorova, Raina N., E-mail: rfichorova@rics.bwh.harvard.edu [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chandra, Neelima; Doncel, Gustavo F. [CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA (United States)

    2015-06-15

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  1. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    International Nuclear Information System (INIS)

    Fichorova, Raina N.; Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan; Chandra, Neelima; Doncel, Gustavo F.

    2015-01-01

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  2. Development of multiplex real-time PCR assay for the detection of Brucella spp., Leptospira spp. and Campylobacter foetus

    Directory of Open Access Journals (Sweden)

    Abdelfattah M. Selim

    2014-12-01

    Full Text Available Abortion among dairy cattle is one of the major causes of economic losses in the livestock industry. This study describes a 1-step multiplex real-time polymerase chain reaction (PCR to detect Brucella spp., Leptospira spp. and Campylobacter foetus, these are significant bacteria commonly implicated in bovine abortion. ß-actin was added to the same PCR reaction as an internal control to detect any extraction failure or PCR inhibition. The detection limit of multiplex real-time PCR using purified DNA from cultured organisms was set to 5 fg for Leptospira spp. and C. foetus and to 50 fg for Brucella spp. The multiplex real-time PCR did not produce any non-specific amplification when tested with different strains of the 3 pathogens. This multiplex real-time PCR provides a valuable tool for diagnosis, simultaneous and rapid detection for the 3 pathogens causing abortion in bovine.

  3. High-throughput multiplex real-time PCR assay for the simultaneous quantification of DNA and RNA viruses infecting cassava plants.

    Science.gov (United States)

    Otti, G; Bouvaine, S; Kimata, B; Mkamillo, G; Kumar, P L; Tomlins, K; Maruthi, M N

    2016-05-01

    To develop a multiplex TaqMan-based real-time PCR assay (qPCR) for the simultaneous detection and quantification of both RNA and DNA viruses affecting cassava (Manihot esculenta) in eastern Africa. The diagnostic assay was developed for two RNA viruses; Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) and two predominant DNA viruses; African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV), which cause the economically important cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) respectively. Our method, developed by analysing PCR products of viruses, was highly sensitive to detect target viruses from very low quantities of 4-10 femtograms. Multiplexing did not diminish sensitivity or accuracy compared to uniplex alternatives. The assay reliably detected and quantified four cassava viruses in field samples where CBSV and UCBSV synergy was observed in majority of mixed-infected varieties. We have developed a high-throughput qPCR diagnostic assay capable of specific and sensitive quantification of predominant DNA and RNA viruses of cassava in eastern Africa. The qPCR methods are a great improvement on the existing methods and can be used for monitoring virus spread as well as for accurate evaluation of the cassava varieties for virus resistance. © 2016 The Society for Applied Microbiology.

  4. Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples.

    Science.gov (United States)

    Wang, Dennis Y; Chang, Chien-Wei; Lagacé, Robert E; Oldroyd, Nicola J; Hennessy, Lori K

    2011-07-01

    The AmpFℓSTR(®) Identifiler(®) Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA(®) card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay's sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA(®) cards, and the assay's specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA(®) substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler(®) Direct Kit for forensic standards and database samples genotyping. © 2011 American Academy of Forensic Sciences.

  5. Development and Validation of a Multiplex PCR-Based Assay for the Upper Respiratory Tract Bacterial Pathogens Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis.

    Science.gov (United States)

    Post; White; Aul; Zavoral; Wadowsky; Zhang; Preston; Ehrlich

    1996-06-01

    Background: Conventional simplex polymerase chain reaction (PCR)-based assays are limited in that they only provide for the detection of a single infectious agent. Many clinical diseases, however, present in a nonspecific, or syndromic, fashion, thereby necessitating the simultaneous assessment of multiple pathogens. Panel-based molecular diagnostic testing can be accomplished by the development of multiplex PCR-based assays, which can detect, individually or severally, different pathogens that are associated with syndromic illness. As part of a larger program of panel development, an assay that can simultaneously detect Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis was developed. These organisms were chosen as they are the most common bacterial pathogens associated with both the acute and chronic forms of otitis media; they are also responsible for a high percentage of sinus infections in both children and adults. In addition, H. influenzae and S. pneumoniae are commonly associated with septic meningitits. Methods and Results: Multiple individual PCR-based assays were developed for each of the three target organisms which were then evaluated for sensitivity and specificity. Utilizing the simplex assays that met our designated performance criteria, a matrix style approach was used to develop a duplex H. influenzae-S. pneumoniae assay. The duplex assay was then used as a single component in the development of a triplex assay, wherein the various M. catarrhalis primer-probe sets were tested for compatibility with the existing assay. A single-step PCR protocol, with species-specific primers for each of the three target organisms and a liquid hybridization-gel retardation amplimer detection system, was developed, which amplifies and then discriminates among each of the amplification products according to size. This assay is able to detect all three organisms in a specific manner, either individually or severally. Dilutional experiments

  6. Multiplex Real-Time PCR Assay Targeting Eight Parasites Customized to the Korean Population: Potential Use for Detection in Diarrheal Stool Samples from Gastroenteritis Patients.

    Directory of Open Access Journals (Sweden)

    Eun Jeong Won

    Full Text Available Intestinal parasitic diseases occur worldwide and can cause diarrhea or gastroenteritis; however, their diagnosis is quite difficult, especially in low-endemism countries. We developed a multiplex real-time PCR assay for detection of eight intestinal parasites and prospectively evaluated it for patients with gastroenteritis. The assay targeted Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, Blastocystis hominis, Dientamoeba fragilis, Clonorchis sinensis, Metagonimus yokogawai, and Gymnophalloides seoi. Performance characteristics were evaluated based on recovery after DNA extraction, analytical sensitivity, specificity, reproducibility, cross-reactivity, and interference characteristics. Clinical performance was validated against microscopy on 123 diarrheal samples. The assay demonstrated strong correlations between DNA concentrations and Ct values (R2, 0.9924-0.9998, and had a high PCR efficiency (83.3%-109.5%. Polymerase chain reactions detected as few as 10-30 copies of genomic DNA, and coefficient of variance was 0-7%. There was no cross-reactivity to the other 54 microorganisms tested. Interference occurred only in presence of high concentrations of erythrocytes or leukocytes. This assay had a higher correct identification rate (100.0% vs. 90.2% and lower incorrect ID rate (0.0% vs. 9.8% when compared to microscopy. Overall, this assay showed a higher sensitivity (100.0%; 95% confidence interval [CI] of 80.5-100.0 than microscopy (29.4%; 95% CI 10.31-55.96, and the specificity levels were comparable for both methods (100.0%; 95% CI 96.58-100.0. This newly developed multiplex real-time PCR assay offers a potential use for detecting intestinal parasitic pathogens customized to the Korean population.

  7. A novel, multiplexed, probe-based quantitative PCR assay for the soybean root- and stem-rot pathogen, Phytophthora sojae, utilizes its transposable element.

    Science.gov (United States)

    Haudenshield, James S; Song, Jeong Y; Hartman, Glen L

    2017-01-01

    Phytophthora root rot of soybean [Glycine max (L.) Merr.] is caused by the oomycete Phytophthora sojae (Kaufm. & Gerd.). P. sojae has a narrow host range, consisting primarily of soybean, and it is a serious pathogen worldwide. It exists in root and stem tissues as mycelium, wherein it can form oospores which subsequently germinate to release motile, infectious zoospores. Molecular assays detecting DNA of P. sojae are useful in disease diagnostics, and for determining the presence of the organism in host tissues, soils, and runoff or ponded water from potentially infested fields. Such assays as published have utilized ITS sequences from the nuclear ribosomal RNA genes in conventional PCR or dye-binding quantitative PCR (Q-PCR) but are not amenable to multiplexing, and some of these assays did not utilize control strategies for type I or type II errors. In this study, we describe primers and a bifunctional probe with specificity to a gypsy-like retroelement in the P. sojae genome to create a fluorogenic 5'-exonuclease linear hydrolysis assay, with a multiplexed internal control reaction detecting an exogenous target to validate negative calls, and with uracil-deglycosylase-mediated protection against carryover contamination. The assay specifically detected 13 different P. sojae isolates, and excluded 17 other Phytophthora species along with 20 non-Phytophthora fungal and oomycete species pathogenic on soybean. A diagnostic limit of detection of 34 fg total P. sojae DNA was observed in serial dilutions, equivalent to 0.3 genome, and a practical detection sensitivity of four zoospores per sample was achieved, despite losses during DNA extraction.

  8. A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in Korean ready-to-eat food.

    Science.gov (United States)

    Lee, Nari; Kwon, Kyung Yoon; Oh, Su Kyung; Chang, Hyun-Joo; Chun, Hyang Sook; Choi, Sung-Wook

    2014-07-01

    A multiplex polymerase chain reaction (PCR) assay was developed for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in various Korean ready-to-eat foods. The six specific primer pairs for multiplex PCR were selected based on the O157 antigen (rfbE) gene of E. coli O157:H7, the DNA gyrase subunit B (gyrB) gene of B. cereus, the toxin regulatory protein (toxR) gene of V. parahaemolyticus, the invasion protein A (invA) gene of Salmonella spp., the hemolysin (hly) gene of L. monocytogenes, and the thermonuclease (nuc) gene of S. aureus. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity assays for multiplex primer pairs were investigated by testing different strains. When this multiplex PCR assay was applied to evaluate the validity of detecting six foodborne pathogens in artificially inoculated several ready-to-eat food samples, the assay was able to specifically simultaneously detect as few as 1 colony-forming unit/mL of each pathogen after enrichment for 12 h. Their presence in naturally contaminated samples also indicates that the developed multiplex PCR assay is an effective and informative supplement for practical use.

  9. Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

    KAUST Repository

    Gangwar, Maulshree; Waters, Alicia M.; Bej, Gautam A.; Bej, Asim K.; Mojib, Nazia

    2012-01-01

    A SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23. 6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84. 27 ± 1. 7 °C; spvB, 88. 76 ± 1. 0 °C; and 16S rRNA gene, 87. 16 ± 0. 8 °C). The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health. © 2012 Springer Science+Business Media, LLC.

  10. Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

    KAUST Repository

    Gangwar, Maulshree

    2012-09-23

    A SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23. 6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84. 27 ± 1. 7 °C; spvB, 88. 76 ± 1. 0 °C; and 16S rRNA gene, 87. 16 ± 0. 8 °C). The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health. © 2012 Springer Science+Business Media, LLC.

  11. Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles.

    Science.gov (United States)

    Lee, Jong-Hwan; Seo, Hyuk Seong; Kwon, Jung-Hyuk; Kim, Hee-Tae; Kwon, Koo Chul; Sim, Sang Jun; Cha, Young Joo; Lee, Jeewon

    2015-07-15

    Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65-90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

    Science.gov (United States)

    Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

    2018-01-01

    The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  13. The current incidence of viral disease in korean sweet potatoes and development of multiplex rt-PCR assays for simultaneous detection of eight sweet potato viruses.

    Science.gov (United States)

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

    2014-12-01

    Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

  14. Development of a multiplex PCR assay for fine-scale population genetic analysis of the Komodo monitor Varanus komodoensis based on 18 polymorphic microsatellite loci.

    Science.gov (United States)

    Ciofi, Claudio; Tzika, Athanasia C; Natali, Chiara; Watts, Phillip C; Sulandari, Sri; Zein, Moch S A; Milinkovitch, Michel C

    2011-05-01

    Multiplex PCR assays for the coamplification of microsatellite loci allow rapid and cost-effective genetic analyses and the production of efficient screening protocols for international breeding programs. We constructed a partial genomic library enriched for di-nucleotide repeats and characterized 14 new microsatellite loci for the Komodo monitor (or Komodo dragon, Varanus komodoensis). Using these novel microsatellites and four previously described loci, we developed multiplex PCR assays that may be loaded on a genetic analyser in three separate panels. We tested the novel set of microsatellites for polymorphism using 69 individuals from three island populations and evaluated the resolving power of the entire panel of 18 loci by conducting (i) a preliminary assignment test to determine population(s) of origin and (ii) a parentage analysis for 43 captive Komodo monitors. This panel of polymorphic loci proved useful for both purposes and thus can be exploited for fine-scale population genetic analyses and as part of international captive breeding programs directed at maintaining genetically viable ex situ populations and reintroductions. © 2011 Blackwell Publishing Ltd.

  15. The Current Incidence of Viral Disease in Korean Sweet Potatoes and Development of Multiplex RT-PCR Assays for Simultaneous Detection of Eight Sweet Potato Viruses

    Directory of Open Access Journals (Sweden)

    Hae-Ryun Kwak

    2014-12-01

    Full Text Available Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV and sweet potato virus C (SPVC were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1, Sweet potato virus G (SPVG, Sweet potato leaf curl virus (SPLCV, Sweet potato virus 2 ( SPV2, Sweet potato chlorotic fleck virus (SPCFV, and Sweet potato latent virus (SPLV were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1 in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

  16. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Science.gov (United States)

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  17. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Directory of Open Access Journals (Sweden)

    Cengiz Ikten

    Full Text Available Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.. Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  18. Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

    Science.gov (United States)

    Singh, Chandra K; Ojha, Abhishek; Bhatanagar, Raj K; Kachru, Devendra N

    2008-01-01

    Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory

  19. Multiplex real-time PCR assay for the detection of extended-spectrum β-lactamase and carbapenemase genes using melting curve analysis.

    Science.gov (United States)

    Singh, Prashant; Pfeifer, Yvonne; Mustapha, Azlin

    2016-05-01

    Real-time PCR melt curve assays for the detection of β-lactamase, extended-spectrum β-lactamase and carbapenemase genes in Gram-negative bacteria were developed. Two multiplex real-time PCR melt curve assays were developed for the detection of ten common β-lactamase genes: blaKPC-like, blaOXA-48-like, blaNDM-like, blaVIM-like, blaIMP-like, blaCTX-M-1+2-group, blaCMY-like, blaACC-like, blaSHV-like and blaTEM-like. The assays were evaluated using 25 bacterial strains and 31 DNA samples (total n=56) comprising different Enterobacteriaceae genera and Pseudomonas spp. These strains were previously characterized at five research institutes. Each resistance gene targeted in this study generated a non-overlapping and distinct melt curve peak. The assay worked effectively and detected the presence of additional resistance genes in 23 samples. The assays developed in this study offer a simple, low cost method for the detection of prevalent β-lactamase, ESBL and carbapenemase genes among Gram-negative pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. A multiplex, internally controlled real-time PCR assay for detection of toxigenic Clostridium difficile and identification of hypervirulent strain 027/ST-1

    DEFF Research Database (Denmark)

    Hoegh, A M; Nielsen, J B; Lester, A

    2012-01-01

    The purpose of this study was to validate a multiplex real-time PCR assay capable of detecting toxigenic Clostridium difficile and simultaneously identifying C. difficile ribotype 027/ST-1 by targeting the toxin genes tcdA, tcdB and cdtA in one reaction and in a separate reaction identifying the Δ...... to confirm the correct identification of the Δ117 deletion in tcdC and C. difficile ribotype 027/ST-1, respectively. The PCR assay displayed a sensitivity, specificity, PPV and NPV of 99.0%, 97.4%, 87.4% and 99.8%, respectively, compared to toxigenic culture on 665 samples evaluable both by PCR and culture....... Sequencing of tcdC, ribotyping and MLST of cultured isolates validated the genotyping assay and confirmed the ability of the assay to correctly identify C. difficile ribotype 027/ST-1 in our current epidemiological setting. We describe the use of a combination of two separate PCR assays for sensitive...

  1. Non-invasive beamforming add-on module

    KAUST Repository

    Bader, Ahmed; Alouini, Mohamed-Slim

    2017-01-01

    An embodiment of a non-invasive beamforming add-on apparatus couples to an existing antenna port and rectifies the beam azimuth in the upstream and downstream directions. The apparatus comprises input circuitry that is configured to receive one

  2. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    Directory of Open Access Journals (Sweden)

    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

  3. Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA.

    Science.gov (United States)

    Song, Y; Kato, N; Liu, C; Matsumiya, Y; Kato, H; Watanabe, K

    2000-06-15

    Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

  4. Evaluation of the Roche LightMix Gastro parasites multiplex PCR assay detecting Giardia duodenalis, Entamoeba histolytica, cryptosporidia, Dientamoeba fragilis, and Blastocystis hominis.

    Science.gov (United States)

    Friesen, J; Fuhrmann, J; Kietzmann, H; Tannich, E; Müller, M; Ignatius, R

    2018-03-23

    Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystishominis, and Dientamoebafragilis with routine laboratory procedures. Stool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR. D. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27). The data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  5. Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

    Science.gov (United States)

    Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

    2016-03-01

    HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

  7. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-01-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

  8. Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

    Science.gov (United States)

    Wu, Qingqing; Xiang, Shengnan; Wang, Wenjun; Zhao, Jinyan; Xia, Jinhua; Zhen, Yueran; Liu, Bang

    2018-05-01

    Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2  > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

  9. Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Noor, M Omair; Krull, Ulrich J

    2013-08-06

    A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

  10. Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

    Science.gov (United States)

    Hill, Ryan C; Oman, Trent J; Shan, Guomin; Schafer, Barry; Eble, Julie; Chen, Cynthia

    2015-08-26

    Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

  11. Development of a multiplex real-time PCR assay for detection of human enteric viruses other than norovirus using samples collected from gastroenteritis patients in Fukui Prefecture, Japan.

    Science.gov (United States)

    Kowada, Kazuaki; Takeuchi, Kenji; Hirano, Eiko; Toho, Miho; Sada, Kiyonao

    2018-01-01

    There are many varieties of gastroenteritis viruses, of which norovirus (NoV) accounts for over 90% of the viral food poisoning incidents in Japan. However, protocols for rapidly identifying other gastroenteritis viruses need to be established to investigate NoV-negative cases intensively. In this study, a multiplex real-time PCR assay targeting rotavirus A, rotavirus C, sapovirus, astrovirus, adenovirus, and enterovirus was developed using stool samples collected from gastroenteritis patients between 2010 and 2013 in Fukui Prefecture, Japan. Of the 126 samples collected sporadically from pediatric patients with suspected infectious gastroenteritis, 51 were positive for non-NoV target viruses, whereas 27 were positive for NoV, showing a high prevalence of non-NoV viruses in pediatric patients. In contrast, testing in 382 samples of 58 gastroenteritis outbreaks showed that non-NoV viruses were detected in 13 samples, with NoV in 267. Of the 267 NoV-positive patients, only two were co-infected with non-NoV target viruses, suggesting that testing for non-NoV gastroenteritis viruses in NoV-positive samples was mostly unnecessary in outbreak investigations. Given these results, multiplex real-time PCR testing for non-NoV gastroenteritis viruses, conducted separately from NoV testing, may be helpful to deal with two types of epidemiological investigations, regular surveillance of infectious gastroenteritis and urgent testing when gastroenteritis outbreaks occur. © 2017 Wiley Periodicals, Inc.

  12. An investigation of genital ulcers in Jackson, Mississippi, with use of a multiplex polymerase chain reaction assay: high prevalence of chancroid and human immunodeficiency virus infection.

    Science.gov (United States)

    Mertz, K J; Weiss, J B; Webb, R M; Levine, W C; Lewis, J S; Orle, K A; Totten, P A; Overbaugh, J; Morse, S A; Currier, M M; Fishbein, M; St Louis, M E

    1998-10-01

    In 1994, an apparent outbreak of atypical genital ulcers was noted by clinicians at the sexually transmitted disease clinic in Jackson, Mississippi. Of 143 patients with ulcers tested with a multiplex polymerase chain reaction (PCR) assay, 56 (39%) were positive for Haemophilus ducreyi, 44 (31%) for herpes simplex virus, and 27 (19%) for Treponema pallidum; 12 (8%) were positive for > 1 organism. Of 136 patients tested for human immunodeficiency virus (HIV) by serology, 14 (10%) were HIV-seropositive, compared with none of 200 patients without ulcers (P chancroid were significantly more likely than male patients without ulcers to report sex with a crack cocaine user, exchange of money or drugs for sex, and multiple sex partners. The strong association between genital ulcers and HIV infection in this population highlights the urgency of preventing genital ulcers in the southern United States.

  13. Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S M; Danganan, L; Tammero, L; Vitalis, B; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed candidate multiplexed assays that may potentially be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the ability to improve our nation's capability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect food and agricultural resources with a diagnostic test which could enhance the nation's capabilities for early detection of a foreign animal disease. In FY2005 with funding from the DHS, LLNL developed the first version (Version 1.0) of a multiplexed (MUX) nucleic-acid-based RT-PCR assay that included signatures for foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases (FADs) of swine, Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease Virus (SVDV), and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus [BPSV], Orf of sheep, and Pseudocowpox). In FY06, LLNL has developed Bovine and Porcine species-specific panel which included existing signatures from Version 1.0 panel as well as new signatures. The MUX RT-PCR porcine assay for detection of FMDV includes the FADs, VESV and SVD in addition to vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome (PRRS). LLNL has also developed a MUX RT-PCR bovine assay for detection of FMDV with rule out tests for the two bovine FADs malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis

  14. A novel one-step real-time multiplex PCR assay to detect Streptococcus agalactiae presence and serotypes Ia, Ib, and III.

    Science.gov (United States)

    Furfaro, Lucy L; Chang, Barbara J; Payne, Matthew S

    2017-09-01

    Streptococcus agalactiae is the leading cause of early-onset neonatal sepsis. Culture-based screening methods lack the sensitivity of molecular assays and do not indicate serotype; a potentially important virulence marker. We aimed to develop a multiplex PCR to detect S. agalactiae while simultaneously identifying serotypes Ia, Ib, and III; commonly associated with infant disease. Primers were designed to target S. agalactiae serotype-specific cps genes and the dltS gene. The assay was validated with 512 vaginal specimens from pregnant women. 112 (21.9%) were dltS positive, with 14.3%, 0.9%, and 6.3% of these identified as cps Ia, Ib, and III, respectively. Our assay is a specific and sensitive method to simultaneously detect S. agalactiae and serotypes Ia, Ib, and III in a single step. It is of high significance for clinical diagnostic applications and also provides epidemiological data on serotype, information that may be important for vaccine development and other targeted non-antibiotic therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. A Multiplex PCR Assay for Differentiating Coconut Rhinoceros Beetle (Coleoptera: Scarabaeidae) From Oriental Flower Beetle (Coleoptera: Scarabaeidae) in Early Life Stages and Excrement.

    Science.gov (United States)

    Watanabe, S; Melzer, M J

    2017-04-01

    The coconut rhinoceros beetle, Oryctes rhinoceros (L.), is a major pest of coconut and other palm trees. An incipient coconut rhinoceros beetle population was recently discovered on the island of Oahu, Hawaii and is currently the target of a large, mutiagency eradication program. Confounding this program is the widespread presence of another scarab beetle on Oahu, the oriental flower beetle, Protaetia orientalis (Gory and Percheron 1833). Eggs, early life stages, and fecal excrement of coconut rhinoceros beetle and oriental flower beetle are morphologically indistinguishable, thereby creating uncertainty when such specimens are discovered in the field. Here, we report the development of a multiplex PCR assay targeting cytochrome oxidase I of coconut rhinoceros beetle and oriental flower beetle that can rapidly detect and distinguish between these insects. This assay also features an internal positive control to ensure DNA of sufficient quantity and quality is used in the assay, increasing its reliability and reducing the chances of false negative results. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Data on serologic inflammatory biomarkers assessed using multiplex assays and host characteristics in the Multicenter AIDS Cohort Study (MACS

    Directory of Open Access Journals (Sweden)

    Heather S. McKay

    2016-12-01

    Full Text Available This article contains data on the associations between fixed and modifiable host characteristics and twenty-three biomarkers of inflammation and immune activation measured longitudinally in a cohort of 250 HIV-uninfected men from the Multicenter AIDS Cohort Study (1984–2009 after adjusting for age, study site, and blood draw time of day using generalized gamma regression. This article also presents associations between each biomarker and each host characteristic in a sample restricted to 2001–2009. These data are supplemental to our original research article entitled “Host factors associated with serologic inflammatory markers assessed using multiplex assays” (McKay, S. Heather, Bream, H. Jay, Margolick, B. Joseph, Martínez-Maza, Otoniel, Phair, P. John, Rinaldo, R. Charles, Abraham, G. Alison, L.P. Jacobson, 2016 [1].

  17. PC add on card for processing of LSC signals

    International Nuclear Information System (INIS)

    Jadhav, S.R.; Nikhare, D.M.; Gurna, R.K.; Paulson, Molly; Kulkarni, C.P.; Vaidya, P.P.

    2001-01-01

    This paper describes PC- add on card developed at Electronics Division for processing of LSC signals. This card uses highly integrated digital and analog circuits, for entire processing of signals available from preamplifiers to get complete beta energy spectrum corresponding to coincident events in Liquid Scintillation Counting. LSC card along with High Voltage PC-add on card gives complete electronics required for LSC system. This card is also used in automatic LSC system along with interface circuits, which are used to control mechanical movements. (author)

  18. Large-scale multiplex polymerase chain reaction assay for diagnosis of viral reactivations after allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Inazawa, Natsuko; Hori, Tsukasa; Hatakeyama, Naoki; Yamamoto, Masaki; Yoto, Yuko; Nojima, Masanori; Suzuki, Nobuhiro; Shimizu, Norio; Tsutsumi, Hiroyuki

    2015-08-01

    Viral reactivations following hematopoietic stem cell transplantation are thought to result from the breakdown of both cell-mediated and humoral immunity. As a result, many viruses could be reactivated individually or simultaneously. Using a multiplex polymerase chain reaction (PCR), we prospectively examined many kinds of viral DNAs at a time in 105 patients who underwent allogeneic hematopoietic stem cell transplantation. In total, 591 whole blood samples were collected weekly from pre- to 42 days post-transplantation and the following 13 viruses were tested; herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, adenovirus, BK virus (BKV), JC virus (JCV), parvovirus B19, and hepatitis B virus (HBV). Several viral DNAs were detected in 12 patients before hematopoietic stem cell transplantation. The detection rate gradually increased after transplantation and peaked at 21 days. The most frequently detected virus was HHV-6 (n = 63; 60.0%), followed by EBV (n = 11; 10.5%), CMV (n = 11; 10.5%), and HHV-7 (n = 9; 8.6%). Adenovirus and HBV were each detected in one patient (1.0%). Detection of HHV-6 DNA was significantly more common among patients undergoing cord blood transplantation or with steroid treatment. EBV DNA tended to be more common in patients treated with anti-thymocyte globulin. Multiplex PCR was useful for detecting many viral reactivations after hematopoietic stem cell transplantation, simultaneously. Cord blood transplantation, steroid treatment, or anti-thymocyte globulin use was confirmed to be risk factors after transplantation. © 2015 Wiley Periodicals, Inc.

  19. A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens.

    Science.gov (United States)

    Rundell, Mark S; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H; Spitzer, Eric D; Golightly, Linnie; Barany, Francis

    2014-06-01

    Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Comparison of one commercial and two in-house TaqMan multiplex real-time PCR assays for detection of enteropathogenic, enterotoxigenic and enteroaggregative Escherichia coli.

    Science.gov (United States)

    Hahn, Andreas; Luetgehetmann, Marc; Landt, Olfert; Schwarz, Norbert Georg; Frickmann, Hagen

    2017-11-01

    Enteropathogenic, enterotoxigenic and enteroaggregative Escherichia coli (EPEC, ETEC, EAEC) are among the most frequent causes of diarrhoea during travel or on military deployments. Cost-efficient and reliable real-time multiplex PCR (mPCR) assays are desirable for surveillance or point prevalence studies in remote and resource-limited tropical settings. We compared one commercial PCR kit and two in-house assays without using a gold standard to estimate sensitivity and specificity of each assay. Residual materials from nucleic acid extractions of stool samples from two groups with presumably different prevalences and increased likelihood of being infected or colonised by diarrhoeagenic E. coli were included in the assessment. One group comprised samples from returnees from tropical deployments, the second group was of migrants and study participants from high-endemicity settings. Each sample was assessed with all of the PCR assays. Cycle threshold (Ct) values were descriptively compared. The calculated sensitivities for the commercial test vs. the in-house tests were for EPEC 0.84 vs. 0.89 and 0.96, for ETEC 0.83 vs. 0.76 and 0.61, and for EAEC 0.69 vs. 0.54 and 0.69. False positive results were rare - specificity was 0.94 and 0.97 for two EPEC tests and 1.0 for all other tests. Most positive samples had late Ct values corresponding to low quantities of pathogens. Discordant test results were associated with late Ct values. As commercial and in-house assays showed comparable results, in-house tests can be assumed to be safe while affording considerable savings, making them a valuable alternative for surveillance testing in resource-limited tropical areas. © 2017 John Wiley & Sons Ltd.

  1. 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.

  2. Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay.

    Science.gov (United States)

    Pabbaraju, Kanti; Wong, Sallene; Gill, Kara; Fonseca, Kevin; Tipples, Graham A; Tellier, Raymond

    2016-10-01

    In the recent past, arboviruses such as Chikungunya (CHIKV) and Zika (ZIKV) have increased their area of endemicity and presented as an emerging global public health threat. To design an assay for the simultaneous detection of ZIKV, CHIKV and Dengue (DENV) 1-4 from patients with symptoms of arboviral infection. This would be advantageous because of the similar clinical presentation typically encountered with these viruses and their co-circulation in endemic areas. In this study we have developed and validated a triplex real time reverse transcription PCR assay using hydrolysis probes targeting the non-structural 5 (NS5) region of ZIKV, non-structural protein 4 (nsP4) from CHIKV and 3' untranslated region (3'UTR) of DENV 1-4. The 95% LOD by the triplex assay was 15 copies/reaction for DENV-1 and less than 10 copies/reaction for all other viruses. The triplex assay was 100% specific and did not amplify any of the other viruses tested. The assay was reproducible and adaptable to testing different specimen types including serum, plasma, urine, placental tissue, brain tissue and amniotic fluid. This assay can be easily implemented for diagnostic testing of patient samples, even in a high throughput laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in Salmonella enterica serovars Typhi and Paratyphi A.

    LENUS (Irish Health Repository)

    Song, Yajun

    2010-08-01

    OBJECTIVES: Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers. METHODS: By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (Nal(R)) and\\/or decreased susceptibility to fluoroquinolones. RESULTS: This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (Nal(R) = 223 and Nal(S) = 69) and 106 isolates of Salmonella Paratyphi A (Nal(R) = 24 and Nal(S) = 82). All of the 247 Nal(R) Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143\\/223 for Salmonella Typhi and 18\\/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight Nal(S) Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies. CONCLUSIONS: The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes.

  4. Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci.

    Science.gov (United States)

    McClure, Jo-Ann; Zaal DeLongchamp, Johanna; Conly, John M; Zhang, Kunyan

    2017-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screening Staphylococcus isolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminating S. aureus from coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including the Staphylococcus 16S rRNA gene (specifically detecting Staphylococcus spp.), nuc (distinguishing S. aureus from CoNS), mecA (distinguishing MRSA from methicillin-susceptible S. aureus ), mupA and mupB (identifying high-level mupirocin resistance), and qac and smr (identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistant Staphylococcus monitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments. Copyright © 2017 American Society for Microbiology.

  5. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Science.gov (United States)

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA). Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD) in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection. PMID:28936257

  6. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Directory of Open Access Journals (Sweden)

    Malin Berggrund

    2016-01-01

    Full Text Available The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA. Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  7. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™.

    Science.gov (United States)

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA). Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD) in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  8. Development of a multiplex PCR assay for the detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei, Burkholderia thailandensis, and Burkholderia cepacia complex.

    Science.gov (United States)

    Zakharova, Irina; Teteryatnikova, Natalya; Toporkov, Andrey; Viktorov, Dmitry

    2017-10-01

    Two species of Burkholderia pseudomallei complex (Bpc), B. pseudomallei and B. mallei, can cause severe life-threatening infections. Rapidly discerning individual species within the group and separating them from other opportunistic pathogens of the Burkholderia cepacia complex (Bcc) is essential to establish a correct diagnosis and for epidemiological surveillance. In this study, a multiplex PCR assay based on the detection of an individual set of chromosomal beta-lactamase genes for single-step identification and differentiation of B. pseudomallei, B. mallei, B. thailandensis, and Bcc was developed. Two pairs of primers specific to a distinct class of B metallo-beta-lactamase genes and a pair of primers specific to the oxacillin-hydrolyzing class D beta-lactamase gene were demonstrated to successfully discriminate species within Bpc and from Bcc. The assay sensitivity was 9561 genomic equivalents (GE) for B. pseudomallei, 7827 GE for B. mallei, 8749 GE for B. thailandensis and 6023 GE for B. cepacia. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening*

    Science.gov (United States)

    AbstractHigh-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may ...

  10. A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

    DEFF Research Database (Denmark)

    Thierry, Simon; Hamidjaja, Raditijo A.; Girault, Guillaume

    2013-01-01

    been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR...

  11. Multiplexed imaging surface plasmon resonance (iSPR) biosensor assay for the detection of Fusarium toxins in wheat

    Science.gov (United States)

    Certain Fusarium species (F. graminearum and F. verticilloides in particular) infest grains and can produce a wide range of fungal (myco)-toxins, causing huge economic losses worldwide. A reproducible and sensitive imaging surface plasmon resonance (iSPR) assay was developed and validated for three ...

  12. Diagnostic multiplex polymerase chain reaction assay for the identification of Pseudomonas aeruginosa from the skin biopsy specimens in burn wound infections and detection of antibiotic susceptibility

    International Nuclear Information System (INIS)

    Mashouf, Rasoul Y.; Farahani, Hadi S.; Zamani, A.

    2008-01-01

    Objective was to identify Pseudomonas aeruginosa (P. aeruginosa) from the skin biopsy specimens in burn wound infections by multiplex polymerase chain reaction (M-PCR) and detection of antimicrobial susceptibility of isolates from culture. We conducted the cross-sectional study in 140 patients with wound infections who admitted to referral burn center of Motahari, Tehran, Iran, during a 12-month period from 2005-2006. Skin biopsy specimens were aseptically taken from each patient, one for PCR and one for bacterial culture. A M-PCR test based on simultaneous amplification of 2 lipoprotein genes: oprI and oprL, was used to directly detect fluorescent pseudomonades and P. aeruginosa in skin biopsy specimens. The susceptibility of P. aeruginosa isolates to 16 antibiotics was determined using the disc diffusion method. Out of 140 biopsy specimens, M-PCR detected 66 (47.2%) isolates, while culture detected 57 (40.7%) isolates as P. aeruginosa. Positive results for both genes which observed only for P. aeruginosa, while only one gene, oprI, was amplified from other fluorescent pseudomonades (n=12) and all other bacterial tested (n=62) were negative by the amplification test. The most effective antibiotics against isolate of P. aeruginosa were cefepime (79%), azetreonam (76%), ticarcillin-clavulanic acid (68%), tobramycin (62%) and amikacin (61%). Multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa from the burned skin biopsy specimens. Simultaneous amplification of 2 lipoprotein genes: oprI and oprL could detect P. aeruginosa and oprI gene only for other fluorescent pseudomonades. (author)

  13. Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening

    International Nuclear Information System (INIS)

    Druwe, Ingrid; Freudenrich, Theresa M.; Wallace, Kathleen; Shafer, Timothy J.; Mundy, William R.

    2015-01-01

    High-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may be used in a battery of tests for detecting chemicals that could result in developmental neurotoxicity. Apoptosis contributes to nervous system development by regulating the size of the neuroprogenitor cell pool, and the balance between cellular proliferation and apoptosis during neuroprogenitor cell proliferation helps to determine the size and shape of the nervous system. Therefore, chemicals that affect apoptosis during neuronal development can have deleterious effects on the developing brain. The present study examined the utility of a high-throughput assay to detect chemical-induced apoptosis in mouse or human neuroprogenitor cells, as well as differentiated human neurons derived from induced pluripotent stem cells. Apoptosis was assessed using an assay that measures enzymatic activity of caspase-3/7 in a rapid and cost efficient manner. The results show that all three commercially available models generated a robust source of proliferating neuroprogenitor cells, and that the assay was sensitive and reproducible when used in a multi-well plate format. There were differences in the response of rodent and human neuroprogenitor cells to a set of chemicals previously shown to induce apoptosis in vitro. Neuroprogenitor cells were more sensitive to chemical-induced apoptosis than differentiated neurons, suggesting that neuroprogenitor cells are one of the cell models that should be considered for use in a developmental neurotoxicity screening battery

  14. A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

    Directory of Open Access Journals (Sweden)

    Sanchita Das

    Full Text Available CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR. The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively. The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus.

  15. A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

    Science.gov (United States)

    Das, Sanchita; Rundell, Mark S; Mirza, Aashiq H; Pingle, Maneesh R; Shigyo, Kristi; Garrison, Aura R; Paragas, Jason; Smith, Scott K; Olson, Victoria A; Larone, Davise H; Spitzer, Eric D; Barany, Francis; Golightly, Linnie M

    2015-01-01

    CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).

  16. MOLECULAR TYPING OF STREPTOCOCCUS PNEUMONIAE BY THE MULTIPLEX POLYMERASE CHAIN REACTION ASSAY IN ACCORDANCE TO THE PREVALENCE OF SEROTYPES IN THE RUSSIAN FEDERATION

    Directory of Open Access Journals (Sweden)

    N. M. Alyab'eva

    2013-01-01

    Full Text Available Aim: to compare two methods of S. pneumoniae molecular typing: classic serological method and the multiplex polymerase chain reaction (M-PCR assay modified in accordance to the date on the serotypes circulating in Russian Federation. Patients and methods: 420 pneumococcal isolates mainly from non-sterile loci were analyzed. After microbiological identification pneumococci were serologically typed with the means of specific antiserum produced by Staten Serum Institute (Denmark in latex agglutination test and/or capsular swelling method. At the same time we performed series of the M-PCR, which consisted of 7 consecutive reactions at the most. Results: serotype was identified by the means of serological method in all 420 strains of S. pneumoniae; in total we determined 24 different serotypes. By the means of the M-PCR we succeeded in identification of 95% (399/420 examined strains, and 90% of them were typed during the first 3 reactions. All isolates failed to be typed by M-PCR (n =21 have serotypes not included into the composition of the M-PCR. The results of serological and molecular typing were identical in 99,2% (396/399 of the isolates; 3 strains showed contradictory results: serotype 19A was found on serological assay and serotype 19F — on PCR assay. Conclusions: the introduced modification of M-PCR allows correct identification of pneumococcal serotype more than in 90% of strains circulating in the Russian Federation, including all serotypes of pneumococcal polysaccharide conjugate vaccine.

  17. Add-on unidirectional elastic metamaterial plate cloak

    Science.gov (United States)

    Lee, Min Kyung; Kim, Yoon Young

    2016-02-01

    Metamaterial cloaks control the propagation of waves to make an object invisible or insensible. To manipulate elastic waves in space, a metamaterial cloak is typically embedded in a base system that includes or surrounds a target object. The embedding is undesirable because it structurally weakens or permanently alters the base system. In this study, we propose a new add-on metamaterial elastic cloak that can be placed over and mechanically coupled with a base structure without embedding. We designed an add-on type annular metamaterial plate cloak through conformal mapping, fabricated it and performed cloaking experiments in a thin-plate with a hole. Experiments were performed in a thin plate by using the lowest symmetric Lamb wave centered at 100 kHz. As a means to check the cloaking performance of the add-on elastic plate cloak, possibly as a temporary stress reliever or a so-called “stress bandage”, the degree of stress concentration mitigation and the recovery from the perturbed wave field due to a hole were investigated.

  18. Evaluation of a Multiplex Real-Time Reverse Transcriptase PCR Assay for Detection and Differentiation of Influenza Viruses A and B during the 2001-2002 Influenza Season in Israel

    Science.gov (United States)

    Hindiyeh, Musa; Levy, Virginia; Azar, Roberto; Varsano, Noemi; Regev, Liora; Shalev, Yael; Grossman, Zehava; Mendelson, Ella

    2005-01-01

    The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories. PMID:15695650

  19. Evaluation of two real-time multiplex PCR screening assays detecting fetal RHD in plasma from RhD negative women to ascertain the requirement for antenatal RhD prophylaxis

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus

    2011-01-01

    and 5. We used the same fluorescent dye for the exon 7 and 10 probes to increase sensitivity; exon 5 was VIC labeled. We evaluated possible inhibition of DNA amplification with dilution experiments. We then tested the two multiplex assays with DNA extracted from 97 plasma samples from 38 RhD negative......OBJECTIVE: To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis. METHODS: We designed a duplex assay for the detection of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10...... assay (exon 7/10), accuracy was 94.2%. Detection of exon 5 was less reliable. CONCLUSION: The duplex assay using exon 7/10 was the most reliable for prenatal prediction of fetal RhD type as a candidate assay for screening of RhD negative women in relation to antenatal RhD prophylaxis. The triplex assay...

  20. Development of an alpha-fetoprotein and Golgi protein 73 multiplex detection assay using xMAP technology.

    Science.gov (United States)

    Wu, Yun; Ma, Jie; Wang, Yipeng; Zhang, Yonghong; Hou, Yongjin; Zhang, Chunming; Sun, Huanqin; Sun, Jianping; Wang, Zikang; Li, Ning

    2018-04-06

    Development of a new method to simultaneously detect Alpha-fetoprotein (AFP) and Golgi protein 73 (GP73) from peripheral blood. Anti human AFP and GP73 monoclonal antibodies was used to develop a sandwich immunity reaction using xMAP technology for the simultaneous detection of plasma AFP and GP73. The assay evaluated the sensitivity, cross reactivity, range of detection, precision, recovery and linearity dilution effect. The assay utilized plasma samples and compared its performance with commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits. The assay was successful in detecting AFP and GP73 simultaneously. Validation experiments demonstrated the limit of detection was AFP 0.006 μg/l and GP73 0.482 μg/l. The functional sensitivity was AFP 0.010 μg/l and GP73 0.640 μg/l. The range of detection was AFP 0.01-50 μg/l and GP73 0.64-100 μg/l. No cross reactivity was observed. The intra- and inter-assay variation for AFP was 0.19-3.46% and 3.1-5.8% and for GP73 was 1.5-3.2% and 1.1-7.6% respectively. The recovery for AFP was 96-106% and GP73 was 89-110%. 80 clinical plasma samples from healthy controls, and patients with liver cirrhosis and Hepatocellular Carcinoma (HCC) were evaluated. For healthy controls (n = 25), the AFP was 2.40 (1.55, 3.30) μg/l and GP73 was 42.60 (39.10, 57.40) μg/l. For patients with liver cirrhosis (n = 19), the AFP was 2.60 (1.70, 4.20) μg/l and GP73 was 136.10 (92.10, 261.70) μg/l, and for HCC patients (n = 36), the AFP was 13.65 (3.35, 158.88) μg/l and GP73 was 186.25 (96.73, 262.03) μg/l. The new assay demonstrated a good correlation with commercially available ELISA kits (correlation coefficients (r) were 0.997 (AFP, p < 0.001) and 0.959 (GP73, p < 0.001). The method demonstrated a sensitive, effective and accurate method for the simultaneous detection of AFP and GP73, and could be used clinically for routine detection and monitoring of patients with chronic hepatitis B

  1. A multiplexed chip-based assay system for investigating the functional development of human skeletal myotubes in vitro.

    Science.gov (United States)

    Smith, A S T; Long, C J; Pirozzi, K; Najjar, S; McAleer, C; Vandenburgh, H H; Hickman, J J

    2014-09-20

    This report details the development of a non-invasive in vitro assay system for investigating the functional maturation and performance of human skeletal myotubes. Data is presented demonstrating the survival and differentiation of human myotubes on microscale silicon cantilevers in a defined, serum-free system. These cultures can be stimulated electrically and the resulting contraction quantified using modified atomic force microscopy technology. This system provides a higher degree of sensitivity for investigating contractile waveforms than video-based analysis, and represents the first system capable of measuring the contractile activity of individual human muscle myotubes in a reliable, high-throughput and non-invasive manner. The development of such a technique is critical for the advancement of body-on-a-chip platforms toward application in pre-clinical drug development screens. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR.

    Science.gov (United States)

    Ramilo, Andrea; Navas, J Ignacio; Villalba, Antonio; Abollo, Elvira

    2013-05-27

    Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a 'gold standard' prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.

  3. Human MLPA Probe Design (H-MAPD: a probe design tool for both electrophoresis-based and bead-coupled human multiplex ligation-dependent probe amplification assays

    Directory of Open Access Journals (Sweden)

    Hatchwell Eli

    2008-09-01

    Full Text Available Abstract Background Multiplex ligation-dependent probe amplification (MLPA is an efficient and reliable technique for gene dosage analysis. Currently MLPA can be conducted on two platforms: traditional electrophoresis-based, and FlexMAP bead-coupled. Since its introduction in 2002, MLPA has been rapidly adopted in both clinical and research situations. However, MLPA probe design is a time consuming process requiring many steps that address multiple criteria. There exist only one or two commercial software packages for traditional electrophoresis-based MLPA probe design. To our knowledge, no software is yet available that performs bead-coupled MLPA probe design. Results We have developed H-MAPD, a web-based tool that automates the generation and selection of probes for human genomic MLPA. The software performs physical-chemical property tests using UNAFold software, and uniqueness tests using the UCSC genome browser. H-MAPD supports both traditional electrophoresis-based assays, as well as FlexMAP bead-coupled MLPA. Conclusion H-MAPD greatly reduces the efforts for human genomic MLPA probe design. The software is written in Perl-CGI, hosted on a Linux server, and is freely available to non-commercial users.

  4. Evaluation of efficiency of nested multiplex allele-specific PCR assay for detection of multidrug resistant tuberculosis directly from sputum samples.

    Science.gov (United States)

    Mistri, S K; Sultana, M; Kamal, S M M; Alam, M M; Irin, F; Nessa, J; Ahsan, C R; Yasmin, M

    2016-05-01

    For an effective control of tuberculosis, rapid detection of multidrug resistant tuberculosis (MDR-TB) is necessary. Therefore, we developed a modified nested multiplex allele-specific polymerase chain reaction (MAS-PCR) method that enables rapid MDR-TB detection directly from sputum samples. The efficacy of this method was evaluated using 79 sputum samples collected from suspected tuberculosis patients. The performance of nested MAS-PCR method was compared with other MDR-TB detection methods like drug susceptibility testing (DST) and DNA sequencing. As rifampicin (RIF) resistance conforms to MDR-TB in greater than 90% cases, only the presence of RIF-associated mutations in rpoB gene was determined by DNA sequencing and nested MAS-PCR to detect MDR-TB. The concordance between nested MAS-PCR and DNA sequencing results was found to be 96·3%. When compared with DST, the sensitivity and specificity of nested MAS-PCR for RIF-resistance detection were determined to be 92·9 and 100% respectively. For developing- and high-TB burden countries, molecular-based tests have been recommended by the World Health Organization for rapid detection of MDR-TB. The results of this study indicate that, nested MAS-PCR assay might be a practical and relatively cost effective molecular method for rapid detection of MDR-TB from suspected sputum samples in developing countries with resource poor settings. © 2016 The Society for Applied Microbiology.

  5. Simultaneous detection of viruses and Toxoplasma gondii in cerebrospinal fluid specimens by multiplex polymerase chain reaction-based reverse hybridization assay.

    Science.gov (United States)

    Del Prete, Raffaele; Di Taranto, Anna Maria; Lipsi, Maria Rosaria; Natalicchio, Maria Iole; Antonetti, Raffaele; Miragliotta, Giuseppe

    2009-04-01

    The lack of rapidity and the low sensitivity and specificity of traditional laboratory methods limits their usefulness in the laboratory diagnosis of viral central nervous system (CNS) infections. This study describes the use of a commercially available multiplex polymerase chain reaction (mPCR)-based reverse hybridization assay (RHA) for the simultaneous detection of the genomes of 8 viruses and Toxoplasma gondii in cerebrospinal fluids (CSF) from 181 patients suspected of having viral meningitis. Twenty-two/181 (12.15%) CSF samples resulted positive by mPCR. Eighteen/22 were positive for 1 viral pathogen, whereas a dual infection was detected in 4/22 samples. Epstein-Barr virus (EBV) was the most commonly detected virus (6/22), followed by herpes simplex virus type-1 (HSV-1) (5/22) and -2 (HSV-2) (4/22). Cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and Epstein-Barr virus (EBV) were detected in 1 specimen each. Two CSF samples were co-infected by HSV-1/HSV-2, 1 sample by HHV-6/T. gondii, and 1 sample by EBV/EV, respectively. Our data support the usefulness of mPCR as a rapid molecular method for the simultaneous detection of major viral pathogens and T. gondii in aseptic meningitis also to allow the earlier application of specific antiviral therapy.

  6. Detection of gene copy number aberrations in mantle cell lymphoma by a single quantitative multiplex PCR assay: clinicopathological relevance and prognosis value.

    Science.gov (United States)

    Jardin, Fabrice; Picquenot, Jean-Michel; Parmentier, Françoise; Ruminy, Philippe; Cornic, Marie; Penther, Dominique; Bertrand, Philippe; Lanic, Hélène; Cassuto, Ophélie; Humbrecht, Catherine; Lemasle, Emilie; Wautier, Agathe; Bastard, Christian; Tilly, Hervé

    2009-09-01

    The t(11;14)(q13;q32) is the hallmark of mantle cell lymphoma (MCL). Additional genetic alterations occur in the majority of cases. This study aimed to design a polymerase chain reaction (PCR) assay to determine the incidence and relevance of recurrent gene copy number aberrations in this disease. Forty-two MCL cases with frozen- or paraffin-embedded (FFPE) tissues were selected. Three different quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) assays were designed to simultaneously analyse eight genes (CDKN2A, RB1, ATM, CDK2, TP53, MYC, CDKN1B, MDM2), to analyse the 9p21 locus (CDKN2A/CDKN2B) and FFPE tissues. Gains of MYC, CDK2, CDKN1B, and MDM2 were observed in 10% of cases. Losses of RB1, CDKN2A, ATM or TP53 were observed in 38%, 31%, 24% and 10% of cases, respectively. Analysis of the 9p21 locus indicated that, in most cases, tumours displayed a complete inactivation of p14(ARF)/p15I(NK4B)/p16I(NK4A). CDKN2A and MYC aberrations were associated with a high MCL international prognostic index (MIPI). CDK2/MDM2 gains and CDKN2A/TP53 losses correlated with an unfavourable outcome. PCR experiments with frozen and FFPE-tissues indicated that our approach is valid in a routine diagnostic setting, providing a powerful tool that could be used for patient stratification in combination with MIPI in future clinical trials.

  7. Effective identification of Lactobacillus casei group species: genome-based selection of the gene mutL as the target of a novel multiplex PCR assay.

    Science.gov (United States)

    Bottari, Benedetta; Felis, Giovanna E; Salvetti, Elisa; Castioni, Anna; Campedelli, Ilenia; Torriani, Sandra; Bernini, Valentina; Gatti, Monica

    2017-07-01

    Lactobacillus casei,Lactobacillus paracasei and Lactobacillusrhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.

  8. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    Directory of Open Access Journals (Sweden)

    Delyan P Ivanov

    Full Text Available Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

  9. Add-on perampanel in Lance-Adams syndrome.

    Science.gov (United States)

    Steinhoff, Bernhard J; Bacher, Matthias; Kurth, Christoph; Staack, Anke M; Kornmeier, Reinhold

    2016-01-01

    Perampanel (PER) is the first-in-class selective, noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist that has been licensed and marketed as antiepileptic drug (AED) indicated for patients with partial-onset and primary generalized tonic-clonic seizures. A positive effect was reported in some patients with epileptic myoclonic jerks in idiopathic generalized epilepsy and in progressive myoclonic epilepsy. We treated a male patient with posthypoxic nonepileptic myoclonus (Lance-Adams syndrome) with add-on PER and achieved an almost complete cessation of jerks. This effect was reproducible and, therefore, we suggest that it might be worth trying PER in comparable cases.

  10. Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

    Science.gov (United States)

    Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

    2017-07-25

    Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an

  11. A subsurface add-on for standard atomic force microscopes

    Energy Technology Data Exchange (ETDEWEB)

    Verbiest, G. J., E-mail: Verbiest@physik.rwth-aachen.de [JARA-FIT and II. Institute of Physics, RWTH Aachen University, 52074 Aachen (Germany); Zalm, D. J. van der; Oosterkamp, T. H.; Rost, M. J., E-mail: Rost@physics.leidenuniv.nl [Kamerlingh Onnes Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden (Netherlands)

    2015-03-15

    The application of ultrasound in an Atomic Force Microscope (AFM) gives access to subsurface information. However, no commercially AFM exists that is equipped with this technique. The main problems are the electronic crosstalk in the AFM setup and the insufficiently strong excitation of the cantilever at ultrasonic (MHz) frequencies. In this paper, we describe the development of an add-on that provides a solution to these problems by using a special piezo element with a lowest resonance frequency of 2.5 MHz and by separating the electronic connection for this high frequency piezo element from all other connections. In this sense, we support researches with the possibility to perform subsurface measurements with their existing AFMs and hopefully pave also the way for the development of a commercial AFM that is capable of imaging subsurface features with nanometer resolution.

  12. Non-invasive beamforming add-on module

    KAUST Repository

    Bader, Ahmed

    2017-02-23

    An embodiment of a non-invasive beamforming add-on apparatus couples to an existing antenna port and rectifies the beam azimuth in the upstream and downstream directions. The apparatus comprises input circuitry that is configured to receive one or more signals from a neighboring node of the linear wireless sensor network; first amplifier circuitry configured to adjust an amplitude of a respective received signal in accordance with a weighting coefficient and invoke a desired phase to a carrier frequency of the received signal thereby forming a first amplified signal; and second amplifier circuitry configured to adjust a gain of the first amplified signal towards upstream and downstream neighbors of the linear wireless sensor in the linear wireless sensor network.

  13. Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

    Science.gov (United States)

    Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

    2014-07-01

    A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

  14. Multiplex assay (Mikrogen recomBead) for detection of serum IgG and IgM antibodies to 13 recombinant antigens of Borrelia burgdorferi sensu lato in patients with neuroborreliosis

    DEFF Research Database (Denmark)

    Dessau, Ram Benny; Møller, Jens K.; Kolmos, Birte

    2015-01-01

    A multiplex-bead-based assay for the detection of serum antibodies to Borrelia burgdorferi sensu lato was evaluated. The assay contained 13 different antigens in both the IgG and the IgM assay; thus, a total of 26 measurement results were available from each sample. A total of 49 Danish patients......, the construction of a diagnostic score, evaluation of the scoring method using an independent dataset and an assessment of the analytical quality of the multiplex assay. The VlsE IgG had the highest diagnostic value with an AUC (area under the curve) of 96% on the receiver operating characteristic curve. The Osp......C IgM had AUCs just above 80%. All the other antigens had both low quantitative reactivity and lower contrast in the patients with LNB compared to controls. The diagnostic value of the assay may be improved by using a logistic model giving a sensitivity of 90 and 79% for the specificities at 92 and 98...

  15. Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order.

    Science.gov (United States)

    Bilodeau, Guillaume J; Martin, Frank N; Coffey, Michael D; Blomquist, Cheryl L

    2014-07-01

    A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic

  16. Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

    Directory of Open Access Journals (Sweden)

    Annika Mohr

    Full Text Available Immunohistochemistry (IHC is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1, progesterone receptor (PGR, prolactin receptor (PRLR and growth hormone receptor (GHR gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

  17. Evaluation of the new AmpliSens multiplex real-time PCR assay for simultaneous detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis.

    Science.gov (United States)

    Rumyantseva, Tatiana; Golparian, Daniel; Nilsson, Christian S; Johansson, Emma; Falk, My; Fredlund, Hans; Van Dam, Alje; Guschin, Alexander; Unemo, Magnus

    2015-10-01

    In this study, we performed an evaluation of the new CE-marked multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay compared to APTIMA tests, i.e., APTIMA COMBO 2 assay, APTIMA Trichomonas vaginalis assay (FDA-approved), and two different APTIMA Mycoplasma genitalium assays (research use only; one of them only used for discrepancy analysis). Vaginal swabs (n = 209) and first-void urine (FVU) specimens from females (n = 498) and males (n = 554), consecutive attendees (n = 1261) at a dermatovenerological clinic in Sweden, were examined. The sensitivity of the AmpliSens PCR assay for detection of C. trachomatis (6.3% prevalence), M. genitalium (5.7% prevalence), N. gonorrhoeae (0.3% prevalence), and T. vaginalis (0.08% prevalence) was 97.5% (95% confidence interval (CI): 91.2-99.6%), 81.9% (95% CI: 70.7-89.7%), 100% (95% CI: 40.2-100%) and 100% (95% CI: 16.5-100%), respectively. The specificity of the AmpliSens PCR assay was 100% (95% CI: 99.6-100%) for all agents. The analytical sensitivity and specificity for N. gonorrhoeae detection was excellent, i.e., 55 international gonococcal strains detected and 135 isolates of 13 non-gonococcal Neisseria species were negative. In conclusion, the multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay demonstrated high sensitivity and excellent specificity for the detection of C. trachomatis, N. gonorrhoeae, and T. vaginalis, and excellent specificity but suboptimal sensitivity for M. genitalium detection. © 2015 APMIS. Published by John Wiley & Sons Ltd.

  18. Lixisenatide as add-on therapy to basal insulin

    Directory of Open Access Journals (Sweden)

    Brown DX

    2013-12-01

    Full Text Available Dominique Xavier Brown, Emma Louise Butler, Marc Evans Diabetes Department, University Hospital Llandough, Cardiff, UK Abstract: Many patients with type 2 diabetes mellitus do not achieve target glycosylated hemoglobin A1c levels despite optimally titrated basal insulin and satisfactory fasting plasma glucose levels. Current evidence suggests that HbA1c levels are dictated by both basal glucose and postprandial glucose levels. This has led to a consensus that postprandial glucose excursions contribute to poor glycemic control in these patients. Lixisenatide is a once-daily, prandial glucagon-like peptide 1 (GLP-1 receptor agonist with a four-fold affinity for the GLP-1 receptor compared with native GLP-1. Importantly, lixisenatide causes a significant delay in gastric emptying time, an important determinant of the once-daily dosing regimen. An exendin-4 mimetic with six lysine residues removed at the C-terminal, lixisenatide has pronounced postprandial glucose-lowering effects, making it a novel incretin agent for use in combination with optimally titrated basal insulin. Lixisenatide exerts profound effects on postprandial glucose through established mechanisms of glucose-dependent insulin secretion and glucagon suppression in combination with delayed gastric emptying. This review discusses the likely place that lixisenatide will occupy in clinical practice, given its profound effects on postprandial glucose and potential to reduce glycemic variability. Keywords: lixisenatide, add-on therapy, insulin, GLP-1 receptor agonist, postprandial glucose, pharmacodynamics

  19. Multiplex Detection and Genotyping of Point Mutations Involved in Charcot-Marie-Tooth Disease Using a Hairpin Microarray-Based Assay

    Directory of Open Access Journals (Sweden)

    Yasser Baaj

    2009-01-01

    Full Text Available We previously developed a highly specific method for detecting SNPs with a microarray-based system using stem-loop probes. In this paper we demonstrate that coupling a multiplexing procedure with our microarray method is possible for the simultaneous detection and genotyping of four point mutations, in three different genes, involved in Charcot-Marie-Tooth disease. DNA from healthy individuals and patients was amplified, labeled with Cy3 by multiplex PCR; and hybridized to microarrays. Spot signal intensities were 18 to 74 times greater for perfect matches than for mismatched target sequences differing by a single nucleotide (discrimination ratio for “homozygous” DNA from healthy individuals. “Heterozygous” mutant DNA samples gave signal intensity ratios close to 1 at the positions of the mutations as expected. Genotyping by this method was therefore reliable. This system now combines the principle of highly specific genotyping based on stem-loop structure probes with the advantages of multiplex analysis.

  20. Analogue multiplexer

    International Nuclear Information System (INIS)

    Gorshkov, V.A.; Kuznetsov, A.N.

    1980-01-01

    In systems of signal recording from several parallel spectrometric channels one can considerably reduce the total apparatus volume using a special unit - an analog multiplexer. A description of the multiplexer in the CAMAC system on the base of fast linear gating circuits which allows one analog-to-code converter to attend four spectrometric channels is given. On the example of the 4-channel spectrometer the logics of interaction of the multiple with analog-to-digital coxernver and signal recorder is shown. Electrical and functional multiplexer flow-sheets are given and its main characteristics are presented

  1. 40 CFR 75.34 - Units with add-on emission controls.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Units with add-on emission controls... add-on emission controls. (a) The owner or operator of an affected unit equipped with add-on SO2 and... assurance/quality control program for the unit, required by section 1 in appendix B of this part. To provide...

  2. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

    Science.gov (United States)

    Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8

  3. Development and Characterization of a Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out Supplemental Materials

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S; Danganan, L; Tammero, L; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed advanced rapid diagnostics that may be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the potential to improve our nation's ability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect animal populations of high economic importance in the United States. Under 2005 DHS funding we have developed multiplexed (MUX) nucleic-acid-based PCR assays that combine foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease (SVD) and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1 or Infectious Bovine Rhinotracheitus IBR), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus BPSV, Orf of sheep, and Pseudocowpox). Under 2006 funding we have developed a Multiplexed PCR [MUX] porcine assay for detection of FMDV with rule out tests for VESV and SVD foreign animal diseases in addition to one other domestic vesicular animal disease vesicular stomatitis virus (VSV) and one domestic animal disease of swine porcine reproductive and respiratory syndrome (PRRS). We have also developed a MUX bovine assay for detection of FMDV with rule out tests for the two bovine foreign animal diseases malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis virus (VSV), bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitus virus (BHV-1), bluetongue virus (BTV), and the Parapox

  4. A novel nested multiplex polymerase chain reaction (PCR assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

    Directory of Open Access Journals (Sweden)

    Parija Subhash C

    2007-05-01

    Full Text Available Abstract Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific. The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.

  5. Naturally-acquired humoral immune responses against the N- and C-termini of the Plasmodium vivax MSP1 protein in endemic regions of Brazil and Papua New Guinea using a multiplex assay

    Directory of Open Access Journals (Sweden)

    Alonso Pedro L

    2010-01-01

    Full Text Available Abstract Background Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. Methods Glutathione S-transferase (GST and GST-fusion proteins representing the N- terminus of the merozoite surface protein 1 of P. vivax, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of P. vivax patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation. Results The results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay (ELISA, showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea (PNG, and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG. Conclusions This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.

  6. Implementation of computerized add-on testing for hospitalized patients in a large academic medical center.

    Science.gov (United States)

    Kim, Ji Yeon; Kamis, Irina K; Singh, Balaji; Batra, Shalini; Dixon, Roberta H; Dighe, Anand S

    2011-05-01

    Physician requests for additional testing on an existing laboratory specimen (add-ons) are resource intensive and generally require a phone call to the laboratory. Verbal orders such as these have been noted to be associated with errors in accuracy. The aim of this study was to compare a novel computerized system for add-on requests to the prior verbal system. We compare the computerized add-on request system to the verbal system with respect to order completeness and workflow. We demonstrate that the computerized add-on system resulted in the complete in-laboratory documentation of the add-on request 100% of the time, compared to 58% with the verbal add-on system. In addition, we show that documentation of a verbal add-on request in the electronic medical record (EMR) occurred for 4% of requests, while in the computerized system EMR documentation occurred 100% of the time. We further demonstrate that the computerized add-on request process was well accepted by providers and did not significantly change the test mix of the add-on requests. In computerized physician order entry (CPOE) implementations, add-on order functionality should be considered so these orders are documented in the EMR.

  7. Development of single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease.

    Science.gov (United States)

    Puenpa, Jiratchaya; Suwannakarn, Kamol; Chansaenroj, Jira; Vongpunsawad, Sompong; Poovorawan, Yong

    2017-10-01

    Real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) has facilitated the rapid and accurate identification of the two most common etiological agents underlying hand, foot, and mouth disease (HFMD). However, the worldwide emergence of CV-A6 infection in HFMD necessitates development of an improved multiplex rRT-PCR method. To rapidly determine the etiology of HFMD, two rRT-PCR assays using TaqMan probes were developed to differentiate among three selected common enteroviruses (EV-A71, CV-A16 and CV-A6) and to enable broad detection of enteroviruses (pan-enterovirus assay). No cross-reactions were observed with other RNA viruses examined. The detection limits of both assays were 10 copies per microliter for EV-A71, CV-A6 and CV-A16, and pan-enterovirus. The methods showed high accuracy (EV-A71, 90.6%; CV-A6, 92.0%; CV-A16, 100%), sensitivity (EV-A71, 96.5%; CV-A6, 95.8%; CV-A16, 99.0%), and specificity (EV-A71, 100%; CV-A6, 99.9%; CV-A16, 99.9%) in testing clinical specimens (n=1049) during 2014-2016, superior to those of conventional RT-PCR. Overall, the multiplex rRT-PCR assays enabled highly sensitive detection and rapid simultaneous typing of EV-A71, CV-A6 and CV-A16, and enteroviruses, rendering them feasible and attractive methods for large-scale surveillance of enteroviruses associated with HFMD outbreaks. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    Directory of Open Access Journals (Sweden)

    Tian-Min Qiao

    2016-10-01

    Full Text Available Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP were developed for detection of C. scoparium based on factor 1-alpha (tef1 and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  9. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    Science.gov (United States)

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

  10. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

    Science.gov (United States)

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-10-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium , has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium . The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  11. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Sirirat Seekhuntod

    Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

  12. NCI-FDA Interagency Oncology Task Force Workshop Provides Guidance for Analytical Validation of Protein-based Multiplex Assays | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    An NCI-FDA Interagency Oncology Task Force (IOTF) Molecular Diagnostics Workshop was held on October 30, 2008 in Cambridge, MA, to discuss requirements for analytical validation of protein-based multiplex technologies in the context of its intended use. This workshop developed through NCI's Clinical Proteomic Technologies for Cancer initiative and the FDA focused on technology-specific analytical validation processes to be addressed prior to use in clinical settings. In making this workshop unique, a case study approach was used to discuss issues related to

  13. Comparison of multiplex reverse transcription-PCR-enzyme ...

    African Journals Online (AJOL)

    Comparison of multiplex reverse transcription-PCR-enzyme hybridization assay with immunofluorescence techniques for the detection of four viral respiratory pathogens in pediatric community acquired pneumonia.

  14. Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay.

    Science.gov (United States)

    Cross, Kristen E; Mercante, Jeffrey W; Benitez, Alvaro J; Brown, Ellen W; Diaz, Maureen H; Winchell, Jonas M

    2016-07-01

    Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5'-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease. Published by Elsevier Inc.

  15. Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2.

    Science.gov (United States)

    Chiapponi, Chiara; Moreno, Ana; Barbieri, Ilaria; Merenda, Marianna; Foni, Emanuela

    2012-09-01

    In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. 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

    2016-01-01

    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 104 CFU mL−1 or 105 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 (R2) 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. PMID:26884128

  17. Validation of a norovirus multiplex real-time RT-PCR assay for the detection of norovirus GI and GII from faeces samples.

    LENUS (Irish Health Repository)

    Jones, S

    2011-01-01

    Norovirus is a leading cause of infectious non-bacterial gastroenteritis. The virus is highly contagious and has multiple modes of transmission, presenting a growing challenge to hospital-based healthcare. In this study, a total of 120 stool samples are tested for the presence of norovirus GI and GII by the Roche two-step Lightcycler 2.0 assay incorporating primers and probes produced by TIB Molbiol, and the results are compared with results from the National Virus Reference Laboratory. The Roche\\/TIB Molbiol assay produced 51 positive results and 69 negative results. Discrepancy analysis was performed for six conflicting results using a second real-time polymerase chain reaction (PCR) assay (Roche\\/TIB Molbiol) and this confirmed that four of the five discrepant positive results were true positives. A single discrepant negative result generated by the Roche assay remained negative using the second assay. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to be 98%, 98.6%, 98.0% and 98.6%, respectively. Melting curve analysis was used to differentiate genogroups I and II and this showed that 92% of strains belonged to genogroup II.

  18. Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

    Science.gov (United States)

    Noguchi, Akio; Nakamura, Kosuke; Sakata, Kozue; Sato-Fukuda, Nozomi; Ishigaki, Takumi; Mano, Junichi; Takabatake, Reona; Kitta, Kazumi; Teshima, Reiko; Kondo, Kazunari; Nishimaki-Mogami, Tomoko

    2016-04-19

    A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

  19. Rapid detection and semi-quantification of IgG-accessible Staphylococcus aureus surface-associated antigens using a multiplex competitive Luminex assay

    NARCIS (Netherlands)

    Hansenova Manaskova, S.; Bikker, F.J.; Veerman, E.C.I.; van Belkum, A.; van Wamel, W.J.B.

    2013-01-01

    The surface characterization of Staphylococcus aureus is currently labor intensive and time consuming. Therefore, we developed a novel method for the rapid yet comprehensive characterization of S. aureus cell-surface-associated proteins and carbohydrates, based on a competitive Luminex assay. In

  20. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

  1. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. © 2011 American Institute of Physics

  2. Detection of enteropathogens associated with travelers’ diarrhea using a multiplex Luminex-based assay performed on stool samples smeared on Whatman FTA Elute cards

    Science.gov (United States)

    Lalani, Tahaniyat; Tisdale, Michele D; Maguire, Jason D; Wongsrichanalai, Chansuda; Riddle, Mark S; Tribble, David R

    2015-01-01

    We evaluated the limits of detection (LoD) for an 11-plex PCR-Luminex assay performed on Whatman FTA Elute cards smeared with stool containing pathogens associated with travelers’ diarrhea. LoDs ranged between 102-105 CFU, PFU or cysts/g for most pathogens except Cryptosporidium. Campylobacter and norovirus LoD increased with prolonged storage of cards. PMID:26072151

  3. Detection of enteropathogens associated with travelers' diarrhea using a multiplex Luminex-based assay performed on stool samples smeared on Whatman FTA Elute cards.

    Science.gov (United States)

    Lalani, Tahaniyat; Tisdale, Michele D; Maguire, Jason D; Wongsrichanalai, Chansuda; Riddle, Mark S; Tribble, David R

    2015-09-01

    We evaluated the limits of detection (LoD) for an 11-plex PCR-Luminex assay performed on Whatman(™) FTA Elute cards smeared with stool containing pathogens associated with travelers' diarrhea. LoDs ranged from 10(2) to 10(5)CFU, PFU, or cysts/g for most pathogens except Cryptosporidium. Campylobacter and norovirus LoDs increased with prolonged storage of cards. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro CardTM

    OpenAIRE

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro cardTM has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro cardTM using the sensitive proximity extension assay (PEA). Among 92 proteins in ...

  5. Carcinoma multiplex

    International Nuclear Information System (INIS)

    Shah, S. A.; Riaz, U.; Zahoor, I.; Jalil, A.; Zubair, M.

    2013-01-01

    Multiple primaries in a single patient are uncommon, though not very rare. The existence of such cancers in two un-related, non-paired organs is even more un-common. Here, we present a case of 55 years old male who presented to us with a mucoepidermoid carcinoma of the parotid gland and was operated. Later on, he presented with a large cystic swelling in the pelvis which turned out to be pseudomyxoma peritonei. A review of slides and immunohistochemistry indicated it to be adenocarcinoma colon. He presented again with recurrent mucoepidermoid carcinoma of the parotid which was operated successfully with the use of myocutaneous flap for wound closure. He is currently undergoing chemotherapy. In order to establish a separate mono-clonal etiology of both tumours, immunohistochemistry was performed. To the best of our knowledge, carcinoma multiplex in the colon and the parotid has never been reported before. (author)

  6. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

    Science.gov (United States)

    Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

    2015-01-01

    Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

  7. Utility of a multiplex reverse transcriptasepolymerase chain reaction assay (HemaVision in the evaluation of genetic abnormalities in Korean children with acute leukemia: a single institution study

    Directory of Open Access Journals (Sweden)

    Hye-Jin kim

    2013-06-01

    Full Text Available &lt;b&gt;Purpose:&lt;/b&gt; In children with acute leukemia, bone marrow genetic abnormalities (GA have prognostic significance, and may be the basis for minimal residual disease monitoring. Since April 2007, we have used a multiplex reverse transcriptase-polymerase chain reaction tool (HemaVision to detect of GA. &lt;b&gt;Methods:&lt;/b&gt; In this study, we reviewed the results of HemaVision screening in 270 children with acute leukemia, newly diagnosed at The Catholic University of Korea from April 2007 to December 2011, and compared the results with those of fluorescence in situ hybridization (FISH, and G-band karyotyping. &lt;b&gt;Results:&lt;/b&gt; Among the 270 children (153 males, 117 females, 187 acute lymphoblastic leukemia and 74 acute myeloid leukemia patients were identified. Overall, GA was detected in 230 patients (85.2%. HemaVision, FISH, and G-band karyotyping identified GA in 125 (46.3%, 126 (46.7%, and 215 patients (79.6%, respectively. TEL-AML1 (20.9%, 39/187 and AML1-ETO (27%, 20/74 were the most common GA in ALL and AML, respectively. Overall sensitivity of HemaVision was 98.4%, with false-negative results in 2 instances: 1 each for TEL-AML1 and MLL-AF4 . An aggregate of diseasesspecific FISH showed 100% sensitivity in detection of GA covered by HemaVision for actual probes utilized. G-band karyotype revealed GA other than those covered by HemaVison screening in 133 patients (49.3%. Except for hyperdiplody and hypodiploidy, recurrent GA as defined by the World Health Organizationthat were not screened by HemaVision, were absent in the karyotype. &lt;b&gt;Conclusion:&lt;/b&gt; HemaVision, supported by an aggregate of FISH tests for important translocations, may allow for accurate diagnosis of GA in Korean children with acute leukemia.

  8. A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World.

    Science.gov (United States)

    Gilligan, Todd M; Tembrock, Luke R; Farris, Roxanne E; Barr, Norman B; van der Straten, Marja J; van de Vossenberg, Bart T L H; Metz-Verschure, Eveline

    2015-01-01

    The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult-adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.

  9. A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae in the New World.

    Directory of Open Access Journals (Sweden)

    Todd M Gilligan

    Full Text Available The Old World bollworm, Helicoverpa armigera (Hübner, and the corn earworm, H. zea (Boddie, are two of the most important agricultural pests in the world. Diagnosing these two species is difficult-adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2 amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.

  10. Evolutionary diversification of protein-protein interactions by interface add-ons.

    Science.gov (United States)

    Plach, Maximilian G; Semmelmann, Florian; Busch, Florian; Busch, Markus; Heizinger, Leonhard; Wysocki, Vicki H; Merkl, Rainer; Sterner, Reinhard

    2017-10-03

    Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein-protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein-protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein-protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein-protein interactions.

  11. Development of a multiplex Luminex assay for detecting swine antibodies to structural and nonstructural proteins of foot-and-mouth disease virus in Taiwan.

    Science.gov (United States)

    Chen, Tsu-Han; Lee, Fan; Lin, Yeou-Liang; Pan, Chu-Hsiang; Shih, Chia-Ni; Tseng, Chun-Hsien; Tsai, Hsiang-Jung

    2016-04-01

    Foot-and-mouth disease (FMD) and swine vesicular disease (SVD) are serious vesicular diseases that have devastated swine populations throughout the world. The aim of this study was to develop a multianalyte profiling (xMAP) Luminex assay for the differential detection of antibodies to the FMD virus of structural proteins (SP) and nonstructural proteins (NSP). After the xMAP was optimized, it detected antibodies to SP-VP1 and NSP-3ABC of the FMD virus in a single serum sample. These tests were also compared with 3ABC polypeptide blocking enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT) methods for the differential diagnosis and assessment of immune status, respectively. To detect SP antibodies in 661 sera from infected naïve pigs and vaccinated pigs, the diagnostic sensitivity (DSn) and diagnostic specificity (DSp) of the xMAP were 90.0-98.7% and 93.0-96.5%, respectively. To detect NSP antibodies, the DSn was 90% and the DSp ranged from 93.3% to 99.1%. The xMAP can detect the immune response to SP and NSP as early as 4 days postinfection and 8 days postinfection, respectively. Furthermore, the SP and NSP antibodies in all 15 vaccinated but unprotected pigs were detected by xMAP. A comparison of SP and NSP antibodies detected in the sera of the infected samples indicated that the results from the xMAP had a high positive correlation with results from the VNT and a 3ABC polypeptide blocking ELISA assay. However, simultaneous quantitation detected that xMAP had no relationship with the VNT. Furthermore, the specificity was 93.3-94.9% with 3ABC polypeptide blocking ELISA for the FMDV-NSP antibody. The results indicated that xMAP has the potential to detect antibodies to FMDV-SP-VP1 and NSP-3ABC and to distinguish FMDV-infected pigs from pigs infected with the swine vesicular disease virus. Copyright © 2014. Published by Elsevier B.V.

  12. A misleading false-negative result using Neisseria gonorrhoeae opa MGB multiplex PCR assay in patient's rectal sample due to partial mutations of the opa gene.

    Science.gov (United States)

    Vahidnia, Ali; van Empel, Pieter Jan; Costa, Sandra; Oud, Rob T N; van der Straaten, Tahar; Bliekendaal, Harry; Spaargaren, Joke

    2015-07-01

    A 53-year-old homosexual man presented at his general practitioner (GP) practice with a suspicion of sexually transmitted infection. Initial NAAT screening was performed for Chlamydia trachomatis and Neisseria gonorrhoeae. The patient was positive for Neisseria gonorrhoeae both for his urine and rectal sample. The subsequent confirmation test for Neisseria gonorrhoeae by a second laboratory was only confirmed for the urine sample and the rectal sample was negative. We report a case of a potential false-negative diagnosis of Neisseria gonorrhoeae due to mutations of DNA sequence in the probe region of opa-MGB assay of the rectal sample. The patient did not suffer any discomfort as diagnosis of Neisseria gonorrhoeae in his urine sample had already led to treatment by prescribing the patient with Ceftriaxone 500 mg IV dissolved in 1 ml lidocaine 2% and 4 mL saline. The patient also received a prescription for Azithromycin (2x500 mg).

  13. Add-on effect of Brahmi in the management of schizophrenia

    Directory of Open Access Journals (Sweden)

    Sukanto Sarkar

    2012-01-01

    Full Text Available Brahmi(Bacopa monnieri, an Ayurvedic herb has primarily been used to enhance cognitive ability, memory and learning skills. We present a case study of schizophrenia in which add-on Brahmi extracts 500 mg/day for a period of one month resulted in reduction in psychopathology without any treatment-emergent adverse effect. Although preliminary, our case study suggests therapeutic efficacy of add-on Brahmi in schizophrenia, thus opening up a new dimension of its role in alternative medicines.

  14. Appropriate statistical methods are required to assess diagnostic tests for replacement, add-on, and triage

    NARCIS (Netherlands)

    Hayen, Andrew; Macaskill, Petra; Irwig, Les; Bossuyt, Patrick

    2010-01-01

    To explain which measures of accuracy and which statistical methods should be used in studies to assess the value of a new binary test as a replacement test, an add-on test, or a triage test. Selection and explanation of statistical methods, illustrated with examples. Statistical methods for

  15. Development of a radiometer based on an Add-ON card for PC

    International Nuclear Information System (INIS)

    Bolanos, L.; Lopez, B.; Fernandez, J.L.; Laria, J.

    1997-01-01

    The design of an add-on card for PC that enables it to record nuclear pulses provided by two channels is described in this paper. This card carries acts as a pulse discriminator, and according to the software it finds many appliances in radioimmunoassay, nuclear medicine, industry and other fields

  16. Ritanserin as add-on medication to neuroleptic therapy for patients with chronic or subchronic schizophrenia

    NARCIS (Netherlands)

    Den Boer, JA; Vahlne, JO; Post, P; Heck, AH; Daubenton, F; Olbrich, R

    The effect of ritanserin, a potent 5HT(2A/2C) receptor antagonist, used as an add-on medication to neuroleptic treatmentin patients with schizophrenia, was compared with that of placebo, in an international, double-blind, parallel-group study. Previously established neuroleptic therapy was

  17. Weight gain with add-on second-generation antipsychotics in bipolar disorder: a naturalistic study.

    Science.gov (United States)

    Najar, H; Joas, E; Kardell, M; Pålsson, E; Landén, M

    2017-06-01

    Our aim was to investigate the prevalence and magnitude of weight gain in-patients with bipolar disorder when treated with a second-generation antipsychotic as an add-on treatment to a mood stabilizer in routine clinical practice. Data were derived from the quality register for bipolar disorder in Sweden (BipoläR). Patients with bipolar disorder who started add-on treatment with a SGA (n = 575) were compared at next yearly follow-up with age and sex matched patients who were only treated with a mood stabilizer (n = 566). The primary outcome measure was change in body weight and body mass index (BMI). We also assessed the prevalence of clinically significant weight gain defined as ≥7% gain in body weight. The group that received add-on treatment with antipsychotics neither gained more weight nor were at higher risk for a clinically significant weight gain than the reference group. Instead, factors associated with clinically significant weight gain were female sex, young age, low-baseline BMI, and occurrence of manic/hypomanic episodes. We found no evidence of an overall increased risk of weight gain for patients with bipolar disorder after receiving add-on SGA to a mood stabilizer in a routine clinical setting. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Pleasure and pedagogy: the consumption of DVD add-ons among Irish teenagers

    OpenAIRE

    Brereton, Pat; O'Connor, Barbara

    2007-01-01

    This article addresses the issue of young people and media use in the digital age, more specifically the interconnection between new media pleasures and pedagogy as they relate to the consumption of DVD add-ons. Arguing against the view of new media as having predominantly detrimental effects on young people, the authors claim that new media can enable young people to develop media literacy skills and are of the view that media literacy strategies must be based on an understanding and legitim...

  19. Hera-FFX: a Firefox add-on for Semi-automatic Web Accessibility Evaluation

    OpenAIRE

    Fuertes Castro, José Luis; González, Ricardo; Gutiérrez, Emmanuelle; Martínez Normand, Loïc

    2009-01-01

    Website accessibility evaluation is a complex task requiring a combination of human expertise and software support. There are several online and offline tools to support the manual web accessibility evaluation process. However, they all have some weaknesses because none of them includes all the desired features. In this paper we present Hera-FFX, an add-on for the Firefox web browser that supports semi-automatic web accessibility evaluation.

  20. Development of an add-on kit for scanning confocal microscopy (Conference Presentation)

    Science.gov (United States)

    Guo, Kaikai; Zheng, Guoan

    2017-03-01

    Scanning confocal microscopy is a standard choice for many fluorescence imaging applications in basic biomedical research. It is able to produce optically sectioned images and provide acquisition versatility to address many samples and application demands. However, scanning a focused point across the specimen limits the speed of image acquisition. As a result, scanning confocal microscope only works well with stationary samples. Researchers have performed parallel confocal scanning using digital-micromirror-device (DMD), which was used to project a scanning multi-point pattern across the sample. The DMD based parallel confocal systems increase the imaging speed while maintaining the optical sectioning ability. In this paper, we report the development of an add-on kit for high-speed and low-cost confocal microscopy. By adapting this add-on kit to an existing regular microscope, one can convert it into a confocal microscope without significant hardware modifications. Compared with current DMD-based implementations, the reported approach is able to recover multiple layers along the z axis simultaneously. It may find applications in wafer inspection and 3D metrology of semiconductor circuit. The dissemination of the proposed add-on kit under $1000 budget could also lead to new types of experimental designs for biological research labs, e.g., cytology analysis in cell culture experiments, genetic studies on multicellular organisms, pharmaceutical drug profiling, RNA interference studies, investigation of microbial communities in environmental systems, and etc.

  1. 40 CFR Table 1 to Subpart Jjjj of... - Operating Limits if Using Add-On Control Devices and Capture System

    Science.gov (United States)

    2010-07-01

    ... Limits if Using Add-On Control Devices and Capture System If you are required to comply with operating... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Operating Limits if Using Add-On Control Devices and Capture System 1 Table 1 to Subpart JJJJ of Part 63 Protection of Environment...

  2. 40 CFR Table 2 to Subpart Oooo of... - Operating Limits if Using Add-On Control Devices and Capture System

    Science.gov (United States)

    2010-07-01

    ... OOOO of Part 63—Operating Limits if Using Add-On Control Devices and Capture System If you are required... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Operating Limits if Using Add-On Control Devices and Capture System 2 Table 2 to Subpart OOOO of Part 63 Protection of Environment...

  3. 40 CFR Table 1 to Subpart Ssss of... - Operating Limits if Using Add-on Control Devices and Capture System

    Science.gov (United States)

    2010-07-01

    ... Using Add-on Control Devices and Capture System If you are required to comply with operating limits by... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Operating Limits if Using Add-on Control Devices and Capture System 1 Table 1 to Subpart SSSS of Part 63 Protection of Environment...

  4. Prospective evaluation of the SeptiFAST multiplex real-time PCR assay for surveillance and diagnosis of infections in haematological patients after allogeneic stem cell transplantation compared to routine microbiological assays and an in-house real-time PCR method.

    Science.gov (United States)

    Elges, Sandra; Arnold, Renate; Liesenfeld, Oliver; Kofla, Grzegorz; Mikolajewska, Agata; Schwartz, Stefan; Uharek, Lutz; Ruhnke, Markus

    2017-12-01

    We prospectively evaluated a multiplex real-time PCR assay (SeptiFast, SF) in a cohort of patients undergoing allo-BMT in comparison to an in-house PCR method (IH-PCR). Overall 847 blood samples (mean 8 samples/patient) from 104 patients with haematological malignancies were analysed. The majority of patients had acute leukaemia (62%) with a mean age of 52 years (54% female). Pathogens could be detected in 91 of 847 (11%) samples by SF compared to 38 of 205 (18.5%) samples by BC, and 57 of 847 (6.7%) samples by IH-PCR. Coagulase-negative staphylococci (n=41 in SF, n=29 in BC) were the most frequently detected bacteria followed by Escherichia coli (n=9 in SF, n=6 in BC). Candida albicans (n=17 in SF, n=0 in BC, n=24 in IH-PCR) was the most frequently detected fungal pathogen. SF gave positive results in 5% of samples during surveillance vs in 26% of samples during fever episodes. Overall, the majority of blood samples gave negative results in both PCR methods resulting in 93% overall agreement resulting in a negative predictive value of 0.96 (95% CI: 0.95-0.97), and a positive predictive value of 0.10 (95% CI: -0.01 to 0.21). SeptiFast appeared to be superior over BC and the IH-PCR method. © 2017 Blackwell Verlag GmbH.

  5. Which thiazide to choose as add-on therapy for hypertension?

    Directory of Open Access Journals (Sweden)

    Barrios V

    2014-07-01

    Full Text Available Vivencio Barrios,1 Carlos Escobar2 1Department of Cardiology, Hospital Ramon y Cajal, 2Department of Cardiology, Hospital La Paz, Madrid, Spain Abstract: Combined therapy is required in the majority of patients with hypertension to achieve blood pressure (BP targets. Although different antihypertensive drugs can be combined, not all combinations are equally effective and safe. In this context, the combination of a renin angiotensin system inhibitor with a diuretic, usually a thiazide, particularly hydrochlorothiazide (HCTZ or thiazide-like diuretics, such as chlorthalidone or indapamide, is recommended. However, not all diuretics are equal. Although HCTZ, chlorthalidone, and indapamide as add-on therapy effectively reduce BP levels, the majority of studies have obtained greater BP reductions with chlorthalidone or indapamide than with HCTZ. Moreover, there are data showing benefits with chlorthalidone or indapamide beyond BP. Thus, chlorthalidone seems to have pleiotropic effects beyond BP reduction. Moreover, compared with placebo, chlorthalidone has small effects on fasting glucose and total cholesterol, and compared with HCTZ, chlorthalidone achieves significantly lower total cholesterol and low-density lipoprotein cholesterol levels. Similarly, indapamide has demonstrated no negative impact on glucose or lipid metabolism. More importantly, although head-to-head clinical trials comparing the effects of indapamide or chlorthalidone with HCTZ are not available, indirect comparisons and post hoc analyses suggest that the use of chlorthalidone or indapamide is associated with a reduction in cardiovascular events. Despite this, the most frequent diuretic used in clinical practice as add-on therapy for hypertension is HCTZ. The purpose of this review is to update the published data on the efficacy and safety of HCTZ, chlorthalidone, and indapamide as add-on therapy in patients with hypertension. Keywords: blood pressure control, hydrochlorothiazide

  6. Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

    Science.gov (United States)

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-05-05

    We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

  7. WITHDRAWN: Oxcarbazepine add-on for drug-resistant partial epilepsy.

    Science.gov (United States)

    Castillo, Sergio M; Schmidt, Dieter B; White, Sarah; Shukralla, Arif

    2016-11-15

    Most people with epilepsy have a good prognosis and their seizures can be well controlled with the use of a single antiepileptic drug, but up to 30% develop refractory epilepsy, especially those with partial seizures. In this review we summarize the current evidence regarding oxcarbazepine when used as an add-on treatment for drug-resistant partial epilepsy. To evaluate the effects of oxcarbazepine when used as an add-on treatment for drug-resistant partial epilepsy. We searched the Cochrane Epilepsy Group's Specialized Register (28 March 2006), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 1, 2006), MEDLINE (1966 to March 2006). No language restrictions were imposed. We checked the reference lists of retrieved studies for additional reports of relevant studies. We also contacted Novartis (manufacturers of oxcarbazepine) and experts in the field. Randomized, placebo-controlled, double-blinded, add-on trials of oxcarbazepine in patients with drug-resistant partial epilepsy. Two review authors independently assessed trials for inclusion and extracted the relevant data. The following outcomes were assessed : (a) 50% or greater reduction in seizure frequency; (b) treatment withdrawal (any reason); (c) side effects. Primary analyses were intention-to-treat. Summary odds ratios were estimated for each outcome. Two trials were included representing 961 randomized patients.Overall Odds Ratio (OR) (95% Confidence Interval (CIs)) for 50% or greater reduction in seizure frequency compared to placebo 2.96 (2.20, 4.00).Treatment withdrawal OR (95% CIs) compared to placebo 2.17 (1.59, 2.97).Side effects: OR (99% CIs) compared to placebo, ataxia 2.93 (1.72, 4.99); dizziness 3.05 (1.99, 4.67); fatigue 1.80 (1.02, 3.19); nausea 2.88 (1.77, 4.69); somnolence 2.55 (1.84, 3.55); diplopia 4.32 (2.65, 7.04), were significantly associated with oxcarbazepine. Oxcarbazepine has efficacy as an add-on treatment in patients with drug

  8. A high resolution 16 k multi-channel analyzer PC add-on card

    International Nuclear Information System (INIS)

    Kulkarni, C.P.; Paulson, Molly; Vaidya, P.P.

    2001-01-01

    This paper describes the system details of a 16 K channel resolution Multi-Channel Analyzer (MCA) developed at Electronics Division, BARC, which is used in high resolution nuclear spectroscopy systems for pulse height analysis. The high resolution data acquisition PC add-on card is architectured using a state of the art digital circuit design technology which makes use of a Field Programmable Gate Array (FPGA), and some of the most modern and advanced analog counterparts like low power, high speed and high precision comparators, Op-amps, ADCs and DACs etc. The 16 K MCA card gives an economic, compact, and low power alternative for nuclear pulse spectroscopy use. (author)

  9. Erythropoietin as an add-on treatment for cognitive side effects of electroconvulsive therapy

    DEFF Research Database (Denmark)

    Schmidt, Lejla Sjanic; Petersen, Jeff Zarp; Vinberg, Maj

    2018-01-01

    trial investigates (1) whether short-term add-on treatment with erythropoietin (EPO) can reduce the cognitive side -effects of ECT and (2) whether such effects are long-lasting. Further, structural and functional magnetic resonance imaging (MRI) will be used to explore the neural underpinnings...... cognitive benefits of EPO are investigated with structural and functional MRI after the final EPO/saline infusion. The primary outcome is change from baseline to after EPO treatment (3 days after eight ECT sessions) in a cognitive composite score spanning attention, psychomotor speed, and executive...... of covariance. Functional MRI data will be preprocessed and analyzed using the FMRIB Software Library....

  10. Theophylline as an add-on to thrombolytic therapy in acute ischaemic stroke (TEA-Stroke)

    DEFF Research Database (Denmark)

    Modrau, Boris; Hjort, Niels; Østergaard, Leif

    2016-01-01

    the collateral supply in acute ischaemic brain tissue and thus facilitate reperfusion despite proximal vessel occlusion. The primary study objective is to evaluate whether theophylline is safe and efficient in acute ischaemic stroke patients as an add-on to thrombolytic therapy.MethodsThe TEA-Stroke Trial...... models, clinical case series and randomized clinical trials are controversial. A Cochrane analysis from 2004 concluded that there was not enough evidence to assess whether theophylline is safe and improves outcomes in patients with acute ischaemic stroke. The TEA-Stroke Trial will clarify whether...

  11. Development of a colonoscopy add-on device for improvement of the intubation process

    Directory of Open Access Journals (Sweden)

    Litten JD

    2011-12-01

    Full Text Available Jonathan D Litten1, JungHun Choi2, David Drozek31Department of Mechanical Engineering; 2Department of Mechanical Engineering and Biomedical Engineering Program; 3College of Osteopathic Medicine, Department of Specialty Medicine, Ohio University, Athens, OH, USAAbstract: A colonoscopy add-on device has been developed to reduce intubation time without modification of the current colonoscope and peripheral devices. One of the main purposes of the system is to minimize trauma caused by the distal tip of the colonoscope. The detachable sensory fixture at the end of the distal tip measures the distance between the distal tip and the colon wall in three directions, and the actuation system attached at the base of the colonoscope controls the distal tip by rotating two dial knobs. The device controls the distal tip to minimize contact between the distal tip and the colon wall, and the distal tip ideally points out the next possible lumen. A compatibility test of the infrared sensory system was carried out, and the design of the actuation system was accomplished. The system is integrated and controlled by a microprocessor. The device was tested in a silicon colon and porcine intestine. The results showed that a colonoscopist successfully reached the cecum with the aid of the colonoscopy add-on device without significant contact between the colon wall and the distal tip. The colonoscopy aid device was very helpful for the novice colonoscopist.Keywords: colonoscope, infrared sensors, intubation, trauma, colonoscopy training model

  12. radEq Add-On Module for CFD Solver Loci-CHEM

    Science.gov (United States)

    McCloud, Peter

    2013-01-01

    Loci-CHEM to be applied to flow velocities where surface radiation due to heating from compression and friction becomes significant. The module adds a radiation equilibrium boundary condition to the computational fluid dynamics (CFD) code to produce accurate results. The module expanded the upper limit for accurate CFD solutions of Loci-CHEM from Mach 4 to Mach 10 based on Space Shuttle Orbiter Re-Entry trajectories. Loci-CHEM already has a very promising architecture and performance, but absence of radiation equilibrium boundary condition limited the application of Loci-CHEM to below Mach 4. The immediate advantage of the add-on module is that it allows Loci-CHEM to work with supersonic flows up to Mach 10. This transformed Loci-CHEM from a rocket engine- heritage CFD code with general subsonic and low-supersonic applications, to an aeroheating code with hypersonic applications. The follow-on advantage of the module is that it is a building block for additional add-on modules that will solve for the heating generated at Mach numbers higher than 10.

  13. [The add-on effect of omalizumab on patients with uncontrolled bronchial asthma].

    Science.gov (United States)

    Minami, Yoshinori; Endo, Satoshi; Okumur, Shunsuke; Sasaki, Takaaki; Yamamoto, Yasushi; Ogasa, Toshiyuki; Osanai, Shinobu; Ohsaki, Yoshinobu

    2011-11-01

    A high-dose administration of inhaled corticosteroid is effective in the majority of patients with bronchial asthma, but is often difficult to attain sufficient control in certain subsets of patients. Omalizumab has recently emerged as a promising drug for bronchial asthma. To assess its add-on effect we administered omalizumab to patients with uncontrolled atopic asthma for more than 16 weeks and gave them questionnaires. The study population comprised 9 patients with frequent asthmatic symptoms despite the administration of high-dose inhaled corticosteroid and other disease controllers. We scored disease control using the Asthma Health Questionnaire-33-Japan and the Asthma Control Test, and evaluated the frequencies of short-acting beta2-agonist use for rescue and drip infusion of theophyllines and/or systemic steroids in a retrospective fashion. Asthmatic scores were significantly improved after 16 weeks of omalizumab therapy. The frequencies of reliever use and drip infusion were also decreased. These trends were present even in patients in whom no aeroallergen-specific IgE antibodies were detected. No statistically significant side effects were observed. Our study confirmed the add-on effect of omalizumab based on evaluation by simple questionnaires. Further studies are needed to clarify whether omalizumab therapy is suitable for patients without specific IgE antibodies.

  14. Integrated photonics : compact multiplexing

    NARCIS (Netherlands)

    Pile, D.; Chen, H.; Uden, van R.G.H.; Okonkwo, C.M.; Koonen, A.M.J.

    2015-01-01

    Spatial multiplexers (SMUXs) for mode division multiplexing often involve multiple strategies for mode-selective excitation and the minimization of insertion and other losses. Haoshuo Chen, Roy van Uden, Chigo Okonkwo and Ton Koonen, working at the COBRA Institute at the Eindhoven University of

  15. Dynamic Optically Multiplexed Imaging

    Science.gov (United States)

    2015-07-29

    Dynamic Optically Multiplexed Imaging Yaron Rachlin, Vinay Shah, R. Hamilton Shepard, and Tina Shih Lincoln Laboratory, Massachusetts Institute of...V. Shah, and T. Shih “Design Architectures for Optically Multiplexed Imaging,” in submission 9 R. Gupta , P. Indyk, E. Price, and Y. Rachlin

  16. Addiction surplus: the add-on margin that makes addictive consumptions difficult to contain.

    Science.gov (United States)

    Adams, Peter J; Livingstone, Charles

    2015-01-01

    Addictive consumptions generate financial surpluses over-and-above non-addictive consumptions because of the excessive consumption of addicted consumers. This add-on margin or 'addiction surplus' provides a powerful incentive for beneficiaries to protect their income by ensuring addicted consumers keep consuming. Not only that, addiction surplus provides the financial base that enables producers to sponsor activities which aim to prevent public health initiatives from reducing consumption. This paper examines the potency of addiction surplus to engage industry, governments and communities in an on-going reliance on addiction surplus. It then explores how neo-liberal constructions of a rational consumer disguise the ethical and exploitative dynamics of addiction surplus by examining ways in which addictive consumptions fail to conform to notions of autonomy and rationality. Four measures are identified to contain the distorting effects of addiction surplus. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Eslicarbazepine acetate add-on for drug-resistant partial epilepsy.

    Science.gov (United States)

    Chang, Xian-Chao; Yuan, Hai; Wang, Yi; Xu, Hui-Qin; Hong, Wen-Ke; Zheng, Rong-Yuan

    2017-10-25

    This is an updated version of the Cochrane Review published in the Cochrane Library 2011, Issue 12.The majority of people with epilepsy have a good prognosis, but up to 30% of people continue to have seizures despite several regimens of antiepileptic drugs. In this review, we summarized the current evidence regarding eslicarbazepine acetate (ESL) when used as an add-on treatment for drug-resistant partial epilepsy. To evaluate the efficacy and tolerability of ESL when used as an add-on treatment for people with drug-resistant partial epilepsy. The searches for the original review were run in November 2011. Subsequently, we searched the Cochrane Epilepsy Group Specialized Register (6 December 2016), the Cochrane Central Register of Controlled Trials (CENTRAL 2016, Issue 11) and MEDLINE (1946 to 6 December 2016). There were no language restrictions. We reviewed the reference lists of retrieved studies to search for additional reports of relevant studies. We also contacted the manufacturers of ESL and experts in the field for information about any unpublished or ongoing studies. Randomized placebo controlled double-blind add-on trials of ESL in people with drug-resistant partial epilepsy. Two review authors independently selected trials for inclusion and extracted data. Outcomes investigated included 50% or greater reduction in seizure frequency, seizure freedom, treatment withdrawal, adverse effects, and drug interactions. Primary analyses were by intention to treat (ITT). The dose-response relationship was evaluated in regression models. We included five trials (1799 participants) rated at low risk of bias; all studies were funded by BIAL. The overall risk ratio (RR) with 95% confidence interval (CI) for 50% or greater reduction in seizure frequency was 1.71 (95% CI 1.42 to 2.05). Dose regression analysis showed evidence that ESL reduced seizure frequency with an increase in efficacy with increasing doses of ESL. ESL was significantly associated with seizure freedom

  18. Circadian rest-activity rhythms during benzodiazepine tapering covered by melatonin versus placebo add-on

    DEFF Research Database (Denmark)

    Baandrup, Lone; Fasmer, Ole Bernt; Glenthøj, Birte Yding

    2016-01-01

    is associated with changes in circadian rhythm parameters. METHOD: Data were derived from a randomized, double-blinded clinical trial with 24 weeks follow-up. Participants were randomized to add-on treatment with prolonged-release melatonin (2 mg) or matching placebo, and usual benzodiazepine dosage...... significantly increased the interdaily stability and at a trend level decreased the intradaily variability compared with placebo. Benzodiazepine dose reduction was not associated with these circadian rhythm parameters. Activity counts were generally higher after benzodiazepine dose reduction compared with pre......BACKGROUND: Patients with severe mental illness often suffer from disruptions in circadian rest-activity cycles, which might partly be attributed to ongoing psychopharmacological medication. Benzodiazepines are frequently prescribed for prolonged periods despite recommendations of only short...

  19. Numerical Study on Self-Cleaning Canister Filter With Add-On Filter Cap

    Directory of Open Access Journals (Sweden)

    Mohammed Akmal Nizam

    2017-01-01

    Full Text Available Filtration in a turbo machinery system such as a gas turbine will ensure that the air entering the inlet is free from contaminants that could bring damage to the main system. Self-cleaning filter systems for gas turbines are designed for continuously efficient flow filtration. A good filter would be able to maintain its effectiveness over a longer time period, prolonging the duration between filter replacements and providing lower pressure drop over its operating lifetime. With this goal in mind, the current study is focused on the difference in pressure loss of the benchmark Salutary Avenue Self-cleaning filter in comparison to a new design with an add-on filter cap. Geometry for the add-on filter cap will be based from Salutary Avenue Manufacturing Sdn.Bhd. SOLIDWORKS software was used to model the geometry of the filter, while simulation analysis on the flow through the filter was done using Computational Fluid Dynamic (CFD software. The simulations are based on a low velocity condition, in which the parameter for the inlet velocity are set at 0.032 m/s, 0.063 m/s, 0.094 m/s and 0.126 m/s respectively. From the simulation data obtained for the inlet velocities considered, the pressure drop reduction of the modified filter compared to the benchmark was found to be between 7.59% and 30.18%. All in all, the modification of the filter cap produced a lower pressure drop in comparison with the benchmark filter; an improvement of 27.02% for the total pressure drop was obtained.

  20. 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.

  1. 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.

  2. Multiplex gas chromatography

    Science.gov (United States)

    Valentin, Jose R.

    1990-01-01

    The principles of the multiplex gas chromatography (GC) technique, which is a possible candidate for chemical analysis of planetary atmospheres, are discussed. Particular attention is given to the chemical modulators developed by present investigators for multiplex GC, namely, the thermal-desorption, thermal-decomposition, and catalytic modulators, as well as to mechanical modulators. The basic technique of multiplex GC using chemical modulators and a mechanical modulator is demonstrated. It is shown that, with the chemical modulators, only one gas stream consisting of the carrier in combination with the components is being analyzed, resulting in a simplified instrument that requires relatively few consumables. The mechanical modulator demonstrated a direct application of multiplex GC for the analysis of gases in atmosphere of Titan at very low pressures.

  3. Multiplexed Engineering in Biology.

    Science.gov (United States)

    Rogers, Jameson K; Church, George M

    2016-03-01

    Biotechnology is the manufacturing technology of the future. However, engineering biology is complex, and many possible genetic designs must be evaluated to find cells that produce high levels of a desired drug or chemical. Recent advances have enabled the design and construction of billions of genetic variants per day, but evaluation capacity remains limited to thousands of variants per day. Here we evaluate biological engineering through the lens of the design–build–test cycle framework and highlight the role that multiplexing has had in transforming the design and build steps. We describe a multiplexed solution to the ‘test’ step that is enabled by new research. Achieving a multiplexed test step will permit a fully multiplexed engineering cycle and boost the throughput of biobased product development by up to a millionfold.

  4. Add-on conservation benefits of marine territorial user rights fishery policies in central Chile.

    Science.gov (United States)

    Gelcich, Stefan; Godoy, Natalio; Prado, Luis; Castilla, Juan Carlos

    2008-01-01

    To combine the rational use of marine benthic resources and economic development of small-scale fishers, Chile passed legislation in 1991 establishing a comanagement policy that grants exclusive territorial user rights for fisheries (TURFs) to artisanal fisher organizations in well-defined inshore coastal areas, known as Management and Exploitation Areas for Benthic Resources (MEABRs). In general the policy has been proclaimed a management and economic success because benthic resource abundances have increased inside MEABRs in comparison with open-access areas. However, there is a lack of studies assessing the impact of this management policy on nontargeted subtidal species and community assemblages and the policy's implications for biodiversity and conservation. This study starts to fill this gap and links the allocation of TURFs for benthic resources with add-on conservation benefits for species that are not directly linked with the fishery policy. Comparative subtidal surveys inside vs. outside MEABRs were used to assess the effects of three MEABRs on managed targeted benthic species, biodiversity (species richness), and community assemblages in central Chile. Surveys focused exclusively on subtidal kelp forest habitats dominated by Lessonia trabeculata, spanning 4-12 m in depth and with similar levels of habitat complexity. The study comprised: (1) quantification of kelp forest complexity, (2) understory survey of sessile species, (3) quantification of conspicuous benthic macroinvertebrates, including those under management, and (4) quantification of reef-fish species inside the kelp habitat. Results showed population enhancement of target-managed invertebrates inside MEABRs. Moreover, reef-fish species were significantly more diverse and abundant inside MEABRs, and community assemblages of nontarget benthic invertebrates and reef fish were significantly different inside vs. outside MEABRs. The comanagement of inshore benthic resources in Chile, through MEABRs

  5. Bilevel alarm monitoring multiplexer

    International Nuclear Information System (INIS)

    Johnson, C.S.

    1977-06-01

    This report describes the operation of the Bilevel Alarm Monitoring Multiplexer used in the Adaptive Intrusion Data System (AIDS) to transfer and control alarm signals being sent to the Nova 2 computer, the Memory Controlled Data Processor, and its own integral Display Panel. The multiplexer can handle 48 alarm channels and format the alarms into binary formats compatible with the destination of the alarm data

  6. ATOM - an OMERO add-on for automated import of image data

    Directory of Open Access Journals (Sweden)

    Lipp Peter

    2011-10-01

    Full Text Available Abstract Background Modern microscope platforms are able to generate multiple gigabytes of image data in a single experimental session. In a routine research laboratory workflow, these data are initially stored on the local acquisition computer from which files need to be transferred to the experimenter's (remote image repository (e.g., DVDs, portable hard discs or server-based storage because of limited local data storage. Although manual solutions for this migration, such as OMERO - a client-server software for visualising and managing large amounts of image data - exist, this import process may be a time-consuming and tedious task. Findings We have developed ATOM, a Java-based and thus platform-independent add-on for OMERO enabling automated transfer of image data from a wide variety of acquisition software packages into OMERO. ATOM provides a graphical user interface and allows pre-organisation of experimental data for the transfer. Conclusions ATOM is a convenient extension of the OMERO software system. An automated interface to OMERO will be a useful tool for scientists working with file formats supported by the Bio-Formats file format library, a platform-independent library for reading the most common file formats of microscope images.

  7. A New and Versatile Adjustable Rigidity Actuator with Add-on Locking Mechanism (ARES-XL

    Directory of Open Access Journals (Sweden)

    Manuel Cestari

    2018-01-01

    Full Text Available Adjustable compliant actuators are being designed and implemented in robotic devices because of their ability to minimize large forces due to impacts, to safely interact with the user, and to store and release energy in passive elastic elements. Conceived as a new force-controlled compliant actuator, an adjustable rigidity with embedded sensor and locking mechanism actuator (ARES-XL is presented in this paper. This compliant system is intended to be implemented in a gait exoskeleton for children with neuro muscular diseases (NMDs to exploit the intrinsic dynamics during locomotion. This paper describes the mechanics and initial evaluation of the ARES-XL, a novel variable impedance actuator (VIA that allows the implementation of an add-on locking mechanism to this system, and in combination with its zero stiffness capability and large deflection range, provides this novel joint with improved properties when compared to previous prototypes developed by the authors and other state-of-the-art (SoA devices. The evaluation of the system proves how this design exceeds the main capabilities of a previous prototype as well as providing versatile actuation that could lead to its implementation in multiple joints.

  8. Final Technical Report: Thermoelectric-Enhanced Cookstove Add-on (TECA) for Clean Biomass Cookstoves

    Energy Technology Data Exchange (ETDEWEB)

    Stokes, David [RTI International, Research Triangle Park, NC (United States)

    2015-09-29

    This program seeks to demonstrate a solution to enhance existing biomass cookstove performance through the use of RTI’s Thermoelectric Enhanced Cookstove Add-on (TECA) device. The self-powered TECA device captures a portion of heat from the stove and converts it to electricity through a thermoelectric (TE) device to power a blower. Colorado State University and Envirofit International are partners to support the air injection design and commercialization to enhance combustion in the stove and reduce emissions. Relevance: By demonstrating a proof of concept of the approach with the Envirofit M-5000 stove and TECA device, we hope to apply this technology to existing stoves that are already in use and reduce emissions for stoves that have already found user acceptance to provide a true health benefit. Challenges: The technical challenges include achieving Tier 4 emissions from a biomass stove and for such a stove to operate reliably in the harsh field environment. Additional challenges include the fact that it is difficult to develop a cost effective solution and insure adoption and proper use in the field. Outcomes: In this program we have demonstrated PM emissions at 82 mg/MJd, a 70% reduction as compared to baseline stove operation. We have also developed a stove optimization approach that reduces the number of costly experiments. We have evaluated component-level reliability and will be testing the stove prototype in the field for performance and reliability.

  9. Studies with lucid dreaming as add-on therapy to Gestalt therapy.

    Science.gov (United States)

    Holzinger, B; Klösch, G; Saletu, B

    2015-06-01

    The aim of the present exploratory clinical study was to evaluate LD as an add-on therapy for treating nightmares. Thirty-two subjects having nightmares (ICD-10: F51.5) at least twice a week participated. Subjects were randomly assigned to group: A) Gestalt therapy group (= GTG), or B) Gestalt and lucid dreaming group therapy (= LDG). Each group lasted ten weeks. Participants kept a sleep/dream diary over the treatment. Examinations with respect to nightmare frequency and sleep quality (Pittsburgh Sleep Quality Index) were carried out at the beginning, after five and ten weeks and at a follow-up three months later. Concerning nightmare frequency, a significant reduction was found in both groups after the ten-week-study and at the follow-up (Wilcoxon test: P ≤ 0.05). Significant reduction in dream recall frequency could only be observed in the GTG (Wilcoxon test: P ≤ 0.05). For subjects having succeeded in learning lucid dreaming, reduction was sooner and higher. Sleep quality improved for both groups at the follow-up (P ≤ 0.05, Wilcoxon test). Only the LDG showed significant improvement at the end of therapy (P ≤ 0.05). Lucid dreaming, in combination with Gestalt therapy, is a potent technique to reduce nightmare frequency and improve the subjective quality of sleep. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Mozart K.448 acts as a potential add-on therapy in children with refractory epilepsy.

    Science.gov (United States)

    Lin, Lung-Chang; Lee, Wei-Te; Wang, Chien-Hua; Chen, Hsiu-Lin; Wu, Hui-Chuan; Tsai, Chin-Lin; Wei, Ruey-Chang; Mok, Hin-Kiu; Weng, Chia-Fen; Lee, Mei-Wen; Yang, Rei-Cheng

    2011-03-01

    Mozart's Sonata for two pianos in D major, K.448 (Mozart K.448), has been shown to improve mental function, leading to what is known as the Mozart effect. Our previous work revealed that epileptiform discharges in children with epilepsy decreased during and immediately after listening to Mozart K.448. In this study, we evaluated the long-term effects of Mozart K.448 on children with refractory epilepsy. Eleven children with refractory epilepsy were enrolled. All of the patients were diagnosed as having had refractory epilepsy for more than 1 year (range =1 year to 6 years 4 months, mean =3 years 11 months) and had been receiving at least two antiepileptic drugs (AED). During the study period, they listened to Mozart K.448 once a day before bedtime for 6 months. Seizure frequencies were recorded 6 months before they started listening to this music and monthly during the study period. All of the patients remained on the same AEDs during the 6-month study period. Frequencies of seizures were compared before and after listening to Mozart K.448. Eight of eleven patients were seizure free (N=2) or had very good responses (N=6) after 6 months of listening to Mozart K.448. The remaining three (27.3%) showed minimal or no effect (effectiveness Mozart K.448 should be further studied as a potential add-on therapy in the treatment of children with refractory epilepsy. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Effects of pitavastatin add-on therapy on chronic kidney disease with albuminuria and dyslipidemia.

    Science.gov (United States)

    Ohsawa, Masato; Tamura, Kouichi; Wakui, Hiromichi; Kanaoka, Tomohiko; Azushima, Kengo; Uneda, Kazushi; Haku, Sona; Kobayashi, Ryu; Ohki, Kohji; Haruhara, Kotaro; Kinguchi, Sho; Toya, Yoshiyuki; Umemura, Satoshi

    2015-12-09

    In non-dialysis chronic kidney disease (CKD) patients with dyslipidemia, statin therapy is recommended to prevent cardiovascular complications. Dyslipidemia has been also shown to be an independent risk factor for the progression of CKD. However, it is still unclear whether statin therapy exerts an inhibitory effect on renal deterioration in CKD patients with dyslipidemia. The purpose of the present study was to examine possible therapeutic effects of statin add-on therapy on renal function as well as parameters of lipid and glucose metabolism, arterial stiffness and oxidative stress, in comparison to diet therapy, in CKD patients with dyslipidemia. This study was a randomized, open-label, and parallel-group trial consisted of a 12-months treatment period in non-dialysis CKD patients with alubuminuria and dyslipidemia. Twenty eight patients were randomly assigned either to receive diet counseling alone (diet therapy group) or diet counseling plus pitavastatin (diet-plus-statin therapy group), to achieve the LDL-cholesterol (LDL-C) target of dyslipidemia.

  12. Add-on deep Transcranial Magnetic Stimulation (dTMS) for the treatment of chronic migraine: A preliminary study.

    Science.gov (United States)

    Rapinesi, Chiara; Del Casale, Antonio; Scatena, Paola; Kotzalidis, Georgios D; Di Pietro, Simone; Ferri, Vittoria Rachele; Bersani, Francesco Saverio; Brugnoli, Roberto; Raccah, Ruggero Nessim; Zangen, Abraham; Ferracuti, Stefano; Orzi, Francesco; Girardi, Paolo; Sette, Giuliano

    2016-06-03

    Deep Transcranial Magnetic Stimulation (dTMS) can be an alternative treatment to relieve pain in chronic migraine (CM). The aim of this study was to evaluate the effect of high-frequency dTMS in add-on to standard treatment for CM in patients not responding to effective abortive or preventive drug treatment. We randomized 14 patients with International Classification of Headache Disorders, 3rd Edition (ICHD-3) treatment-resistant CM to add-on dTMS (n=7) or standard abortive or preventive antimigraine treatment (n=7). Three sessions of alternate day 10Hz dTMS consisting of 600 pulses in 10 trains were delivered to the dorsolateral prefrontal cortex (DLPFC), bilaterally, but with left hemisphere prevalence, for 12 sessions spread over one month. The add-on dTMS treatment was well tolerated. Patients treated with dTMS showed significant reduction of pain intensity, frequency of attacks, analgesic overuse, and depressive symptoms during treatment and one month later, compared to the month preceding treatment and at the same time-points compared to the control group. As compared to standard pharmacological treatment alone, add-on high-frequency dTMS of the bilateral DLPFC reduced the frequency and intensity of migraine attack, drug overuse, and depressive symptoms. This study supports the add-on dTMS treatment in treatment-resistant CM. Copyright © 2016. Published by Elsevier Ireland Ltd.

  13. 40 CFR 63.4567 - How do I establish the emission capture system and add-on control device operating limits during...

    Science.gov (United States)

    2010-07-01

    ... system and add-on control device operating limits during the performance test? 63.4567 Section 63.4567... Emission Rate with Add-on Controls Option § 63.4567 How do I establish the emission capture system and add-on control device operating limits during the performance test? During the performance test required...

  14. 40 CFR 63.3546 - How do I establish the emission capture system and add-on control device operating limits during...

    Science.gov (United States)

    2010-07-01

    ... system and add-on control device operating limits during the performance test? 63.3546 Section 63.3546... device or system of multiple capture devices. The average duct static pressure is the maximum operating... Add-on Controls Option § 63.3546 How do I establish the emission capture system and add-on control...

  15. 40 CFR 63.4767 - How do I establish the emission capture system and add-on control device operating limits during...

    Science.gov (United States)

    2010-07-01

    ... system and add-on control device operating limits during the performance test? 63.4767 Section 63.4767... Rate with Add-on Controls Option § 63.4767 How do I establish the emission capture system and add-on control device operating limits during the performance test? During the performance test required by § 63...

  16. 40 CFR 63.4167 - How do I establish the emission capture system and add-on control device operating limits during...

    Science.gov (United States)

    2010-07-01

    ... system and add-on control device operating limits during the performance test? 63.4167 Section 63.4167... with Add-on Controls Option § 63.4167 How do I establish the emission capture system and add-on control device operating limits during the performance test? During the performance test required by § 63.4160...

  17. 40 CFR 63.4966 - How do I establish the emission capture system and add-on control device operating limits during...

    Science.gov (United States)

    2010-07-01

    ... system and add-on control device operating limits during the performance test? 63.4966 Section 63.4966... outlet gas temperature is the maximum operating limit for your condenser. (e) Emission capture system... with Add-on Controls Option § 63.4966 How do I establish the emission capture system and add-on control...

  18. 40 CFR 63.3169 - What are the requirements for a capture system or add-on control device which is not taken into...

    Science.gov (United States)

    2010-07-01

    ... systems or add-on control devices which you choose not to take into account when demonstrating compliance... system or add-on control device which is not taken into account when demonstrating compliance with the....3169 What are the requirements for a capture system or add-on control device which is not taken into...

  19. Add-On Effect of Probucol in Atherosclerotic, Cholesterol-Fed Rabbits Treated with Atorvastatin

    Science.gov (United States)

    Keyamura, Yuka; Nagano, Chifumi; Kohashi, Masayuki; Niimi, Manabu; Nozako, Masanori; Koyama, Takashi; Yasufuku, Reiko; Imaizumi, Ayako; Itabe, Hiroyuki; Yoshikawa, Tomohiro

    2014-01-01

    Objective Lowering the blood concentration of low-density lipoprotein (LDL) cholesterol is the primary strategy employed in treating atherosclerotic disorders; however, most commonly prescribed statins prevent cardiovascular events in just 30% to 40% of treated patients. Therefore, additional treatment is required for patients in whom statins have been ineffective. In this study of atherosclerosis in rabbits, we examined the effect of probucol, a lipid-lowering drug with potent antioxidative effects, added to treatment with atorvastatin. Methods and Results Atherosclerosis was induced by feeding rabbits chow containing 0.5% cholesterol for 8 weeks. Probucol 0.1%, atorvastatin 0.001%, and atorvastatin 0.003% were administered solely or in combination for 6 weeks, beginning 2 weeks after the start of atherosclerosis induction. Atorvastatin decreased the plasma concentration of non-high-density lipoprotein cholesterol (non-HDLC) dose-dependently; atorvastatin 0.003% decreased the plasma concentration of non-HDLC by 25% and the area of atherosclerotic lesions by 21%. Probucol decreased the plasma concentration of non-HDLC to the same extent as atorvastatin (i.e., by 22%) and the area of atherosclerotic lesions by 41%. Probucol with 0.003% atorvastatin decreased the plasma concentration of non-HDLC by 38% and the area of atherosclerotic lesions by 61%. Co-administration of probucol with atorvastatin did not affect the antioxidative effects of probucol, which were not evident on treatment with atorvastatin alone, such as prevention of in vitro LDL-oxidation, increase in paraoxonase-1 activity of HDL, and decreases in plasma and plaque levels of oxidized-LDL in vivo. Conclusions Probucol has significant add-on anti-atherosclerotic effects when combined with atorvastatin treatment; suggesting that this combination might be beneficial for treatment of atherosclerosis. PMID:24810608

  20. Establishing a Near Term Lunar Farside Gravity Model via Inexpensive Add-on Navigation Payload

    Science.gov (United States)

    Folta, David; Mesarch, Michael; Miller, Ronald; Bell, David; Jedrey, Tom; Butman, Stanley; Asmar, Sami

    2007-01-01

    The Space Communications and Navigation, Constellation Integration Project (SCIP) is tasked with defining, developing, deploying and operating an evolving multi-decade communications and navigation (C/N) infrastructure including services and subsystems that will support both robotic and human exploration activities at the Moon. This paper discusses an early far side gravitational mapping service and related telecom subsystem that uses an existing spacecraft (WIND) and the Lunar Reconnaissance Orbiter (LRO) to collect data that would address several needs of the SCIP. An important aspect of such an endeavor is to vastly improve the current lunar gravity model while demonstrating the navigation and stationkeeping of a relay spacecraft. We describe a gravity data acquisition activity and the trajectory design of the relay orbit in an Earth-Moon L2 co-linear libration orbit. Several phases of the transfer from an Earth-Sun to the Earth-Moon region are discussed along with transfers within the Earth-Moon system. We describe a proposed, but not integrated, add-on to LRO scheduled to be launched in October of 2008. LRO provided a real host spacecraft against which we designed the science payload and mission activities. From a strategic standpoint, LRO was a very exciting first flight opportunity for gravity science data collection. Gravity Science data collection requires the use of one or more low altitude lunar polar orbiters. Variations in the lunar gravity field will cause measurable variations in the orbit of a low altitude lunar orbiter. The primary means to capture these induced motions is to monitor the Doppler shift of a radio signal to or from the low altitude spacecraft, given that the signal is referenced to a stable frequency reference. For the lunar far side, a secondary orbiting radio signal platform is required. We provide an in-depth look at link margins, trajectory design, and hardware implications. Our approach posed minimum risk to a host mission while

  1. Inflammation in Patients with Schizophrenia: the Therapeutic Benefits of Risperidone Plus Add-On Dextromethorphan

    Science.gov (United States)

    Chen, Shiou-Lan; Lee, Sheng-Yu; Chang, Yun-Hsuan; Chen, Shih-Heng; Chu, Chun-Hsieh; Tzeng, Nian-Sheng; Lee, I-Hui; Chen, Po-See; Yeh, Tzung Lieh; Huang, San-Yuan; Yang, Yen-Kuang; Lu, Ru-Band; Hong, Jau-Shyong

    2013-01-01

    Objectives Increasing evidence suggests that inflammation contributes to the etiology and progression of schizophrenia. Molecules that initiate inflammation, such as virus- and toxin-induced cytokines, are implicated in neuronal degeneration and schizophrenia-like behavior. Using therapeutic agents with anti-inflammatory or neurotrophic effects may be beneficial for treating schizophrenia. Methods One hundred healthy controls and 95 Han Chinese patients with schizophrenia were tested in this double-blind study. Their PANSS scores, plasma interleukin (IL)-1β, TNF-α and brain-derived neurotrophic factor (BDNF) levels were measured before and after pharmacological treatment. Results Pretreatment, plasma levels of IL-1β and TNF-α were significantly higher in patients with schizophrenia than in controls, but plasma BDNF levels were significantly lower. Patients were treated with the atypical antipsychotic risperidone (Risp) only or with Risp+add-on dextromethorphan (DM). PANSS scores and plasma IL-1β levels significantly decreased, but plasma TNF-α and BDNF levels significantly increased after 11 weeks of Risp treatment. Patients in the Risp+DM group showed a greater and earlier reduction of symptoms than did those in the Risp-only group. Moreover, Risp+DM treatment attenuated Risp-induced plasma increases in TNF-α. Conclusion Patients with schizophrenia had a high level of peripheral inflammation and a low level of peripheral BDNF. Long-term Risp treatment attenuated inflammation and potentiated the neurotrophic function but also produced a certain degree of toxicity. Risp+DM was more beneficial and less toxic than Risp-only treatment. PMID:22730040

  2. Effects of Vildagliptin Add-on Insulin Therapy on Nocturnal Glycemic Variations in Uncontrolled Type 2 Diabetes.

    Science.gov (United States)

    Li, Feng-Fei; Shen, Yun; Sun, Rui; Zhang, Dan-Feng; Jin, Xing; Zhai, Xiao-Fang; Chen, Mao-Yuan; Su, Xiao-Fei; Wu, Jin-Dan; Ye, Lei; Ma, Jian-Hua

    2017-10-01

    To investigate whether vildagliptin add-on insulin therapy improves glycemic variations in patients with uncontrolled type 2 diabetes (T2D) compared to patients with placebo therapy. This was a 24-week, single-center, double-blind, placebo-controlled trial. Inadequately controlled T2D patients treated with insulin therapy were recruited between June 2012 and April 2013. The trial included a 2-week screening period and a 24-week randomized period. Subjects were randomly assigned to a vildagliptin add-on insulin therapy group (n = 17) or a matched placebo group (n = 16). Scheduled visits occurred at weeks 4, 8, 12, 16, 20, and 24. Continuous glucose monitoring (CGM) was performed before and at the endpoint of the study. A total of 33 subjects were admitted, with 1 patient withdrawing from the placebo group. After 24 weeks of therapy, HbA1c values were significantly reduced at the endpoint in the vildagliptin add-on group. CGM data showed that patients with vildagliptin add-on therapy had a significantly lower 24-h mean glucose concentration and mean amplitude of glycemic excursion (MAGE). At the endpoint of the study, patients in the vildagliptin add-on group had a significantly lower MAGE and standard deviation compared to the control patients during the nocturnal period (0000-0600). A severe hypoglycemic episode was not observed in either group. Vildagliptin add-on therapy to insulin has the ability to improve glycemic variations, especially during the nocturnal time period, in patients with uncontrolled T2D.

  3. Randomized, controlled trial of rasagiline as an add-on to dopamine agonists in Parkinson's disease.

    Science.gov (United States)

    Hauser, Robert A; Silver, Dee; Choudhry, Azhar; Eyal, Eli; Isaacson, Stuart

    2014-07-01

    Dopamine agonists (DA) are often used as first-line monotherapy for the symptomatic control of Parkinson's disease (PD). However, DA monotherapy typically becomes inadequate within a few years, at which time the DA dosage must be increased or other antiparkinsonian medications added. Adding a monoamine oxidase-B (MAO-B) inhibitor to DA monotherapy might improve symptomatic control while maintaining good safety and tolerability. We conducted an 18-week, randomized, double-blind, placebo-controlled trial of rasagiline 1 mg/d as an add-on to DA therapy (ropinirole ≥ 6 mg/d or pramipexole ≥ 1.0 mg/d) in early PD patients whose conditions were not adequately controlled on their current treatment regimen. The primary efficacy variable was the change in total Unified Parkinson Disease Rating Scale (UPDRS) score (sum of parts I, II, and III) from baseline to week 18, comparing rasagiline and placebo groups. The modified intent-to-treat (ITT) population included 321 subjects whose mean ± SD age was 62.6 ± 9.7, and duration of PD was 2.1 ± 2.1 years. Results demonstrated a significantly greater improvement in total UPDRS scores from baseline to week 18 in the rasagiline group compared with the placebo group (least squares [LS] mean difference ± SE, -2.4 ± 0.95; 95% confidence interval [CI], -4.3, -0.5; P = 0.012). Mean improvement (LS mean ± SE) was -3.6 ± 0.68 in the rasagiline group and -1.2 ± 0.68 in the placebo group. Rasagiline was well tolerated, and the most common adverse events (AEs; rasagiline vs. placebo) were dizziness (7.4% vs. 6.1%), somnolence (6.8% vs. 6.7%), and headache (6.2% vs. 4.3%). Rasagiline 1 mg/d provided statistically significant improvement when added to dopamine agonist therapy and was well tolerated. © 2014 International Parkinson and Movement Disorder Society.

  4. 40 CFR 63.4566 - How do I determine the add-on control device emission destruction or removal efficiency?

    Science.gov (United States)

    2010-07-01

    ... by volume (ppmv), dry basis. Qsd = Volumetric flow rate of gases entering or exiting the add-on...). 0.0416 = Conversion factor for molar volume, kg-moles per cubic meter (mol/m3) (@ 293 Kelvin (K) and... to 40 CFR part 60, as appropriate, to measure gas volumetric flow rate. (3) Use Method 3, 3A, or 3B...

  5. 40 CFR Table 1 to Subpart IIIi of... - Operating Limits for Capture Systems and Add-On Control Devices

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Operating Limits for Capture Systems... 63—Operating Limits for Capture Systems and Add-On Control Devices If you are required to comply with operating limits by § 63.3093, you must comply with the applicable operating limits in the following table...

  6. Measurement of the Economic Growth and Add-on of the R.M. Solow Adjusted Model

    Directory of Open Access Journals (Sweden)

    Ion Gh. Rosca

    2007-08-01

    Full Text Available Besides the models of M. Keynes, R.F. Harrod, E. Domar, D. Romer, Ramsey-Cass-Koopmans model etc., the R.M. Solow model is part of the category which characterizes the economic growth.The paper aim is the economic growth measurement and add-on of the R.M. Solow adjusted model.

  7. A high-throughput multiplex method adapted for GMO detection.

    Science.gov (United States)

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  8. Coherence Multiplex System Topologies

    NARCIS (Netherlands)

    Meijerink, Arjan; Taniman, R.O.; Heideman, G.H.L.M.; van Etten, Wim

    2007-01-01

    Coherence multiplexing is a potentially inexpensive form of optical code-division multiple access, which is particularly suitable for short-range applications with moderate bandwidth requirements, such as access networks, LANs, or interconnects. Various topologies are known for constructing an

  9. Microprocessorized message multiplexer

    International Nuclear Information System (INIS)

    Ejzman, S.; Guglielmi, L.; Jaeger, J.J.

    1980-07-01

    The 'Microprocessorized Message Multiplexer' is an elementary development tool used to create and debug the software of a target microprocessor (User Module: UM). It connects together four devices: a terminal, a cassette recorder, the target microprocessor and a host computer where macro and editor for the M 6800 microprocessor are resident [fr

  10. 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....

  11. 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.

  12. Characterization of an add-on multileaf collimator for electron beam therapy

    International Nuclear Information System (INIS)

    Gauer, T; Sokoll, J; Cremers, F; Schmidt, R; Harmansa, R; Luzzara, M

    2008-01-01

    An add-on multileaf collimator for electrons (eMLC) has been developed that provides computer-controlled beam collimation and isocentric dose delivery. The design parameters result from the design study by Gauer et al (2006 Phys. Med. Biol. 51 5987-6003) and were configured such that a compact and light-weight eMLC with motorized leaves can be industrially manufactured and stably mounted on a conventional linear accelerator. In the present study, the efficiency of an initial computer-controlled prototype was examined according to the design goals and the performance of energy- and intensity-modulated treatment techniques. This study concentrates on the attachment and gantry stability as well as the dosimetric characteristics of central-axis and off-axis dose, field size dependence, collimator scatter, field abutment, radiation leakage and the setting of the accelerator jaws. To provide isocentric irradiation, the eMLC can be placed either 16 or 28 cm above the isocentre through interchangeable holders. The mechanical implementation of this feature results in a maximum field displacement of less than 0.6 mm at 90 0 and 270 0 gantry angles. Compared to a 10 x 10 cm applicator at 6-14 MeV, the beam penumbra of the eMLC at a 16 cm collimator-to-isocentre distance is 0.8-0.4 cm greater and the depth-dose curves show a larger build-up effect. Due to the loss in energy dependence of the therapeutic range and the much lower dose output at small beam sizes, a minimum beam size of 3 x 3 cm is necessary to avoid suboptimal dose delivery. Dose output and beam symmetry are not affected by collimator scatter when the central axis is blocked. As a consequence of the broader beam penumbra, uniform dose distributions were measured in the junction region of adjacent beams at perpendicular and oblique beam incidence. However, adjacent beams with a high difference in a beam energy of 6 to 14 MeV generate cold and hot spots of approximately 15% in the abutting region. In order to

  13. Characterization of an add-on multileaf collimator for electron beam therapy

    Energy Technology Data Exchange (ETDEWEB)

    Gauer, T; Sokoll, J; Cremers, F; Schmidt, R [Department of Radiotherapy and Radio-Oncology, University Medical Center Hamburg-Eppendorf, Hamburg (Germany); Harmansa, R [3D Line, Schwarzenbruck (Germany); Luzzara, M [3D Line, Milan (Italy)], E-mail: t.gauer@uke.uni-hamburg.de

    2008-02-21

    An add-on multileaf collimator for electrons (eMLC) has been developed that provides computer-controlled beam collimation and isocentric dose delivery. The design parameters result from the design study by Gauer et al (2006 Phys. Med. Biol. 51 5987-6003) and were configured such that a compact and light-weight eMLC with motorized leaves can be industrially manufactured and stably mounted on a conventional linear accelerator. In the present study, the efficiency of an initial computer-controlled prototype was examined according to the design goals and the performance of energy- and intensity-modulated treatment techniques. This study concentrates on the attachment and gantry stability as well as the dosimetric characteristics of central-axis and off-axis dose, field size dependence, collimator scatter, field abutment, radiation leakage and the setting of the accelerator jaws. To provide isocentric irradiation, the eMLC can be placed either 16 or 28 cm above the isocentre through interchangeable holders. The mechanical implementation of this feature results in a maximum field displacement of less than 0.6 mm at 90{sup 0} and 270{sup 0} gantry angles. Compared to a 10 x 10 cm applicator at 6-14 MeV, the beam penumbra of the eMLC at a 16 cm collimator-to-isocentre distance is 0.8-0.4 cm greater and the depth-dose curves show a larger build-up effect. Due to the loss in energy dependence of the therapeutic range and the much lower dose output at small beam sizes, a minimum beam size of 3 x 3 cm is necessary to avoid suboptimal dose delivery. Dose output and beam symmetry are not affected by collimator scatter when the central axis is blocked. As a consequence of the broader beam penumbra, uniform dose distributions were measured in the junction region of adjacent beams at perpendicular and oblique beam incidence. However, adjacent beams with a high difference in a beam energy of 6 to 14 MeV generate cold and hot spots of approximately 15% in the abutting region. In

  14. 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 Θ ˜ 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.

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

    KAUST Repository

    Harrison, Hugo B.; Feldheim, Kevin Andrew; Jones, Geoffrey P.; Mansour, Hicham; Perumal, Sadhasivam; Williamson, David H.; Berumen, Michael L.

    2014-01-01

    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

  16. Lutzomyia (Pintomyia) fischeri (Diptera: Psychodidae: Phlebotominae), a probable vector of American cutaneous leishmaniasis: detection of natural infection by Leishmania (Viannia) DNA in specimens from the municipality of Porto Alegre (RS), Brazil, using multiplex PCR assay.

    Science.gov (United States)

    Pita-Pereira, Daniela de; Souza, Getúlio D; Pereira, Thaís de Araújo; Zwetsch, Adriana; Britto, Constança; Rangel, Elizabeth F

    2011-12-01

    In order to determine natural Leishmania (Viannia) infection in Lutzomyia (Pintomyia) fischeri, a multiplex PCR methodology coupled to non-isotopic hybridization was adopted for the analysis of sand fly samples collected by CDC light traps in an endemic area of American Cutaneous Leishmaniasis (ACL) in the periurban region of the municipality of Porto Alegre, Rio Grande do Sul State, Brazil. We analyzed by PCR methodology 560 specimens of Lutzomyia (Pintomyia) fischeri (520 females and 40 males). The wild sand flies were grouped into 56 pools (52 females and 4 males) of 10 each, and positive results were detected in 2 of the 52 female pools, representing a minimum infection rate of 0.38% based on the presence of at least 1 infected insect in the pool. This result associated with some local evidence such as anthopophily, spatial distribution in accordance with the transmission area and human case incidence, suggests that L. (P.)fischeri may be considered as a secondary vector of ACL in the studied locality. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Stereotaxic core needle biopsy of breast microcalcifications obtained using a standard mammography table with an add-on unit

    International Nuclear Information System (INIS)

    Ward, S.E.; Taves, D.H.; McCurdy, L.I.

    2000-01-01

    To demonstrate the reliability of stereotaxic biopsy of indeterminate microcalcifications using a standard mammography table with an add-on unit. In 121 cases of indeterminate microcalcifications, core biopsy was performed using a standard mammography table with an add-on stereotaxic unit. Microcalcifications were identified on radiography of core specimens. Microcalcifications and a definitive histologic diagnosis were obtained in 112 core biopsies (92.6%), with no significant complications. In 23 lesions frank malignancy was diagnosed, and all of these diagnoses were confirmed on surgery. Pathologic examination suggested carcinoma in 4 lesions, and open biopsy confirmed malignancy in 3 of these cases. Four lesions showed atypical ductal hyperplasia. Benign disease was diagnosed in 81 lesions, of which 78 remained stable on mammographic follow-up (mean 16 months later) and 3 were subjected to surgical biopsy (of which 1 was malignant and 2 were benign). Nine cases were technically unsatisfactory because microcalcifications were not sampled. Stereotaxic core biopsy performed with an add-on unit is a safe and reliable technique for biopsy of indeterminate microcalcifications. For successful biopsy, microcalcifications must be harvested. Pathologic results should be correlated with mammographic findings. The accuracy rate compares favourably with results reported using prone biopsy tables. In an era of cost containment, this alternative to prone biopsy tables could result m significant savings in terms of capital investment and use of hospital rooms. In this study, surgical biopsy could have been avoided in 64.5% of cases. (author)

  18. Monitoring bacterial faecal contamination in waters using multiplex ...

    African Journals Online (AJOL)

    Monitoring of sanitary quality or faecal pollution in water is currently based on quantifying some bacterial indicators such as Escherichia coli and faecal enterococci. Using a multiplex real-time PCR assay for faecal enterococci and Bacteroides spp., the detection of faecal contamination in non-treated water can be done in a ...

  19. Bright light treatment as add-on therapy for depression in 28 adolescents: a randomized trial.

    Science.gov (United States)

    Niederhofer, Helmut; von Klitzing, Kai

    2011-01-01

    In the last decade, a significant incidence of depression in the younger population has been observed. Bright light therapy, an effective therapeutic option for depressed adults, could also provide safe, economical, and effective rapid recovery in adolescents. The randomized trial included 28 inpatients (18 females and 10 males) between 14 and 17 years old with depressive complaints. The study was conducted between February and December of 2010 in Rodewisch, Germany. Half of the patients (n = 14) first received placebo (50 lux) 1 hour a day in the morning from 9:00 am to 10:00 am for 1 week and then received bright light therapy (2,500 lux) for 1 week in the morning from 9:00 am to 10:00 am. The other half (n = 14) first received bright light therapy and then received placebo. Patients were encouraged to continue ongoing treatment (fluoxetine 20 mg/day and 2 sessions of psychotherapy/week) because there were no changes in medication/dosage and psychotherapy since 1 month before the 4-week study period. For assessment of depressive symptoms, the Beck Depression Inventory (BDI) was administered 1 week before and 1 day before placebo treatment, on the day between placebo and bright light treatment, and on the day after and 1 week after bright light treatment. Saliva samples of melatonin and cortisol were collected at 8:00 am and 8:00 pm 1 week before and 1 day before placebo treatment, on the day between placebo and bright light treatment, on the day after bright light treatment, and 1 week after bright light treatment and were assayed for melatonin and cortisol to observe any change in circadian timing. The BDI scores improved significantly (P = .015). The assays of saliva showed significant differences between treatment and placebo for evening melatonin (P = .040). No significant adverse reactions were observed. Antidepressant response to bright light treatment in this age group was statistically superior to placebo. World Health Organization International Clinical

  20. Multiplexed Western Blotting Using Microchip Electrophoresis.

    Science.gov (United States)

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-05

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

  1. A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma

    DEFF Research Database (Denmark)

    Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John

    2008-01-01

    -based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both...... reactivity levels to twenty eight different recombinant PfEMP1 proteins were simultaneously measured using a single microliter of plasma. Thus, the assay reported here provides a useful tool for rapid and efficient quantification of antibody reactivity against PfEMP1 variants in human plasma....... of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1...

  2. 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.

  3. Multiplex measuring systems in physics

    International Nuclear Information System (INIS)

    Soroko, L.M.

    1980-01-01

    The principles of operation of multiplex devices used in different spheres of physics are discussed. The ''multiplex'' notion means that the data output of the device is an integral image of the functional dependence under investigation, but not its readings as in usual instruments. The analysis of the present state of developments of the multiplex systems in optics, nuclear magnetic resonance spectroscopy, in time-of-flight spectrometers for slow and fast neutrons, as well as elementary particle detectors, is given. The construction algorithms for the digital codes are presented, the history of development of the multiplex measuring principle is given [ru

  4. 40 CFR 63.3555 - How do I determine the outlet THC emissions and add-on control device emission destruction or...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 12 2010-07-01 2010-07-01 true How do I determine the outlet THC.../outlet Concentration Option § 63.3555 How do I determine the outlet THC emissions and add-on control... section to determine either the outlet THC emissions or add-on control device emission destruction or...

  5. Evaluation of homoeopathic medicines as add-on to institutional management protocol in Acute Encephalitis Syndrome: An exploratory observational comparative study

    Directory of Open Access Journals (Sweden)

    Raj K Manchanda

    2015-01-01

    Conclusion: This exploratory observational study suggests reduction of mortality and morbidity with add-on homoeopathic medicine. Further randomized controlled trial study with comparable groups is desirable. If findings are confirmed by subsequent research, add-on Homoeopathy might have relevant implication for its management.

  6. 40 CFR Table 4 to Subpart Kkkk of... - Operating Limits If Using the Emission Rate With Add-on Controls Option or the Control Efficiency...

    Science.gov (United States)

    2010-07-01

    ... Rate With Add-on Controls Option or the Control Efficiency/Outlet Concentration Compliance Option 4... Add-on Controls Option or the Control Efficiency/Outlet Concentration Compliance Option If you are... volumetric flow rate at a location upstream of the control device, or duct static pressure at a location...

  7. 40 CFR 63.3556 - How do I establish the emission capture system and add-on control device operating limits during...

    Science.gov (United States)

    2010-07-01

    ... system and add-on control device operating limits during the performance test? 63.3556 Section 63.3556... of key parameters of the valve operating system (e.g., solenoid valve operation, air pressure.../outlet Concentration Option § 63.3556 How do I establish the emission capture system and add-on control...

  8. 40 CFR 63.3967 - How do I establish the emission capture system and add-on control device operating limits during...

    Science.gov (United States)

    2010-07-01

    ... system and add-on control device operating limits during the performance test? 63.3967 Section 63.3967... capture system and add-on control device operating limits during the performance test? During the... the operating limits required by § 63.3892 according to this section, unless you have received...

  9. 40 CFR 63.9324 - How do I establish the emission capture system and add-on control device operating limits during...

    Science.gov (United States)

    2010-07-01

    ... capture system and add-on control device operating limits during the performance test? 63.9324 Section 63... Requirements § 63.9324 How do I establish the emission capture system and add-on control device operating... the operating limits required by § 63.9302 according to this section, unless you have received...

  10. 40 CFR 63.3174 - What are the requirements for a capture system or add-on control device which is not taken into...

    Science.gov (United States)

    2010-07-01

    ... control device which is not taken into account when demonstrating compliance with the applicable emission limitations? You may have capture systems or add-on control devices which you choose not to take into account... system or add-on control device which is not taken into account when demonstrating compliance with the...

  11. Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

    Science.gov (United States)

    Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

    2010-02-01

    An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

  12. A simple electron multiplexer

    International Nuclear Information System (INIS)

    Dobrzynski, L; Akjouj, A; Djafari-Rouhani, B; Al-Wahsh, H; Zielinski, P

    2003-01-01

    We present a simple multiplexing device made of two atomic chains coupled by two other transition metal atoms. We show that this simple atomic device can transfer electrons at a given energy from one wire to the other, leaving all other electron states unaffected. Closed-form relations between the transmission coefficients and the inter-atomic distances are given to optimize the desired directional electron ejection. Such devices can be adsorbed on insulating substrates and characterized by current surface technologies. (letter to the editor)

  13. The COMT Val158Met Polymorphism Is Associated With Response to Add-on Dextromethorphan Treatment in Bipolar Disorder.

    Science.gov (United States)

    Lee, Sheng-Yu; Chen, Shiou-Lan; Wang, Tzu-Yun; Chang, Yun-Hsuan; Chen, Po See; Huang, San-Yuan; Tzeng, Nian-Sheng; Wang, Liang-Jen; Lee, I Hui; Chen, Kao Ching; Yang, Yen Kuang; Lu, Ru-Band

    2017-02-01

    We previously conducted a randomized, double-blind, controlled, 12-week study evaluating the effect of add-on dextromethorphan (DM), a noncompetitive N-methyl-D-aspartate receptor antagonist, on patients with bipolar disorder (BD) treated using valproate (VPA), which showed negative clinical differences. The genetic variation between each individual may be responsible for interindividual differences. The catechol-O-methyltransferase (COMT) gene has been a candidate gene for BD. In the current study, we investigated whether the COMT Val158Met polymorphism predicts treatment response to VPA + add-on DM and to VPA + placebo. Patients with BD (n = 309) undergoing regular VPA treatments were randomly assigned to groups given either add-on DM (30 mg/d) (n = 102), DM (60 mg/d) (n = 101), or placebo (n = 106) for 12 weeks. The Hamilton Depression Rating Scale and Young Mania Rating Scale were used to evaluate clinical response during weeks 0, 1, 2, 4, 8, and 12. The genotypes of the COMT Val158Met polymorphism were determined using polymerase chain reaction plus restriction fragment length polymorphism analysis. To adjust for within-subject dependence over repeated assessments, multiple linear regression with generalized estimating equation methods was used. When stratified by the COMT Val158Met genotypes, significantly greater decreases in Hamilton Depression Rating Scale scores were found in the VPA + DM (30 mg/d) group in patients with the Val/Met genotype (P = 0.008). We conclude that the COMT Val158Met polymorphism may influence responses to DM (30 mg/d) by decreasing depressive symptoms in BD patients.

  14. Cost-effectiveness of add-on treatments to metformin in a Swedish setting: liraglutide vs sulphonylurea or sitagplitin.

    Science.gov (United States)

    Steen Carlsson, Katarina; Persson, Ulf

    2014-09-01

    To evaluate long-run cost-effectiveness in a Swedish setting for liraglutide compared with sulphonylureas (glimepiride) or sitagliptin, all as add-on to metformin for patients with type 2 diabetes insufficiently controlled with metformin in monotherapy. The IHE Cohort Model of Type 2 Diabetes was used to evaluate clinical and economic outcomes from a societal perspective. Model input data were obtained from two clinical trials, the Swedish National Diabetes Register and the literature. Cost data reflected year 2013 price level. The robustness of results was checked with one-way-sensitivity analysis and probability sensitivity analysis. The cost per QALY gained for liraglutide (1.2 mg) compared to SU (glimepiride 4 mg), both as add-on to metformin, ranged from SEK 226,000 to SEK 255,000 in analyzed patient cohorts. The cost per QALY for liraglutide (1.2 mg) vs sitagliptin (100 mg) as second-line treatment was lower, ranging from SEK 149,000 to SEK 161,000. Costs of preventive treatment were driving costs, but there was also a cost offset from reduced costs of complications of ∼ 20%. Notable cost differences were found for nephropathy, stroke, and heart failure. The predicted life expectancy with liraglutide increased the cost of net consumption for liraglutide. The analysis was an ex-ante analysis using model input data from clinical trials which may not reflect effectiveness in real-world clinical practice in broader patient populations. This limitation was explored in the sensitivity analysis. The lack of specific data on loss of production due to diabetes complications implied that these costs may be under-estimated. Treatment strategies with liraglutide 1.2 mg improved the expected quality-of-life and increased costs when compared to SU and to sitagliptin for second-line add-on treatments. The cost per QALY for liraglutide was in the range considered medium by Swedish authorities.

  15. Usage of OpenStack Virtual Machine and MATLAB HPC Add-on leads to faster turnaround

    KAUST Repository

    Van Waveren, Matthijs

    2017-03-16

    We need to run hundreds of MATLAB® simulations while changing the parameters between each simulation. These simulations need to be run sequentially, and the parameters are defined manually from one simulation to the next. This makes this type of workload unsuitable for a shared cluster. For this reason we are using a cluster running in an OpenStack® Virtual Machine and are using the MATLAB HPC Add-on for submitting jobs to the cluster. As a result we are now able to have a turnaround time for the simulations of the order of a few hours, instead of the 24 hours needed on a local workstation.

  16. Multiplex serology of paraneoplastic antineuronal antibodies.

    Science.gov (United States)

    Maat, Peter; Brouwer, Eric; Hulsenboom, Esther; VanDuijn, Martijn; Schreurs, Marco W J; Hooijkaas, Herbert; Smitt, Peter A E Sillevis

    2013-05-31

    Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diagnosing a neurological syndrome as paraneoplastic (70% is not yet known to have cancer) and in directing the search for the underlying neoplasm. At present six onconeural antibodies are considered 'well characterized' and recognize the antigens HuD, CDR62 (Yo), amphiphysin, CRMP-5 (CV2), NOVA-1 (Ri), and Ma2. The gold standard of detection is the characteristic immunohistochemical staining pattern on brain tissue sections combined with confirmation by immunoblotting using recombinant purified proteins. Since all six onconeural antibodies are usually analyzed simultaneously and objective cut-off values for these analyses are warranted, we developed a multiplex assay based on Luminex technology. Reaction of serial dilutions of six onconeural standard sera with microsphere-bound antigens showed lower limits of detection than with Western blotting. Using the six standard sera at a dilution of 1:200, the average within-run coefficient of variation (CV) was 4% (range 1.9-7.3%). The average between-run within-day CV was 5.1% (range 2.9-6.7%) while the average between-day CV was 8.1% (range 2.8-11.6%). The shelf-life of the antigen coupled microspheres was at least two months. The sensitivity of the multiplex assay ranged from 83% (Ri) to 100% (Yo, amphiphysin, CV2) and the specificity from 96% (CV2) to 100% (Ri). In conclusion, Luminex-based multiplex serology is highly reproducible with high sensitivity and specificity for the detection of onconeural antibodies. Conventional immunoblotting for diagnosis of onconeural antibodies in the setting of a routine laboratory may be replaced by this novel, robust technology. Copyright © 2013 Elsevier B.V. All rights

  17. 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.

  18. Omega-3 fatty acids decreased irritability of patients with bipolar disorder in an add-on, open label study

    Directory of Open Access Journals (Sweden)

    Baldassano Claudia F

    2005-02-01

    Full Text Available Abstract This is a report on a 37-patient continuation study of the open ended, Omega-3 Fatty Acid (O-3FA add-on study. Subjects consisted of the original 19 patients, along with 18 new patients recruited and followed in the same fashion as the first nineteen. Subjects carried a DSM-IV-TR diagnosis of Bipolar Disorder and were visiting a Mood Disorder Clinic regularly through the length of the study. At each visit, patients' clinical status was monitored using the Clinical Monitoring Form. Subjects reported on the frequency and severity of irritability experienced during the preceding ten days; frequency was measured by way of percentage of days in which subjects experienced irritability, while severity of that irritability was rated on a Likert scale of 1 – 4 (if present. The irritability component of Young Mania Rating Scale (YMRS was also recorded quarterly on 13 of the 39 patients consistently. Patients had persistent irritability despite their ongoing pharmacologic and psychotherapy. Omega-3 Fatty Acid intake helped with the irritability component of patients suffering from bipolar disorder with a significant presenting sign of irritability. Low dose (1 to 2 grams per day, add-on O-3FA may also help with the irritability component of different clinical conditions, such as schizophrenia, borderline personality disorder and other psychiatric conditions with a common presenting sign of irritability.

  19. ALDH2 polymorphism, associated with attenuating negative symptoms in patients with schizophrenia treated with add-on dextromethorphan.

    Science.gov (United States)

    Lee, Sheng-Yu; Chen, Shiou-Lan; Chang, Yun-Hsuan; Chen, Po-See; Huang, San-Yuan; Tzeng, Nian-Sheng; Wang, Liang-Jen; Lee, I-Hui; Wang, Tzu-Yun; Chen, Kao-Chin; Yang, Yen-Kuang; Hong, Jau-Shyong; Lu, Ru-Band

    2015-10-01

    Increasing the evidence of inflammation's contribution to schizophrenia; using anti-inflammatory or neurotrophic therapeutic agents to see whether they improve schizophrenia treatment. Dextromethorphan (DM), a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, might protect monoamine neurons. Whether treating schizophrenia with risperidone plus add-on DM is more effective than risperidone (RISP) alone, and the association between the ALDH2 polymorphism and treatment response were investigated. A double-blind study in which patients with schizophrenia were randomly assigned to the RISP + DM (60 mg/day; n = 74) or the RISP + Placebo (n = 75) group. The Positive and Negative Syndrome Scale (PANSS) and the Scale for the Assessment of Negative Symptoms (SANS) scores were used to evaluate clinical response during weeks 0, 1, 2, 4, 6, 8, and 11. The genotypes of the ALDH2 polymorphism were determined using polymerase chain reactions plus restriction fragment length polymorphism analysis. A generalized estimating equation was used to analyze the effects of ALDH2 polymorphism on the clinical performance of DM. PANSS and SANS scores were significantly lower in both groups after 11 weeks of treatment. SANS total scores were significantly lower in the RISP + DM group in patients with the ALDH2*2*2 genotype. RISP plus add-on DM treatment reduced negative schizophrenia symptoms in patients with the ALDH2 polymorphism. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Lacosamide as add-on treatment of focal symptomatic epilepsy in a patient with alcoholic liver cirrhosis

    Directory of Open Access Journals (Sweden)

    A. Romigi

    2014-01-01

    Full Text Available The occurrence of epileptic seizures in the presence of hepatic disease is not uncommon in clinical practice. Selecting an appropriate AED for patients affected by liver failure who have new-onset epileptic seizures can be challenging. We describe a 64-year-old man affected by liver cirrhosis. The patient developed partial epilepsy with secondary generalization because of an intracerebral hemorrhage in the left parieto-occipital regions. After the neurosurgery procedure, seizures reappeared and were initially managed with levetiracetam. After one month, the patient experienced clusters of seizures while on stable treatment with levetiracetam. Pregabalin as add-on was not tolerated; therefore, he received a low dose of phenobarbital as add-on treatment. The patient developed hepatic encephalopathy. Phenobarbital was immediately stopped, and oral lacosamide was added. A rapid recovery of encephalopathy with a 6-month seizure freedom was obtained. The patient died 6 months later because of progressive impairment of liver function. Lacosamide may represent an alternative to other AEDs in patients with liver failure; however, further prospective evaluation of its efficacy and safety in this clinical setting is needed.

  1. Antidiabetic Effects of Add-On Gynostemma pentaphyllum Extract Therapy with Sulfonylureas in Type 2 Diabetic Patients

    Directory of Open Access Journals (Sweden)

    V. T. T. Huyen

    2012-01-01

    Full Text Available Aims. To investigate the antidiabetic effect of the traditional Vietnamese herb Gynostemma pentaphyllum (GP together with sulfonylurea (SU in 25 drug-naïve type 2 diabetic patients. Methods. After 4-week treatment with gliclazide (SU, 30 mg daily, all patients were randomly assigned into 2 groups to add on GP extract or placebo extract, 6 g daily, during eight weeks. Results. After 4-week SU treatment, fasting plasma glucose (FPG and HbA1C decreased significantly (P<0.001. FPG was further reduced after add-on therapy with 2.9 ± 1.7 and 0.9 ± 0.6 mmol/L in the GP and placebo groups, respectively (P<0.001. Therapy with GP extract also reduced 30- and 120-minute oral glucose tolerance test postload values. HbA1C levels decreased approximately 2% units in the GP group compared to 0.7% unit in the placebo group (P<0.001. Conclusion. GP extract in addition to SU offers an alternative to addition of other oral medication to treat type 2 diabetic patients.

  2. MPprimer: a program for reliable multiplex PCR primer design

    Directory of Open Access Journals (Sweden)

    Wang Xiaolei

    2010-03-01

    Full Text Available Abstract Background Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. Results A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy, which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions. Conclusions MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

  3. Forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault-validating an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and D-dimer.

    Science.gov (United States)

    Holtkötter, Hannah; Dias Filho, Claudemir Rodrigues; Schwender, Kristina; Stadler, Christian; Vennemann, Marielle; Pacheco, Ana Claudia; Roca, Gabriela

    2018-05-01

    Sexual assault is a serious offense and identification of body fluids originating from sexual activity has been a crucial aspect of forensic investigations for a long time. While reliable tests for the detection of semen and saliva have been successfully implemented into forensic laboratories, the detection of other body fluids, such as vaginal or menstrual fluid, is more challenging. Especially, the discrimination between peripheral and menstrual blood can be highly relevant for police investigations because it provides potential evidence regarding the issue of consent. We report the forensic validation of an immunochromatographic test that allows for such discrimination in forensic stains, the SERATEC PMB test, and its performance on real casework samples. The PMB test is a duplex test combining human hemoglobin and D-dimer detection and was developed for the identification of blood and menstrual fluid, both at the crime scene and in the laboratory. The results of this study showed that the duplex D-dimer/hemoglobin assay reliably detects the presence of human hemoglobin and identifies samples containing menstrual fluid by detecting the presence of D-dimers. The method distinguished between menstrual and peripheral blood in a swab from a historical artifact and in real casework samples of alleged sexual assaults. Results show that the development of the new duplex test is a substantial progress towards analyzing and interpreting evidence from sexual assault cases.

  4. 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

  5. Globes from global data: Charting international research networks with the GRASS GIS r.out.polycones add-on module.

    Science.gov (United States)

    Löwe, Peter

    2015-04-01

    Many Free and Open Source Software (FOSS) tools have been created for the various application fields within geoscience. While FOSS allows re-implementation of functionalities in new environments by access to the original codebase, the easiest approach to build new software solutions for new problems is the combination or merging of existing software tools. Such mash-ups are implemented by embedding and encapsulating FOSS tools within each another, effectively focusing the use of the embedded software to the specific role it needs to perform in the given scenario, while ignoring all its other capabilities. GRASS GIS is a powerful and established FOSS GIS for raster, vector and volume data processing while the Generic Mapping Tools (GMT) are a suite of powerful Open Source mapping tools, which exceed the mapping capabilities of GRASS GIS. This poster reports on the new GRASS GIS add-on module r.out.polycones. It enables users to utilize non-continuous projections for map production within the GRASS production environment. This is implemented on the software level by encapsulating a subset of GMT mapping capabilities into a GRASS GIS (Version 6.x) add-on module. The module was developed at the German National Library of Science and Technology (TIB) to provide custom global maps of scientific collaboration networks, such as the DataCite consortium, the registration agency for Digital Object Identifiers (DOI) for research data. The GRASS GIS add-on module can be used for global mapping of raster data into a variety of non continuous sinosoidal projections, allowing the creation of printable biangles (gores) to be used for globe making. Due to the well structured modular nature of GRASS modules, technical follow-up work will focus on API-level Python-based integration in GRASS 7 [1]. Based on this, GMT based mapping capabilities in GRASS will be extended beyond non-continuous sinosoidal maps and advanced from raster-layers to content GRASS display monitors. References

  6. Polarization-multiplexing ghost imaging

    Science.gov (United States)

    Dongfeng, Shi; Jiamin, Zhang; Jian, Huang; Yingjian, Wang; Kee, Yuan; Kaifa, Cao; Chenbo, Xie; Dong, Liu; Wenyue, Zhu

    2018-03-01

    A novel technique for polarization-multiplexing ghost imaging is proposed to simultaneously obtain multiple polarimetric information by a single detector. Here, polarization-division multiplexing speckles are employed for object illumination. The light reflected from the objects is detected by a single-pixel detector. An iterative reconstruction method is used to restore the fused image containing the different polarimetric information by using the weighted sum of the multiplexed speckles based on the correlation coefficients obtained from the detected intensities. Next, clear images of the different polarimetric information are recovered by demultiplexing the fused image. The results clearly demonstrate that the proposed method is effective.

  7. Signal multiplexing scheme for LINAC

    International Nuclear Information System (INIS)

    Sujo, C.I.; Mohan, Shyam; Joshi, Gopal; Singh, S.K.; Karande, Jitendra

    2004-01-01

    For the proper operation of the LINAC some signals, RF (radio frequency) as well as LF (low frequency) have to be available at the Master Control Station (MCS). These signals are needed to control, calibrate and characterize the RF fields in the resonators. This can be achieved by proper multiplexing of various signals locally and then routing the selected signals to the MCS. A multiplexing scheme has been designed and implemented, which will allow the signals from the selected cavity to the MCS. High isolation between channels and low insertion loss for a given signal are important issues while selecting the multiplexing scheme. (author)

  8. Treatment of traumatised refugees with basic body awareness therapy versus mixed physical activity as add-on treatment

    DEFF Research Database (Denmark)

    Nordbrandt, Maja Sticker; Carlsson, Jessica; Lindberg, Laura Glahder

    2015-01-01

    . In clinical studies, physical activity has shown a positive effect on psychiatric illnesses such as depression and anxiety and for patients with chronic pain. However, scientific knowledge about physical activity as part of the treatment for traumatised refugees is very limited and no guidelines exist......-based Cognitive Behavioural Therapy. The first group only receives treatment as usual while the second and the third groups receive either Basic-Body Awareness Therapy or mixed physical activity as add-on treatments. Each physical activity is provided for an individual 1-hour consultation per week...... as well as quality of life, functional capacity, coping with pain, body awareness and physical fitness. DISCUSSION: This study will examine the effect of physical activity for traumatised refugees. This has not yet been done in a randomised controlled setting on such a large scale before. Hereby the study...

  9. Long-term efficacy and safety of vildagliptin add-on therapy in type 2 diabetes mellitus with insulin treatment.

    Science.gov (United States)

    Kanazawa, Ippei; Tanaka, Ken-Ichiro; Notsu, Masakazu; Tanaka, Sayuri; Kiyohara, Nobuaki; Koike, Sayo; Yamane, Yuko; Tada, Yuko; Sasaki, Motofumi; Yamauchi, Mika; Sugimoto, Toshitsugu

    2017-01-01

    The use of dipeptidyl peptidase (DPP)-4 inhibitors in patients with type 2 diabetes treated with insulin may be beneficial. However, the long-term efficacy and safety of vildagliptin add-on therapy in these patients remains unclear. A total of 73 patients with type 2 diabetes treated with insulin were randomly assigned to receive either add-on therapy of vildagliptin (n=37) or conventional therapy without DPP-4 inhibitors (n=36) for glucose control. Hemoglobin A1c (HbA1c) levels, dose and number of insulin injections, number of hypoglycemia episodes, and liver and renal function were monitored for 2years. The baseline characteristics of subjects, including age, dose of insulin injections, or HbA1c levels, did not differ between the two groups. In the vildagliptin group, HbA1c levels significantly decreased and the significance of HbA1c reduction was maintained for 24months (from 8.0±1.2% to 7.4±1.0%, pinsulin injections significantly reduced (-5.6units, p1, and -0.9 times, p1). However, these parameters were unchanged in the control group. The number of patients who experienced three or more episodes of hypoglycemia per year was significantly lower in the vildagliptin group (n=4) than in the control group (n=11) (odds ratio, 0.28; 95% confidence interval, 0.08-0.97; pinsulin treatment for 24months was well tolerated and led to sustained reductions in HbA1c, the dose and number of insulin injections, and the risk of hypoglycemia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. A multiplex PCR for detection of Listeria monocytogenes and its lineages.

    Science.gov (United States)

    Rawool, Deepak B; Doijad, Swapnil P; Poharkar, Krupali V; Negi, Mamta; Kale, Satyajit B; Malik, S V S; Kurkure, Nitin V; Chakraborty, Trinad; Barbuddhe, Sukhadeo B

    2016-11-01

    A novel multiplex PCR assay was developed to identify genus Listeria, and discriminate Listeria monocytogenes and its major lineages (LI, LII, LIII). This assay is a rapid and inexpensive subtyping method for screening and characterization of L. monocytogenes. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Percolation in real multiplex networks

    Science.gov (United States)

    Bianconi, Ginestra; Radicchi, Filippo

    2016-12-01

    We present an exact mathematical framework able to describe site-percolation transitions in real multiplex networks. Specifically, we consider the average percolation diagram valid over an infinite number of random configurations where nodes are present in the system with given probability. The approach relies on the locally treelike ansatz, so that it is expected to accurately reproduce the true percolation diagram of sparse multiplex networks with negligible number of short loops. The performance of our theory is tested in social, biological, and transportation multiplex graphs. When compared against previously introduced methods, we observe improvements in the prediction of the percolation diagrams in all networks analyzed. Results from our method confirm previous claims about the robustness of real multiplex networks, in the sense that the average connectedness of the system does not exhibit any significant abrupt change as its individual components are randomly destroyed.

  12. Simvastatin as add-on therapy to interferon β-1a for relapsing-remitting multiple sclerosis (SIMCOMBIN study): a placebo-controlled randomised phase 4 trial

    DEFF Research Database (Denmark)

    Sorensen, Per Soelberg; Lycke, Jan; Erälinna, Juha-Pekka

    2011-01-01

    Treatment of relapsing-remitting multiple sclerosis with interferon beta is only partly effective. We aimed to establish whether add-on of simvastatin, a statin with anti-inflammatory properties, improves this efficacy.......Treatment of relapsing-remitting multiple sclerosis with interferon beta is only partly effective. We aimed to establish whether add-on of simvastatin, a statin with anti-inflammatory properties, improves this efficacy....

  13. Determination of Sperm Sex Ratio in Bovine Semen Using Multiplex Real-time Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Trisadee Khamlor

    2014-10-01

    Full Text Available Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP gene and sex-determining region Y (SRY were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99% comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05. The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90% as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

  14. 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.

  15. Evaluation of add-on devices for the prevention of phlebitis and other complications associated with the use of peripheral catheters in hospitalised adults: a randomised controlled study.

    Science.gov (United States)

    Martínez, J A; Piazuelo, M; Almela, M; Blecua, P; Gallardo, R; Rodríguez, S; Escalante, Z; Robau, M; Trilla, A

    2009-10-01

    The aim of this study was to assess the role of add-on devices for the prevention of phlebitis and other complications associated with the use of peripheral catheters. Patients admitted to an infectious diseases ward and requiring the insertion of a peripheral catheter for at least 24h were randomly allocated to be managed with or without add-on devices. Incidence of phlebitis and all complications were the primary outcomes. Extravasation, inadvertent withdrawal, obstruction and rupture were considered to be mechanical complications, and analysis was performed using survival methods. Of 683 evaluated catheters, 351 were allocated to the add-on device arm and 332 to the control arm. Despite randomisation, patients in the add-on device group were older (P=0.048), less likely to have human immunodeficiency virus (P=0.02) and more likely to have received antibiotics (P=0.05). After adjustment for these variables, the hazard ratio for phlebitis remained non-significant (hazard ratio: 0.95; 95% confidence interval: 0.7-1.3), but the risk of mechanical complications became lower in the add-on device arm (0.68; 0.5-0.94). This translated into a trend towards a lower risk of any complication (0.83; 0.67-1.01). The beneficial effect on mechanical or all complications was noticeable after six days of catheterisation. Add-on devices do not reduce the incidence of phlebitis but may prevent mechanical complications. However, the impact of add-on devices on the incidence of all complications is at most small and only apparent after the sixth day of catheter use.

  16. Low- vs high- frequency Repetitive Transcranial Magnetic Stimulation as an add-on treatment for refractory depression

    Directory of Open Access Journals (Sweden)

    julien eeche

    2012-03-01

    Full Text Available Objectives: Repetitive transcranial magnetic stimulation (rTMS seems to be effective as an antidepressant treatment, however, some confusion remain about the best parameters to apply and the efficacy of its association with pharmacological antidepressant treatments.Method: In a single blind randomized study14 patients with unipolar resistant depression to one antidepressant treatment were enrolled to received, in combination with venlafaxine (150 mg, either 20 sessions of 10Hz rTMS (2 000 pulses per session applied over le left dorsolateral prefrontal cortex (DLPFC or 20 sessions of 1 Hz rTMS (120 stimulations per sessions applied over the right DLPFC. Results: A similar antidepressant effect was observed in both groups with a comparable antidepressant delay of action (2 weeks and a comparable number of patients in remission after 4 weeks of daily rTMS sessions (66 vs 50 %.Conclusion: Low- and high- frequency rTMS seem to be effective as an add-on treatment to venlafaxine in pharmacological refractory major depression. Due to its short duration and its safety, low frequency rTMS may be a useful alternative treatment for patients with refractory depression.

  17. Ozone therapy as add-on treatment in fibromyalgia management by rectal insufflation: an open-label pilot study.

    Science.gov (United States)

    Hidalgo-Tallón, Javier; Menéndez-Cepero, Silvia; Vilchez, Juan S; Rodríguez-López, Carmen M; Calandre, Elena P

    2013-03-01

    The objectives of this study were to evaluate the effectiveness and tolerability of ozone therapy by rectal insufflation as add-on therapy in fibromyalgia management. Patients with fibromyalgia received 24 sessions of ozone therapy during a 12-week period. At each session, the administered dose of ozone was 8 mg (200 mL of gas, at a concentration of 40 μg/mL). Ozone sessions were given 5 days a week during the first 2 weeks, twice a week from weeks 3-6, and weekly from weeks 7-12. Fibromyalgia Impact Questionnaire (FIQ) was the main outcome measure, and was administered at baseline and at weeks 4, 8, and 12. Secondary outcome measures, administered at baseline and at endpoint, were the Pittsburgh Sleep Quality Index, the Beck Depression Inventory, the State and Trait Anxiety Inventory, and the SF-12, the abbreviated form of the Short Form Health Survey. Emergent adverse reactions to treatment were recorded. FIQ total scores decreased significantly during the study period, with the decrease being observed in the first 4 weeks of the study. Significant improvement was also seen both in depression scores and in the Physical Summary Score of the SF-12. Transient meteorism after ozone therapy sessions was the most frequently reported side-effect. At the dose and number of sessions used in this study, ozone therapy by rectal insufflation seems to be beneficial for physical symptoms and depression of fibromyalgia.

  18. Use of asenapine as add-on therapy in the treatment of bipolar disorder: a comprehensive review and case series.

    Science.gov (United States)

    Dell'Osso, Bernardo; Cremaschi, Laura; Palazzo, Maria Carlotta; Spagnolin, Gregorio; Cattaneo, Alma; Grancini, Benedetta; Maggi, Matteo; Altamura, Alfredo Carlo

    2014-09-01

    Several randomized controlled trials (RCTs), conducted in schizophrenic and bipolar patients, have documented the efficacy and tolerability of asenapine as monotherapy both for short- and long-term treatment. However, evidence on its augmentative use is more limited and related to the manic/mixed phase of bipolar disorder (BD). The present article reviews augmentative asenapine efficacy and safety/tolerability in the treatment of BD. It also includes some original cases of bipolar patients treated with add-on asenapine in the short- and long-term. To date, only a single RCT with manic/mixed patients with partial response to mood-stabilizer monotherapy supports the efficacy and safety/tolerability of augmentative asenapine to lithium/valproate, both in acute and long-term treatment. Additionally, two case reports confirm the overall effectiveness of augmentative asenapine to clozapine and valproate. Our case series, consisting of 4 bipolar patients treated with adjunctive asenapine to mood stabilizers and atypical antipsychotics - with treatment duration ranging from 1 to 14 months - provided clinical results that are consistent with literature data. Taken as a whole, available evidence seems to support the efficacy and safety of adjunctive asenapine in bipolar patients, though additional studies with active comparators are requested to confirm the current body of evidence.

  19. Flexible add-on solution for MR image-guided interventions in a closed-bore scanner environment.

    Science.gov (United States)

    Busse, Harald; Garnov, Nikita; Thörmer, Gregor; Zajonz, Dirk; Gründer, Wilfried; Kahn, Thomas; Moche, Michael

    2010-09-01

    MRI is of great clinical utility for the guidance of various diagnostic and therapeutic procedures. In a standard closed-bore scanner, the simplest approach is to manipulate the instrument outside the bore and move the patient into the bore for reference and control imaging only. Without navigational assistance, however, such an approach can be difficult, inaccurate, and time consuming. Therefore, an add-on navigation solution is described that addresses these limitations. Patient registration is established by an automatic, robust, and fast (<30 sec) localization of table-mounted MR reference markers and the instrument is tracked optically. Good hand-eye coordination is provided by following the virtual instrument on MR images that are reconstructed in real time from the reference data. Needle displacements of 2.2 +/- 0.6 mm and 3.9 +/- 2.4 mm were determined in a phantom (P < 0.05), depending on whether the reference markers were placed at smaller (98-139 mm) or larger (147-188 mm) distances from the isocenter. Clinical functionality of the navigation concept is demonstrated by a double oblique, subscapular hook-wire insertion in a patient with a body mass index of 30.1 kg/m(2). Ease of use, compactness, and flexibility of this technique suggest that it can be used for many other procedures in different body regions. More patient cases are needed to evaluate clinical performance and workflow. 2010 Wiley-Liss, Inc.

  20. 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.

  1. Thermally multiplexed polymerase chain reaction.

    Science.gov (United States)

    Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

    2015-07-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.

  2. Multiplex polymerase chain reaction (PCR) assay for simultaneous ...

    African Journals Online (AJOL)

    To evaluate the etiology of shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC) and enteroinvasive E. coli (EIEC) in children with diarrhea in Iran a total 300 stool specimens from children with diarrhea were tested for the detection of E.coli. Out of 300 samples, 39 were identified as E. coli by biochemical ...

  3. Multiplex polymerase chain reaction (PCR) assay for simultaneous ...

    African Journals Online (AJOL)

    user

    2011-02-28

    Feb 28, 2011 ... To evaluate the etiology of shiga toxin-producing E. coli (STEC), enteropathogenic E. ... National Center for Biotechnology Information; BLAST, Basic ... Poor sanitation, personal ..... Mem Inst Oswaldo Cruz, Rio de Janeiro.

  4. 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.

  5. Laguerre Gaussian beam multiplexing through turbulence

    CSIR Research Space (South Africa)

    Trichili, A

    2014-08-17

    Full Text Available We analyze the effect of atmospheric turbulence on the propagation of multiplexed Laguerre Gaussian modes. We present a method to multiplex Laguerre Gaussian modes using digital holograms and decompose the resulting field after encountering a...

  6. 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.

  7. Semi-Individualized Homeopathy Add-On Versus Usual Care Only for Premenstrual Disorders: A Randomized, Controlled Feasibility Study.

    Science.gov (United States)

    Klein-Laansma, Christien T; Jong, Mats; von Hagens, Cornelia; Jansen, Jean Pierre C H; van Wietmarschen, Herman; Jong, Miek C

    2018-03-22

    Premenstrual syndrome and premenstrual dysphoric disorder (PMS/PMDD) bother a substantial number of women. Homeopathy seems a promising treatment, but it needs investigation using reliable study designs. The feasibility of organizing an international randomized pragmatic trial on a homeopathic add-on treatment (usual care [UC] + HT) compared with UC alone was evaluated. A multicenter, randomized, controlled pragmatic trial with parallel groups. The study was organized in general and private homeopathic practices in the Netherlands and Sweden and in an outpatient university clinic in Germany. Women diagnosed as having PMS/PMDD, based on prospective daily rating by the daily record of severity of problems (DRSP) during a period of 2 months, were included and randomized. Women were to receive UC + HT or UC for 4 months. Homeopathic medicine selection was according to a previously tested prognostic questionnaire and electronic algorithm. Usual care was as provided by the women's general practitioner according to their preferences. Before and after treatment, the women completed diaries (DRSP), the measure yourself concerns and well-being, and other questionnaires. Intention-to-treat (ITT) and per protocol (PP) analyses were performed. In Germany, the study could not proceed because of legal limitations. In Sweden, recruitment proved extremely difficult. In the Netherlands and Sweden, 60 women were randomized (UC + HT: 28; UC: 32), data of 47/46 women were analyzed (ITT/PP). After 4 months, relative mean change of DRSP scores in the UC + HT group was significantly better than in the UC group (p = 0.03). With respect to recruitment and different legal status, it does not seem feasible to perform a larger, international, pragmatic randomized trial on (semi-)individualized homeopathy for PMS/PMDD. Since the added value of HT compared with UC was demonstrated by significant differences in symptom score changes, further studies are warranted.

  8. Two-year, randomized, controlled study of safinamide as add-on to levodopa in mid to late Parkinson's disease.

    Science.gov (United States)

    Borgohain, Rupam; Szasz, Jozsef; Stanzione, Paolo; Meshram, Chandrashekhar; Bhatt, Mohit H; Chirilineau, Dana; Stocchi, Fabrizio; Lucini, Valentina; Giuliani, Rodolfo; Forrest, Emma; Rice, Patricia; Anand, Ravi

    2014-09-01

    In a 6-month double-blind, placebo-controlled study of Parkinson's disease patients with motor fluctuations, safinamide 50 and 100 mg/d significantly increased ON-time without increasing dyskinesia. Further long-term safinamide use in these patients was evaluated over an additional 18 months. Patients continued on their randomized placebo, 50, or 100 mg/d safinamide. The primary endpoint was change in Dyskinesia Rating Scale total score during ON-time over 24 months. Other efficacy endpoints included change in ON-time without troublesome dyskinesia, changes in individual diary categories, depressive symptoms, and quality of life measures. Change in Dyskinesia Rating Scale was not significantly different in safinamide versus placebo groups, despite decreased mean total Dyskinesia Rating Scale with safinamide compared with an almost unchanged score in placebo. Ad hoc subgroup analysis of moderate to severe dyskinetic patients at baseline (36% of patients) showed a decrease with safinamide 100 mg/d compared with placebo (P = 0.0317). Improvements in motor function, activities of daily living, depressive symptoms, clinical status, and quality of life at 6 months remained significant at 24 months. Adverse events and discontinuation rates were similar with safinamide and placebo. This 2-year, controlled study of add-on safinamide in mid-to-late Parkinson's disease with motor fluctuations, although not demonstrating an overall difference in dyskinesias between patients and controls, showed improvement in dyskinesia in patients at least moderately dyskinetic at baseline. The study additionally demonstrated significant clinical benefits in ON-time (without troublesome dyskinesia), OFF-time, activities of daily living, motor symptoms, quality of life, and symptoms of depression. © 2014 International Parkinson and Movement Disorder Society.

  9. Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers.

    Science.gov (United States)

    Iliuk, Anton; Li, Li; Melesse, Michael; Hall, Mark C; Tao, W Andy

    2016-05-17

    Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. On-chip mode division multiplexing technologies

    DEFF Research Database (Denmark)

    Ding, Yunhong; Frellsen, Louise Floor; Guan, Xiaowei

    2016-01-01

    Space division multiplexing (SDM) is currently widely investigated in order to provide enhanced capacity thanks to the utilization of space as a new degree of multiplexing freedom in both optical fiber communication and on-chip interconnects. Basic components allowing the processing of spatial...... photonic integrated circuit mode (de) multiplexer for few-mode fibers (FMFs)....

  11. EMDR as Add-On Treatment for Psychiatric and Traumatic Symptoms in Patients with Substance Use Disorder

    Directory of Open Access Journals (Sweden)

    Sara Carletto

    2018-01-01

    significant improvement of post-traumatic and dissociative symptoms, accompanied by a reduction in anxiety and overall psychopathology levels, whereas TAU group showed a significant reduction only in post-traumatic symptoms. Although our results can only be considered preliminary, this study suggests that a combined TF- and AF- EMDR protocol is an effective and well-accepted add-on treatment for patients with SUD.

  12. Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads

    Directory of Open Access Journals (Sweden)

    Dae Hong Jeong

    2012-03-01

    Full Text Available Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.

  13. Efficacy and Safety of Lamotrigine as Add-On Treatment to Lithium in Bipolar Depression : A Multicenter, Double-Blind, Placebo-Controlled Trial

    NARCIS (Netherlands)

    van der Loos, Marc L. M.; Mulder, Paul G. H.; Hartong, Erwin G. Th. M.; Blom, Marc B. J.; Vergouwen, Anton C.; de Keyzer, Herman J. U. E. M.; Notten, Peter J. H.; Luteijn, Marijke L.; Timmermans, Manuela A.; Nolen, Willem A.

    Objective: Lamotrigine is one of the pharmacologic options for the treatment of bipolar depression but has only been studied as monotherapy. This study compared the acute effects of lamotrigine and placebo as add-on therapy to ongoing treatment with lithium in patients with bipolar depression.

  14. N-Acetylcysteine in the Treatment of Pediatric Trichotillomania: A Randomized, Double-Blind, Placebo-Controlled Add-On Trial

    Science.gov (United States)

    Bloch, Michael H.; Panza, Kaitlyn E.; Grant, Jon E.; Pittenger, Christopher; Leckman, James F.

    2013-01-01

    Objective: To examine the efficacy of N-acetylcysteine (NAC) for the treatment of pediatric trichotillomania (TTM) in a double-blind, placebo-controlled, add-on study. Method: A total of 39 children and adolescents aged 8 to 17 years with pediatric trichotillomania were randomly assigned to receive NAC or matching placebo for 12 weeks. Our primary…

  15. Acupuncture and related therapies used as add-on or alternative to prokinetics for functional dyspepsia: overview of systematic reviews and network meta-analysis.

    Science.gov (United States)

    Ho, Robin S T; Chung, Vincent C H; Wong, Charlene H L; Wu, Justin C Y; Wong, Samuel Y S; Wu, Irene X Y

    2017-09-04

    Prokinetics for functional dyspepsia (FD) have relatively higher number needed to treat values. Acupuncture and related therapies could be used as add-on or alternative. An overview of systematic reviews (SRs) and network meta-analyses (NMA) were performed to evaluate the comparative effectiveness of different acupuncture and related therapies. We conducted a comprehensive literature search for SRs of randomized controlled trials (RCTs) in eight international and Chinese databases. Data from eligible RCTs were extracted for random effect pairwise meta-analyses. NMA was used to explore the most effective treatment among acupuncture and related therapies used alone or as add-on to prokinetics, compared to prokinetics alone. From five SRs, 22 RCTs assessing various acupuncture and related therapies were included. No serious adverse events were reported. Two pairwise meta-analyses showed manual acupuncture has marginally stronger effect in alleviating global FD symptoms, compared to domperidone or itopride. Results from NMA showed combination of manual acupuncture and clebopride has the highest probability in alleviating patient reported global FD symptom. Combination of manual acupuncture and clebopride has the highest probability of being the most effective treatment for FD symptoms. Patients who are contraindicated for prokinetics may use manual acupuncture or moxibustion as alternative. Future confirmatory comparative effectiveness trials should compare clebopride add-on manual acupuncture with domperidone add-on manual acupuncture and moxibustion.

  16. Safety and tolerability of once-daily tiotropium Respimat(®) as add-on to at least inhaled corticosteroids in adult patients with symptomatic asthma

    DEFF Research Database (Denmark)

    Dahl, Ronald; Engel, Michael; Dusser, Daniel

    2016-01-01

    BACKGROUND: Tiotropium, a long-acting anticholinergic bronchodilator, has demonstrated efficacy and safety as add-on therapy to inhaled corticosteroids (ICS), with or without other maintenance therapies, in patients with symptomatic asthma. OBJECTIVE: To evaluate safety and tolerability of tiotro...

  17. Simultaneous Expression of GUS and Actin Genes by Using the Multiplex RT-PCR and Multiplex Gold Nanoparticle Probes.

    Science.gov (United States)

    Ghazi, Yaser; Vaseghi, Akbar; Ahmadi, Sepideh; Haddadi, Fatemeh

    2018-04-23

    Gene expression analysis is considered to be extremely important in many different biological researches. DNA-based diagnostic test, which contributes to DNA identification, has higher specificity, cost, and speed than some biochemical and molecular methods. In this study, we try to use the novel nano technology approach with Multiplex RT-PCR and Gold nano particular probes (GNPs-probes) in order to get gene expression in Curcumas melons. We used Agrobacterium tumefactions for gene transfer and GUS reporter gene as a reporter. After cDNA synthesis, Multiplex PCR and Multiplex RT-PCR techniques were used. Finally, probes were designed for RNA of GUS and Actin genes, and then the analysis of the gene expression using the probes attached to GNPs was carried out and the color changes in the GNPs were applied. In the following, probes hybridization was checked with DNA between 400 to 700 nm wavelengths and the highest rate was observed in the 550 to 650 nm. The results show that the simultaneous use of GNP-attached detectors and Multiplex RT-PCRcan reduce time and costmore considerably than somelaboratory methods for gene expiration investigation. Additionally, it can be seen thatthere is an increase in sensitivity and specificity of our investigation. Based on our findings, this can bea novel study doneusingMultiplex RT-PCRand unmodified AuNPs for gene transfer and expression detection to plants. We can claim that this assay has a remarkable advantage including rapid, cost-effectiveness, specificity and accuracy to detect transfer and expression genes in plants. Also,we can use this technique from other gene expressionsin many different biology samples.

  18. Rate and associated factors of solifenacin add-on after tamsulosin monotherapy in men with voiding and storage lower urinary tract symptoms.

    Science.gov (United States)

    Lee, H N; Lee, K-S; Kim, J C; Chung, B H; Kim, C-S; Lee, J G; Kim, D K; Park, C H; Park, J K; Hong, S J

    2015-04-01

    To explore the rate of add-on therapy with solifenacin in men with voiding and storage lower urinary tract symptoms (LUTS) after tamsulosin monotherapy and to explore predictive factors for starting solifenacin add-on therapy. Men aged ≥ 45 years with IPSS ≥ 12 and symptoms of OAB (OAB-V8 ≥ 8, micturition ≥ 8/24 h, urgency ≥ 2/24 h) were enrolled to receive tamsulosin 0.2 mg once daily. After 4 weeks, men with residual symptoms of OAB and reported 'dissatisfied' or 'a little satisfied' were received solifenacin 5 mg in combination with tamsulosin monotherapy. Subjects completed an IPSS, a Quality of life (QoL) index, OAB V8, and an International Consultation of Incontinence Questionnaire (ICIQ)-Male LUTS, and patient perception of bladder condition (PPBC) at baseline and week 4. Of a total of 305 patients, 254 patients completed 4 weeks of tamsulosin treatment. For 176 patients, solifenacin was added (69.3%). Significant predictive factors of solifenacin add-on therapy included long LUTS duration, high IPSS, number of micturitions per 24 h, more urgency episodes, high urgency severity score in a voiding diary and high OAB V8 score. Based on multivariable analysis, potential predictive factors of solifenacin add-on therapy included long LUTS duration (OR = 1.008, 95% CI: 1.001-1.014), high serum PSA (OR = 1.543, 95% CI: 1.136-2.095) and small prostate size (OR = 0.970, 95% CI: 0.947-0.994) (p tamsulosin monotherapy. Two thirds of men with voiding and storage LUTS needed to add anticholinergics after 4 weeks of tamsulosin monotherapy. Patients with longer lasting symptoms and storage symptoms with small prostate volume may require the anticholinergic add-on. © 2014 John Wiley & Sons Ltd.

  19. Gold nanoparticle-enhanced multiplexed imaging surface plasmon resonance (iSPR) detection of Fusarium mycotoxins in wheat

    Science.gov (United States)

    A rapid, sensitive and multiplexed imaging surface plasmon resonance (iSPR) biosensor assay was developed and validated for three Fusarium toxins, deoxynivalenol (DON), zearalenone (ZEA) and T-2 toxin. The iSPR assay was based on a competitive inhibition format with secondary antibodies (Ab2) conjug...

  20. Barcoded microchips for biomolecular assays.

    Science.gov (United States)

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  1. A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience

    DEFF Research Database (Denmark)

    Nielsen, A. C. Y.; Bottiger, B.; Midgley, S. E.

    2013-01-01

    As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay....... The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel...... testing and routine usage. During 15 months of routine use, from October 2008 to December 2009, we received and analysed 2187 samples (stool samples, cerebrospinal fluids, blood samples, respiratory samples and autopsy samples) were tested, from 1546 patients and detected enteroviruses and parechoviruses...

  2. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

    Energy Technology Data Exchange (ETDEWEB)

    Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

    2000-05-05

    Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.

  3. Development of multiplex polymerase chain reaction for detection of Ehrlichia canis, Babesia spp and Hepatozoon canis in canine blood.

    Science.gov (United States)

    Kledmanee, Kan; Suwanpakdee, Sarin; Krajangwong, Sakranmanee; Chatsiriwech, Jarin; Suksai, Parut; Suwannachat, Pongpun; Sariya, Ladawan; Buddhirongawatr, Ruangrat; Charoonrut, Phingphol; Chaichoun, Kridsada

    2009-01-01

    A multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of canine blood parasites, Ehrlichia canis, Babesia spp and Hepatozoon canis, from blood samples in a single reaction. The multiplex PCR primers were specific to E. canis VirB9, Babesia spp 16S rRNA and H. canis 16S rRNA genes. Specificity of the amplicons was confirmed by DNA sequencing. The assay was evaluated using normal canine and infected blood samples, which were detected by microscopic examination. This multiplex PCR offers scope for simultaneous detection of three important canine blood parasites and should be valuable in monitoring parasite infections in dogs and ticks.

  4. Centrality in earthquake multiplex networks

    Science.gov (United States)

    Lotfi, Nastaran; Darooneh, Amir Hossein; Rodrigues, Francisco A.

    2018-06-01

    Seismic time series has been mapped as a complex network, where a geographical region is divided into square cells that represent the nodes and connections are defined according to the sequence of earthquakes. In this paper, we map a seismic time series to a temporal network, described by a multiplex network, and characterize the evolution of the network structure in terms of the eigenvector centrality measure. We generalize previous works that considered the single layer representation of earthquake networks. Our results suggest that the multiplex representation captures better earthquake activity than methods based on single layer networks. We also verify that the regions with highest seismological activities in Iran and California can be identified from the network centrality analysis. The temporal modeling of seismic data provided here may open new possibilities for a better comprehension of the physics of earthquakes.

  5. A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR.

    Science.gov (United States)

    Nikolaou, Anastasios; Saxami, Georgia; Kourkoutas, Yiannis; Galanis, Alex

    2011-02-01

    In this study we present a novel multiplex PCR assay for rapid and efficient detection of Lactobacillus delbrueckii subsp. bulgaricus. The accuracy of our method was confirmed by the successful identification of L. delbrueckii subsp. bulgaricus in commercial yoghurts and food supplements and it may be readily applied to the food industry. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system.

    Science.gov (United States)

    Sankuntaw, Nipaporn; Sukprasert, Saovaluk; Engchanil, Chulapan; Kaewkes, Wanlop; Chantratita, Wasun; Pairoj, Vantanit; Lulitanond, Viraphong

    2011-01-01

    Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%-3.67% and 0.79%-4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. 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.

  8. Consideration for wavelength multiplexing versus time multiplexing in optical transport network

    DEFF Research Database (Denmark)

    Limal, Emmanuel; Stubkjær, Kristian Elmholdt

    1999-01-01

    We compare optical wavelength multiplexing and time multiplexing techniquesfor optical transport network by studying the space switch sizes of OXCs andtheir interfaces as a function of the fraction of add/drop traffic....

  9. Multiplex Immunoassay Profiling of Hormones Involved in Metabolic Regulation.

    Science.gov (United States)

    Stephen, Laurie; Guest, Paul C

    2018-01-01

    Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

  10. Strain measurement using multiplexed fiber optic sensors

    International Nuclear Information System (INIS)

    Kwon, Il Bum; Kim, Chi Yeop; Yoon, Dong Jin; Lee, Seung Seok

    2003-01-01

    FBG(Fiber Bragg grating) sensor, which is one of the fiber optic sensors for the application of smart structures, can not only measure one specific point but also multiple points by multiplexing techniques. We have proposed a novel multiplexing technique of FBG sensor by the intensity modulation of light source. This technique is applicable to WDM(Wavelength Division Multiplexing) technique and number of sensors in this system can be increased by using this technique with WDM technique.

  11. Effects of vildagliptin as add-on treatment in patients with type 2 diabetes mellitus: insights from long-term clinical studies in Japan.

    Science.gov (United States)

    Odawara, Masato; Sagara, Rieko

    2015-01-01

    Vildagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, is wildly used to treat type 2 diabetes mellitus (T2DM) with mono- or combination-therapy. We review two previously published open-label studies to extract insights on the long-term efficacy and safety of vildagliptin. Two studies were conducted in Japan to assess the efficacy and safety of vildagliptin as an add-on to other oral antidiabetes drugs (OADs) for 52 weeks. These studies were performed under the similar protocol in Japanese patients with T2DM who were inadequately controlled with OAD monotherapy [excluding other dipeptidyl peptidase-4 (DPP-4) inhibitors]. Addition of vildagliptin (50 mg twice daily) to other OAD monotherapy [sulfonylurea (SU), metformin, thiazolidinedione, alpha-glucosidase inhibitor and glinide] reduced glycated hemoglobin (HbA1c) levels by -0.64 %,-0.75 %,-0.92 %,-0.94 % and - 0.64 %, respectively, over 52 weeks of treatment. Overall, the incidence of hypoglycemia was low and was slightly higher in the add-on to SU treatment group compared with the other groups. The incidences of adverse events were comparable among the treatment groups, and vildagliptin was well-tolerated as add-on therapy to other OADs. The evidence from the two studies indicates that vildagliptin as an add-on therapy to other OADs is a clinically reasonable option for Japanese patients with T2DM who respond inadequately to other OAD monotherapy.

  12. Acupuncture and related therapies used as add-on or alternative to prokinetics for functional dyspepsia: overview of systematic reviews and network meta-analysis

    OpenAIRE

    Ho, Robin S. T.; Chung, Vincent C. H.; Wong, Charlene H. L.; Wu, Justin C. Y.; Wong, Samuel Y. S.; Wu, Irene X. Y.

    2017-01-01

    Prokinetics for functional dyspepsia (FD) have relatively higher number needed to treat values. Acupuncture and related therapies could be used as add-on or alternative. An overview of systematic reviews (SRs) and network meta-analyses (NMA) were performed to evaluate the comparative effectiveness of different acupuncture and related therapies. We conducted a comprehensive literature search for SRs of randomized controlled trials (RCTs) in eight international and Chinese databases. Data from ...

  13. Frequency multiplexing for readout of spin qubits

    Energy Technology Data Exchange (ETDEWEB)

    Hornibrook, J. M.; Colless, J. I.; Mahoney, A. C.; Croot, X. G.; Blanvillain, S.; Reilly, D. J., E-mail: david.reilly@sydney.edu.au [ARC Centre of Excellence for Engineered Quantum Systems, School of Physics, University of Sydney, Sydney, NSW 2006 (Australia); Lu, H.; Gossard, A. C. [Materials Department, University of California, Santa Barbara, California 93106 (United States)

    2014-03-10

    We demonstrate a low loss, chip-level frequency multiplexing scheme for readout of scaled-up spin qubit devices. By integrating separate bias tees and resonator circuits on-chip for each readout channel, we realise dispersive gate-sensing in combination with charge detection based on two radio frequency quantum point contacts. We apply this approach to perform multiplexed readout of a double quantum dot in the few-electron regime and further demonstrate operation of a 10-channel multiplexing device. Limitations for scaling spin qubit readout to large numbers of multiplexed channels are discussed.

  14. Add-on deep transcranial magnetic stimulation (dTMS) in patients with dysthymic disorder comorbid with alcohol use disorder: a comparison with standard treatment.

    Science.gov (United States)

    Girardi, Paolo; Rapinesi, Chiara; Chiarotti, Flavia; Kotzalidis, Georgios D; Piacentino, Daria; Serata, Daniele; Del Casale, Antonio; Scatena, Paola; Mascioli, Flavia; Raccah, Ruggero N; Brugnoli, Roberto; Digiacomantonio, Vittorio; Ferri, Vittoria Rachele; Ferracuti, Stefano; Zangen, Abraham; Angeletti, Gloria

    2015-01-01

    Dorsolateral prefrontal cortex (DLPFC) is dysfunctional in mood and substance use disorders. We predicted higher efficacy for add-on bilateral prefrontal high-frequency deep transcranial magnetic stimulation (dTMS), compared with standard drug treatment (SDT) in patients with dysthymic disorder (DD)/alcohol use disorder (AUD) comorbidity. We carried-out a 6-month open-label study involving 20 abstinent patients with DSM-IV-TR AUD comorbid with previously developed DD. Ten patients received SDT for AUD with add-on bilateral dTMS (dTMS-AO) over the DLPFC, while another 10 received SDT alone. We rated alcohol craving with the Obsessive Compulsive Drinking Scale (OCDS), depression with the Hamilton Depression Rating Scale (HDRS), clinical status with the Clinical Global Impressions scale (CGI), and global functioning with the Global Assessment of Functioning (GAF). At the end of the 20-session dTMS period (or an equivalent period in the SDT group), craving scores and depressive symptoms in the dTMS-AO group dropped significantly more than in the SDT group (P < 0.001 and P < 0.02, respectively). High frequency bilateral DLPFC dTMS with left preference was well tolerated and found to be effective as add-on in AUD. The potential of dTMS for reducing craving in substance use disorder patients deserves to be further investigated.

  15. Effect of omega-3 fatty acids versus 5-hydroxytryptophan as add on therapy to sertraline in controlling suicidal ideation in patients with depression: A comparative study

    Directory of Open Access Journals (Sweden)

    Jyoti Prakash Sahoo

    2016-04-01

    Full Text Available Background: Omega-3 fatty acids and 5-hydroxytryptophan have been gaining attention as promising alternative treatment for depressive illness. These agents are given as as add on treatment to conventional antidepressant drugs. The present study was carried out to evaluate efficacy of omega-3 fatty acids versus 5-hydroxytryptophan as add on therapy in controlling suicidal ideation in depressive patients on sertraline.Methods: This was a prospective, open label, randomized, parallel group study conducted in department of Psychiatry. Ninety treatment naïve patients (18-65 years age were divided into 3 groups of 30 each. Group I: Sertraline, Group II: Sertraline plus omega-3 fatty acids, Group III: Sertraline plus 5-hydroxytryptophan. Suicidal ideations were assessed with Beck’s scale for suicidal ideation (BSI at weeks 0, 4 and 8. Data were analyzed using repeated measures ANOVA (SPSS version 20.0. Post hoc analysis was done using Bonferroni test.Results: Baseline parameters in patients of all groups were comparable. Administration of sertraline resulted in reduction of Beck’s scale for suicidal ideation scores as compared to baseline. Addition of omega-3 fatty acids and 5-hydroxytryptophan also showed reduction in BSI scores. Effect of sertraline monotherapy was more as compared to omega-3 fatty acids or 5-hydroxytryptophan as add on therapy, which was statistically significant (p value < 0.05.Conclusion: Both omega-3 fatty acids and 5-hydroxytryptophan produce significant effect in controlling suicidal ideations in patients with depression.

  16. Who and How: Comprehensive RNA-Based BodyfluID Assay to Provide Context to a Recovered DNA Profile

    Science.gov (United States)

    2015-08-25

    capillary electrophoresis (CE) and high resolution melt ( HRM ) assays 3 were developed and fully validated. The results of this study demonstrate...Multiplex High Resolution Melt ( HRM ) Assays ................................ 11  D. Developmental Validation of the CE and HRM Assays...Sequences (Top) and Primer Mix Composition (Bottom) for the Blood-Menstrual Blood HRM Assay

  17. Steatocystoma multiplex hos 39-årig kvinde

    DEFF Research Database (Denmark)

    Duffy, Jonas Raymond; Siersen, Hans Erik; Bonde, Christian T

    2011-01-01

    -coloured cystic lesions on the chest, abdomen, axillae and back. The patient's clinical presentations and history were compatible with steatocystoma multiplex. Various treatment options for steatocystoma multiplex and steatocystoma multiplex suppurativum have been published and include oral antibiotics...

  18. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

    Science.gov (United States)

    Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

    2016-10-01

    The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

  19. An alarm multiplexer communication system

    International Nuclear Information System (INIS)

    Herrera, G.V.

    1986-01-01

    A low cost Alarm Multiplexer Communication System (AMCS) has been developed to perform the security sensor monitoring and control functions and to provide remote relay control capability for integrated security systems. AMCS has a distributed multiplexer/repeater architecture with up to four dual communication loops and dual control computers that guarantee total system operation under any single point failure condition. Each AMCS can control up to 4096 sensors and 2048 remote relays. AMCS reports alarm status information to and is controlled by either one or two Host computers. This allows for independent operation of primary and backup security command centers. AMCS communicates with the Host computers over an asynchronous serial communication link and has a message protocol which allows AMCS to fully recover from lost messages or large blocks of data communication errors. This paper describes the AMCS theory of operation, AMCS fault modes, and AMCS system design methodology. Also, cost and timing information is presented. AMCS is being used and considered for several DOE and DOD facilities

  20. EDRN Pre-Validation of Multiplex Biomarker in Urine — EDRN Public Portal

    Science.gov (United States)

    The goal of this proposal is to begin to establish an EDRN “pre-validation” trial of a multiplex set of transcripts, including the ETS gene fusions, in post-DRE urine sediments. As can be evidenced by our preliminary data, we have established the utility of this multiplex urine test (which includes TMPRSS-ERG, SPINK1, PCA3 and GOLPH2) in a cohort of prospectively collected urine sediments from the University of Michigan EDRN CEVC site (collected by co-I, Dr. John Wei). In this proposal, we will run this multiplex assay on prospectively collected post-DRE urines collected from other EDRN sites. The idea is to couple this “pre-validation” study with an EDRN validation trial under consideration for the Gen-Probe PCA3 urine test (directed by Drs. John Wei and Harry Rittenhouse).

  1. A laser ablation ICP-MS based method for multiplexed immunoblot analysis

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... 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....

  2. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    Directory of Open Access Journals (Sweden)

    Ratthaphol Charlermroj

    Full Text Available Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac, chilli vein-banding mottle virus (CVbMV, potyvirus, watermelon silver mottle virus (WSMoV, tospovirus serogroup IV and melon yellow spot virus (MYSV, tospovirus. An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour was much shorter than that of ELISA (4 hours. This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

  3. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    Science.gov (United States)

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Oplatowska, Michalina; Gajanandana, Oraprapai; Grant, Irene R; Karoonuthaisiri, Nitsara; Elliott, Christopher T

    2013-01-01

    Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

  4. Internet-based attentional bias modification training as add-on to regular treatment in alcohol and cannabis dependent outpatients: a study protocol of a randomized control trial.

    Science.gov (United States)

    Heitmann, Janika; van Hemel-Ruiter, Madelon E; Vermeulen, Karin M; Ostafin, Brian D; MacLeod, Colin; Wiers, Reinout W; DeFuentes-Merillas, Laura; Fledderus, Martine; Markus, Wiebren; de Jong, Peter J

    2017-05-23

    The automatic tendency to attend to and focus on substance-related cues in the environment (attentional bias), has been found to contribute to the persistence of addiction. Attentional bias modification (ABM) interventions might, therefore, contribute to treatment outcome and the reduction of relapse rates. Based on some promising research findings, we designed a study to test the clinical relevance of ABM as an add-on component of regular intervention for alcohol and cannabis patients. The current protocol describes a study which will investigate the effectiveness and cost-effectiveness of a newly developed home-delivered, multi-session, internet-based ABM (iABM) intervention as an add-on to treatment as usual (TAU). TAU consists of cognitive behavioural therapy-based treatment according to the Dutch guidelines for the treatment of addiction. Participants (N = 213) will be outpatients from specialized addiction care institutions diagnosed with alcohol or cannabis dependency who will be randomly assigned to one of three conditions: TAU + iABM; TAU + placebo condition; TAU-only. Primary outcome measures are substance use, craving, and rates of relapse. Changes in attentional bias will be measured to investigate whether changes in primary outcome measures can be attributed to the modification of attentional bias. Indices of cost-effectiveness and secondary physical and psychological complaints (depression, anxiety, and stress) are assessed as secondary outcome measures. This randomized control trial will be the first to investigate whether a home-delivered, multi-session iABM intervention is (cost-) effective in reducing relapse rates in alcohol and cannabis dependency as an add-on to TAU, compared with an active and a waiting list control group. If proven effective, this ABM intervention could be easily implemented as a home-delivered component of current TAU. Netherlands Trial Register, NTR5497 , registered on 18th September 2015.

  5. Long-term efficacy and safety of sodium-glucose cotransporter-2 inhibitors as add-on to metformin treatment in the management of type 2 diabetes mellitus

    Science.gov (United States)

    Li, Jian; Gong, Yanping; Li, Chunlin; Lu, Yanhui; Liu, Yu; Shao, Yinghong

    2017-01-01

    Abstract Background: Drug intensification is often required for patients with type 2 diabetes mellitus on stable metformin therapy. Among the potential candidates for a combination therapy, sodium-glucose transporter-2 (SGLT2) inhibitors have shown promising outcomes. This meta-analysis was performed to compare the efficacy and safety of SGLT2 inhibitors with non-SGLT2 combinations as add-on treatment to metformin. Methods: Literature search was carried out in multiple electronic databases for the acquisition of relevant randomized controlled trials (RCTs) by following a priori eligibility criteria. After the assessment of quality of the included RCTs, meta-analyses of mean differences or odds ratios (OR) were performed to achieve overall effect sizes of the changes from baseline in selected efficacy and safety endpoints reported in the individual studies. Between-studies heterogeneity was estimated with between-studies statistical heterogeneity (I2) index. Results: Six RCTs fulfilled the eligibility criteria. SGLT2 inhibitors as add-on to metformin treatment reduced % HbA1c significantly more than non-SGLT2 combinations after 52 weeks (P = .002) as well as after 104 weeks (P SGLT2 inhibitors also reduced fasting plasma glucose levels, body weight, systolic, and diastolic blood pressures after 52 weeks and 104 weeks significantly (P SGLT2 combinations. Incidence of hypoglycemia was significantly lower (P = .02) but incidence of suspected or confirmed genital tract infections was significantly higher (P SGLT2 inhibitors treated in comparison with non-SGLT2 combinations. Conclusion: As add-on to metformin treatment, SGLT2 inhibitors are found significantly more efficacious than non-SGLT2 inhibitor combinations in the management of type 2 diabetes mellitus, although, SGLT2 inhibitor therapy is associated with significantly higher incidence of suspected or confirmed genital tract infections. PMID:28682870

  6. Renal function preservation with pioglitazone or with basal insulin as an add-on therapy for patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Chang, Yu-Hung; Hwu, Der-Wei; Chang, Dao-Ming; An, Ling-Wang; Hsieh, Chang-Hsun; Lee, Yau-Jiunn

    2017-06-01

    Clinical outcome may differ owing to the distinct pharmacological characteristics of insulin sensitizers and insulin. This study was performed to compare the metabolic and renal function changes with add-on pioglitazone treatment versus basal insulin in patients with type 2 diabetes mellitus (DM) in whom sulfonylurea and metformin regimens failed. Patients who were consecutively managed in the diabetes comprehensive program with add-on pioglitazone or detemir/glargine treatment for at least 2 years following sulfonylurea and metformin treatment failure were included. A total of 1002 patients were enrolled (pioglitazone: 559, detemir: 264, glargine: 179). After propensity score matching, there were 105 patients with matchable baseline characteristics in each group. After a mean of 3.5 years of follow-up, the pioglitazone group showed a greater HbA1c reduction than the detemir group and the glargine group. Despite patients in all three groups exhibiting significant body weight gain, those in the pioglitazone group and the glargine group showed greater body weight increases than the patients in the detemir group (2.1, 1.6 and 0.8 kg, respectively, p 1.79-3.88) and 3.13 (95% CI 2.01-4.87), respectively. Our study first showed that treatment with both pioglitazone and basal insulin improved glycemic control, while only pioglitazone treatment was observed to be advantageous in terms of preserving renal function when used as an add-on therapy for patients with type 2 DM in whom sulfonylurea and metformin regimens failed.

  7. Add-on treatment of benzoate for schizophrenia: a randomized, double-blind, placebo-controlled trial of D-amino acid oxidase inhibitor.

    Science.gov (United States)

    Lane, Hsien-Yuan; Lin, Ching-Hua; Green, Michael F; Hellemann, Gerhard; Huang, Chih-Chia; Chen, Po-Wei; Tun, Rene; Chang, Yue-Cung; Tsai, Guochuan E

    2013-12-01

    In addition to dopaminergic hyperactivity, hypofunction of the N-methyl-d-aspartate receptor (NMDAR) has an important role in the pathophysiology of schizophrenia. Enhancing NMDAR-mediated neurotransmission is considered a novel treatment approach. To date, several trials on adjuvant NMDA-enhancing agents have revealed beneficial, but limited, efficacy for positive and negative symptoms and cognition. Another method to enhance NMDA function is to raise the levels of d-amino acids by blocking their metabolism. Sodium benzoate is a d-amino acid oxidase inhibitor. To examine the clinical and cognitive efficacy and safety of add-on treatment of sodium benzoate for schizophrenia. A randomized, double-blind, placebo-controlled trial in 2 major medical centers in Taiwan composed of 52 patients with chronic schizophrenia who had been stabilized with antipsychotic medications for 3 months or longer. Six weeks of add-on treatment of 1 g/d of sodium benzoate or placebo. The primary outcome measure was the Positive and Negative Syndrome Scale (PANSS) total score. Clinical efficacy and adverse effects were assessed biweekly. Cognitive functions were measured before and after the add-on treatment. Benzoate produced a 21% improvement in PANSS total score and large effect sizes (range, 1.16-1.69) in the PANSS total and subscales, Scales for the Assessment of Negative Symptoms-20 items, Global Assessment of Function, Quality of Life Scale and Clinical Global Impression and improvement in the neurocognition subtests as recommended by the National Institute of Mental Health's Measurement and Treatment Research to Improve Cognition in Schizophrenia initiative, including the domains of processing speed and visual learning. Benzoate was well tolerated without significant adverse effects. Benzoate adjunctive therapy significantly improved a variety of symptom domains and neurocognition in patients with chronic schizophrenia. The preliminary results show promise for d-amino acid oxidase

  8. Cost-effectiveness of exenatide twice daily vs insulin glargine as add-on therapy to oral antidiabetic agents in patients with type 2 diabetes in China.

    Science.gov (United States)

    Gu, Shuyan; Wang, Xiaoyong; Qiao, Qing; Gao, Weiguo; Wang, Jian; Dong, Hengjin

    2017-12-01

    To estimate the long-term cost-effectiveness of exenatide twice daily vs insulin glargine once daily as add-on therapy to oral antidiabetic agents (OADs) for Chinese patients with type 2 diabetes (T2DM). The Cardiff Diabetes Model was used to simulate disease progression and estimate the long-term effects of exenatide twice daily vs insulin glargine once daily. Patient profiles and treatment effects required for the model were obtained from literature reviews (English and Chinese databases) and from a meta-analysis of 8 randomized controlled trials comparing exenatide twice daily with insulin glargine once daily add-on to OADs for T2DM in China. Medical expenditure data were collected from 639 patients with T2DM (aged ≥18 years) with and without complications incurred between January 1, 2014 and December 31, 2015 from claims databases in Shandong, China. Costs (2014 Chinese Yuan [¥]) and benefits were estimated, from the payers' perspective, over 40 years at a discount rate of 3%. A series of sensitivity analyses were performed. Patients on exenatide twice daily + OAD had a lower predicted incidence of most cardiovascular and hypoglycaemic events and lower total costs compared with those on insulin glargine once daily + OAD. A greater number of quality-adjusted life years (QALYs; 1.94) at a cost saving of ¥117 706 gained was associated with exenatide twice daily vs insulin glargine once daily. (i.e. cost saving of ¥60 764/QALY) per patient. In Chinese patients with T2DM inadequately controlled by OADs, exenatide twice daily is a cost-effective add-on therapy alternative to insulin glargine once daily, and may address the problem of an excess of medical needs resulting from weight gain and hypoglycaemia in T2DM treatment. © 2017 John Wiley & Sons Ltd.

  9. Efficacy and safety of dipeptidyl peptidase-4 inhibitors as an add-on to insulin treatment in patients with Type 2 diabetes

    DEFF Research Database (Denmark)

    Frandsen, Christian S.; Madsbad, S

    2014-01-01

    diabetes. METHODS: We searched the MEDLINE and PubMed databases to identify all randomized controlled clinical trials evaluating dipeptidyl peptidase-4 inhibitors as an add-on to insulin in patients with Type 2 diabetes, which were selected for review. The abstracts and posters of the recent annual...... and placebo treatment. CONCLUSION: Adding a dipeptidyl peptidase-4 inhibitor treatment to insulin has a moderate effect on HbA1c , a weight-neutral effect and a good safety profile. The risk of hypoglycaemia is not increased despite a significant improvement in HbA1c ....

  10. A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience.

    Science.gov (United States)

    Nielsen, Alex Christian Yde; Böttiger, Blenda; Midgley, Sofie Elisabeth; Nielsen, Lars Peter

    2013-11-01

    As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay. The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel testing and routine usage. During 15 months of routine use, from October 2008 to December 2009, we received and analysed 2187 samples (stool samples, cerebrospinal fluids, blood samples, respiratory samples and autopsy samples) were tested, from 1546 patients and detected enteroviruses and parechoviruses in 171 (8%) and 66 (3%) of the samples, respectively. 180 of the positive samples could be genotyped by PCR and sequencing and the most common genotypes found were human parechovirus type 3, echovirus 9, enterovirus 71, Coxsackievirus A16, and echovirus 25. During 2009 in Denmark, both enterovirus and human parechovirus type 3 had a similar seasonal pattern with a peak during the summer and autumn. Human parechovirus type 3 was almost invariably found in children less than 4 months of age. In conclusion, a multiplex assay was developed allowing simultaneous detection of 2 viruses, which can cause similar clinical symptoms. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. All Inkjet-Printed Amperometric Multiplexed Biosensors Based on Nanostructured Conductive Hydrogel Electrodes.

    Science.gov (United States)

    Li, Lanlan; Pan, Lijia; Ma, Zhong; Yan, Ke; Cheng, Wen; Shi, Yi; Yu, Guihua

    2018-02-12

    Multiplexing, one of the main trends in biosensors, aims to detect several analytes simultaneously by integrating miniature sensors on a chip. However, precisely depositing electrode materials and selective enzymes on distinct microelectrode arrays remains an obstacle to massively produced multiplexed sensors. Here, we report on a "drop-on-demand" inkjet printing process to fabricate multiplexed biosensors based on nanostructured conductive hydrogels in which the electrode material and several kinds of enzymes were printed on the electrode arrays one by one by employing a multinozzle inkjet system. The whole inkjet printing process can be finished within three rounds of printing and only one round of alignment. For a page of sensor arrays containing 96 working electrodes, the printing process took merely ∼5 min. The multiplexed assays can detect glucose, lactate, and triglycerides in real time with good selectivity and high sensitivity, and the results in phosphate buffer solutions and calibration serum samples are comparable. The inkjet printing process exhibited advantages of high efficiency and accuracy, which opens substantial possibilities for massive fabrication of integrated multiplexed biosensors for human health monitoring.

  12. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    Science.gov (United States)

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. © 2016 Society for Laboratory Automation and Screening.

  13. Cooperative epidemics on multiplex networks

    Science.gov (United States)

    Azimi-Tafreshi, N.

    2016-04-01

    The spread of one disease, in some cases, can stimulate the spreading of another infectious disease. Here, we treat analytically a symmetric coinfection model for spreading of two diseases on a two-layer multiplex network. We allow layer overlapping, but we assume that each layer is random and locally loopless. Infection with one of the diseases increases the probability of getting infected with the other. Using the generating function method, we calculate exactly the fraction of individuals infected with both diseases (so-called coinfected clusters) in the stationary state, as well as the epidemic spreading thresholds and the phase diagram of the model. With increasing cooperation, we observe a tricritical point and the type of transition changes from continuous to hybrid. Finally, we compare the coinfected clusters in the case of cooperating diseases with the so-called "viable" clusters in networks with dependencies.

  14. The Add-On Effect of Solifenacin for Patients with Remaining Overactive Bladder after Treatment with Tamsulosin for Lower Urinary Tract Symptoms Suggestive of Benign Prostatic Obstruction

    Directory of Open Access Journals (Sweden)

    Naoya Masumori

    2010-01-01

    Full Text Available Objectives. To investigate the add-on effect of solifenacin for Japanese men with remaining overactive bladder (OAB symptoms after tamsulosin monotherapy for lower urinary tract symptoms (LUTS suggestive of benign prostatic obstruction (BPO in real-life clinical practice. Methods. Patients aged ≥ 50 having remaining OAB symptoms (≥ 3 of OAB symptom score (OABSS with ≥2 of urgency score after at least 4 weeks treatment by 0.2 mg of tamsulosin for BPO/LUTS received 2.5 or 5.0 mg of solifenacin for 12 weeks. The International Prostate Symptom Score (IPSS, QOL index and OABSS, maximum flow rate (Qmax and postvoid residual urine volume (PVR were determined. Results. A total of 48 patients (mean age 72.5 years completed the study. There were significant improvement in IPSS (15.1 to 11.2 and QOL index (4.2 to 3.0 by add-on of solifenacin. Although the IPSS storage symptom score was significantly improved, there were no changes observed in the IPSS voiding symptom score. The OABSS showed significant improvement (8.0 to 4.8. No changes were observed in Qmax and PVR. Conclusions. Under the supervision of an experienced urologist, the additional administration of solifenacin to patients with BPO/LUTS treated with tamsulosin, is effective in controlling remaining OAB symptoms.

  15. Development of 8K-MCA Add-on based on FPGA technique with a control of VB6 software application under windows environment

    International Nuclear Information System (INIS)

    Dang Lanh; Nguyen Nhi Dien; Nguyen Xuan Hai; Nguyen Bach Viet; Pham Ngoc Son; Huynh Minh; Vuong Huu Tan; Pham Dinh Khang; Phan Nam Anh

    2003-01-01

    The multichannel analyzer is the heart of most experimental measurements. Today, it becomes popular and convenient. It can play a role of getting, processing data and easily interfacing to PC. Some standard-alone multichannel analysis systems can be replaced by Add-on MCA card. Especially, MCA card is necessary for application in nuclear physics research. The main aim of this sub-project is to focus on a design and construction of an 8K-MCA Add-on served for studying nuclear structure, for development of Gamma spectroscopy system, and for a production of low-cost electronics instruments as well. Some experimental results were obtained through the aforementioned card combining with other needed functional analog units. A multichannel data processing (MCD) circuit for nuclear spectroscopy application was developed using a field programmable gate arrays (FPGA) as the central processing element. In addition to the first role, A 13-bit analog-to-digital converter (8k ADC) circuit for nuclear spectroscopy application was also developed using a successive approximation ADC with a control of FPGA technology. The FPGA operates a program that builds the distributed functions of data collected by the ADC and then corrects the ADC differential non-linearity (DNL) via the sliding scale method. The acquisition routine runs in 3.7μ s. the conversion time is approximated 2.2μ s, and the integral non-linearity ≤ 0.14%. (author)

  16. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  17. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  18. Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

    Directory of Open Access Journals (Sweden)

    Sorette M

    2004-12-01

    Full Text Available Abstract Background Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. Results Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. Conclusion The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

  19. Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella.

    Science.gov (United States)

    Singh, Prashant; Mustapha, Azlin

    2013-09-02

    Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5 ng-50 fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 10(4) CFU/g, this was improved to 10 CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food. © 2013.

  20. Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR.

    Science.gov (United States)

    Wagner, Karoline; Springer, Burkard; Imkamp, Frank; Opota, Onya; Greub, Gilbert; Keller, Peter M

    2018-04-01

    Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was

  1. Diagnosis of Cutaneous Leishmaniasis by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    M Heiat

    2010-07-01

    Full Text Available Introduction: Annually, more than 14 million people are reported to be infected with Leishmaniasis all over the world. In Iran, this disease is seen in the form of cutaneous and visceral leishmaniasis, of which the cutaneous form is more wide spread. In recent years, cutaneous leishmaniaisis is diagnosed by PCR utilizing specific primers in order to amplify different parasite genes including ribosomal RNA genes, kinetoplast DNA or tandem repeating sequences. The aim of this research was to detect early stage cutaneous leishmaniasis using Multiplex-PCR technique. Methods: In this study, 67 samples were prepared from patients with cutaneous leishmaniasis. DNA was extracted with phenolchloroform. Each specimen was analyzed using two different pairs of PCR primers. The sensitivity of each PCR was optimized on pure Leishmania DNA prior to use for diagnosis. Two standard parasites L. major and L. tropica were used as positive control. Results: DNA amplification fragments were two 115 bp and 683 bp for AB and UL primers, respectively. The sensitivity of two primers was not equal for detection of L. major and L. tropica. The sensivity of PCR with AB primer was 35 cells, while that for UL primer was 40 cells. Conclusion: The results of this study indicate that PCR is a sensitive diagnostic assay for cutaneous leishmaniasis and could be employed as the new standard for routine diagnosis when species identification is not required. However, the ability to identify species is especially important in prognosis of the disease and in deciding appropriate therapy, especially in regions where more than one type of species and disease are seen by clinicians.

  2. Multiplex detection of pathogen biomarkers in human blood, serum, and saliva using silicon photonic microring resonators

    Science.gov (United States)

    Estrada, I. A.; Burlingame, R. W.; Wang, A. P.; Chawla, K.; Grove, T.; Wang, J.; Southern, S. O.; Iqbal, M.; Gunn, L. C.; Gleeson, M. A.

    2015-05-01

    Genalyte has developed a multiplex silicon photonic chip diagnostics platform (MaverickTM) for rapid detection of up to 32 biological analytes from a drop of sample in just 10 to 20 minutes. The chips are manufactured with waveguides adjacent to ring resonators, and probed with a continuously variable wavelength laser. A shift in the resonant wavelength as mass binds above the ring resonators is measured and is directly proportional to the amount of bound macromolecules. We present here the ability to multiplex the detection of hemorrhagic fever antigens in whole blood, serum, and saliva in a 16 minute assay. Our proof of concept testing of a multiplex antigencapture chip has the ability to detect Zaire Ebola (ZEBOV) recombinant soluble glycoprotein (rsGP), Marburg virus (MARV) Angola recombinant glycoprotein (rGP) and dengue nonstructural protein I (NS1). In parallel, detection of 2 malaria antigens has proven successful, but has yet to be incorporated into multiplex with the others. Each assay performs with sensitivity ranging from 1.6 ng/ml to 39 ng/ml depending on the antigen detected, and with minimal cross-reactivity.

  3. Simultaneous detection of enteropathogenic viruses in buffalos faeces using multiplex reverse transcription-polymerase chain reaction (mRT-PCR

    Directory of Open Access Journals (Sweden)

    U. Pagnini

    2010-02-01

    Full Text Available A multiplex reverse transcription- polymerase chain reaction (mRT-PCR assay that detects Bovine Viral Diarrhoea Virus, Bovine Coronavirus, and Group A Rotaviruses in infected cell-culture fluids and clinical faecal samples is described. One hundred twenty faecal samples from buffalo calves with acute gastroenteritis were tested. The mRT-PCR was validated against simplex RT-PCR with published primers for Pestivirus, Coronavirus and Rotavirus. The multiplex RT-PCR was equally sensitive and specific in detecting viral infections compared with simplex RT-PCR. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. This mRT-PCR may be a sensitive and rapid assay for surveillance of buffalo enteric viruses in field specimens. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of acute gastroenteritis.

  4. Immunization of Epidemics in Multiplex Networks

    Science.gov (United States)

    Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

    2014-01-01

    Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks. PMID:25401755

  5. 16-channel analog store and multiplexer unit

    Energy Technology Data Exchange (ETDEWEB)

    Brossard, M; Kulka, Z [Clermont-Ferrand-2 Univ., 63 - Aubiere (France). Lab. de Physique Corpusculaire

    1979-03-15

    A 16-channel analog store and multiplexer unit is described. The unit enables storing and selection of analog information which is then digitally encoded by single ADC. This solution becomes economically attractive particularly in multidetector pulse height analysis systems.

  6. Packaged mode multiplexer based on silicon photonics

    NARCIS (Netherlands)

    Chen, H.; Koonen, A.M.J.; Snyder, B.; Raz, O.; Boom, van den H.P.A.; Chen, X.

    2012-01-01

    A silicon photonics based mode multiplexer is proposed. Four chirped grating couplers structure can support all 6 channels in a two-mode fiber and realize LP01 and LP11 mode selective exciting. The packaged device is tested.

  7. Immunization of epidemics in multiplex networks.

    Science.gov (United States)

    Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

    2014-01-01

    Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks.

  8. Immunization of epidemics in multiplex networks.

    Directory of Open Access Journals (Sweden)

    Dawei Zhao

    Full Text Available Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted immunization and layer node-based random (targeted immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF networks.

  9. Multiplex Biosensing Based on Highly Sensitive Magnetic Nanolabel Quantification: Rapid Detection of Botulinum Neurotoxins A, B, and E in Liquids.

    Science.gov (United States)

    Orlov, Alexey V; Znoyko, Sergey L; Cherkasov, Vladimir R; Nikitin, Maxim P; Nikitin, Petr I

    2016-11-01

    We present a multiplex quantitative lateral flow (LF) assay for simultaneous on-site detection of botulinum neurotoxin (BoNT) types A, B, and E in complex matrixes, which is innovative by virtually no sacrifice in performance while transition from the single-plex assays and by characteristics on the level of laboratory quantitative methods. The novel approach to easy multiplexing is realized via joining an on-demand set of single-plex LF strips, which employ magnetic nanolabels, into a miniature cylinder cartridge that mimics LF strip during all assay stages. The cartridge is read out by an original portable multichannel reader based on the magnetic particle quantification technique. The developed reader offers the unmatched 60 zmol detection limit and 7-order linear dynamic range for volumetric registration of magnetic labels inside a cartridge of several millimeters in diameter regardless of its optical transparency. Each of the test strips, developed here as building blocks for the multiplex assay, can be used "as is" for autonomous quantitative single-plex detection with the same measuring setup, exhibiting the limits of detection (LOD) of 0.22, 0.11, and 0.32 ng/mL for BoNT-A, -B, and -E, respectively. The proposed multiplex assay has demonstrated the remarkably similar LOD values of 0.20, 0.12, 0.35 ng/mL under the same conditions. The multiplex assay performance was successfully validated by BoNT detection in milk and apple and orange juices. The developed methods can be extended to other proteins and used for rapid multianalyte tests for point-of-care in vitro diagnostics, food analysis, biosafety and environmental monitoring, forensics, and security, etc.

  10. Multiplex detection of tumor markers with photonic suspension array

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Yuanjin; Zhao Xiangwei [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Pei Xiaoping [Department of Hematology, Affiliated Zhongda Hospital, Southeast University, Nanjing 210009 (China); Hu Jing; Zhao Wenju [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Chen Baoan [Department of Hematology, Affiliated Zhongda Hospital, Southeast University, Nanjing 210009 (China); Gu Zhongze [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Laboratory of Environment and Biosafety, Research Institute of Southeast University in Suzhou, Dushu Lake Higher Education Town, Suzhou 215123 (China)], E-mail: gu@seu.edu.cn

    2009-02-02

    A novel photonic suspension array was developed for multiplex immunoassay. The carries of this array were silica colloidal crystal beads (SCCBs). The codes of these carriers are the characteristic reflection peak originated from their structural periodicity, and therefore they do not suffer from fading, bleaching, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are included, the fluorescence background of SCCBs is very low. With a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the four tumor markers, {alpha}-fetoprotein (AFP), carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125) and carcinoma antigen 19-9 (CA 19-9) could be assayed in the ranges of 1.0-500 ng mL{sup -1}, 1.0-500 ng mL{sup -1}, 1.0-500 U mL{sup -1} and 3.0-500 U mL{sup -1} with limits of detection of 0.68 ng mL{sup -1}, 0.95 ng mL{sup -1}, 0.99 U mL{sup -1} and 2.30 U mL{sup -1} at 3{sigma}, respectively. The proposed array showed acceptable accuracy, detection reproducibility, storage stability and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassay.

  11. Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children.

    Science.gov (United States)

    Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep

    2017-08-01

    Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant. © 2017 Wiley Periodicals, Inc.

  12. Effect of heart failure reversal treatment as add-on therapy in patients with chronic heart failure: A randomized, open-label study.

    Science.gov (United States)

    Sane, Rohit; Aklujkar, Abhijeet; Patil, Atul; Mandole, Rahul

    The present study was designed to evaluate effect of heart failure reversal therapy (HFRT) using herbal procedure (panchakarma) and allied therapies, as add-on to standard CHF treatment (SCT) in chronic heart failure (CHF) patients. This open-label, randomized study conducted in CHF patients (aged: 25-65 years, ejection fraction: 30-65%), had 3-phases: 1-week screening, 6-week treatment (randomized [1:1] to HFRT+SCT or SCT-alone) and follow-up (12-week). Twice weekly HFRT (60-75min) consisting of snehana (external oleation), swedana (passive heat therapy), hrudaydhara (concoction dripping treatment) and basti (enema) was administered. Primary endpoints included evaluation of change in metabolic equivalents of task (MET) and peak oxygen uptake (VO 2peak ) from baseline, at end of 6-week treatment and follow-up at week-18 (non-parametric rank ANCOVA analysis). Safety and quality of life (QoL) was assessed. Seventy CHF patients (n=35, each treatment-arm; mean [SD] age: 53.0 [8.6], 80% men) were enrolled in the study. All patients completed treatment phase. Add-on HFRT caused a significant increase in METs (least square mean difference [LSMD], 6-week: 1.536, p=0.0002; 18-week: -1.254, p=0.0089) and VO 2peak (LSMD, 6-week: -5.52, p=0.0002; 18-week: -4.517, p=0.0089) as compared with SCT-alone. Results were suggestive of improved functional capacity in patients with HFRT (QoL; Mean [SD] HFRT+SCT vs. SCT-alone; 6-week: -0.44 [0.34] vs. -0.06 [0.25], p<0.0001 and 18-week: -0.53 [0.35] vs. -0.29 [0.26], p=0.0013). Seven treatment-emergent adverse events (mild severity) were reported in HFRT-arm. Findings of this study highlight therapeutic efficacy of add-on HFRT vs. SCT-alone in CHF patients. The non-invasive HFRT showed no safety concerns. Copyright © 2016. Published by Elsevier B.V.

  13. Real-life efficacy and safety of vildagliptin compared with sulfonylureas as add-on to metformin in patients with type 2 diabetes mellitus in Germany.

    Science.gov (United States)

    Göke, R; Gruenberger, J B; Bader, G; Dworak, M

    2014-05-01

    Metformin is an established first-line treatment for type 2 diabetes mellitus (T2DM) patients, but intensification of oral anti-diabetes therapy is usually required over time. A large observational study of 45,868 T2DM patients in 27 countries (EDGE) was conducted to compare the effectiveness and safety of vildagliptin as add-on therapy to another oral anti-diabetes drug (OAD) vs other dual OAD combinations. This report presents results from a post-hoc analysis of patients in Germany who received vildagliptin or a sulfonylurea (SU) in combination with metformin. Patients inadequately controlled with monotherapy became eligible only after the add-on treatment was finalized. Patients included were assigned to receive either vildagliptin or another OAD (SUs, thiazolidinediones, glinides, α-glucosidase inhibitors, or metformin; DPP-4 inhibitors or glucagon-like peptide-1 [GLP-1] mimetics/analogs were excluded). The primary end-point was the proportion of patients achieving a reduction in HbA1c >0.3% without peripheral edema, hypoglycemia, discontinuation due to gastrointestinal event, or weight gain ≥5%. Of 8887 patients enrolled in Germany, 6439 received vildagliptin and 971 received SUs as add-on to metformin. The primary end-point was reached in 34.9% and 29.6% of patients in the vildagliptin and SU groups, respectively, with an unadjusted odds ratio of 1.27 (95% CI = 1.09, 1.47; p = 0.001). HbA1c decreased in both cohorts from baseline (-0.7% with vildagliptin vs -0.5% with SUs), with a mean between-group difference of -0.2% (95% CI = -0.22, -0.09). The number of hypoglycemic events was 4-fold higher in the SU group than in the vildagliptin group (vildagliptin = 0.11%; SU = 0.41%). In a real-life setting, vildagliptin was associated with a numerically greater reduction in HbA1c, less hypoglycemia, and more patients reaching target HbA1c without hypoglycemia or weight gain compared with SUs. Open-label design and under reporting of adverse

  14. 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.

  15. Colloid-based multiplexed method for screening plant biomass-degrading glycoside hydrolase activities in microbial communities

    Energy Technology Data Exchange (ETDEWEB)

    Reindl, W.; Deng, K.; Gladden, J.M.; Cheng, G.; Wong, A.; Singer, S.W.; Singh, S.; Lee, J.-C.; Yao, J.-S.; Hazen, T.C.; Singh, A.K; Simmons, B.A.; Adams, P.D.; Northen, T.R.

    2011-05-01

    The enzymatic hydrolysis of long-chain polysaccharides is a crucial step in the conversion of biomass to lignocellulosic biofuels. The identification and characterization of optimal glycoside hydrolases is dependent on enzyme activity assays, however existing methods are limited in terms of compatibility with a broad range of reaction conditions, sample complexity, and especially multiplexity. The method we present is a multiplexed approach based on Nanostructure-Initiator Mass Spectrometry (NIMS) that allowed studying several glycolytic activities in parallel under diverse assay conditions. Although the substrate analogs carried a highly hydrophobic perfluorinated tag, assays could be performed in aqueous solutions due colloid formation of the substrate molecules. We first validated our method by analyzing known {beta}-glucosidase and {beta}-xylosidase activities in single and parallel assay setups, followed by the identification and characterization of yet unknown glycoside hydrolase activities in microbial communities.

  16. Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction

    Science.gov (United States)

    Kim, Ji Yeun; Lee, Jung-Lim

    2014-01-01

    Background This study describes the first multiplex real-time polymerase chain reaction assay developed, as a multipurpose assessment, for the simultaneous quantification of total bacteria and three Vibrio spp. (V. parahaemolyticus, V. vulnificus and V. anguillarum) in fish and seawater. The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers. Freshness and quality of fishery products also depend on the total bacterial populations present. Results The detection sensitivity of the specific targets for the multiplex assay was 1 CFU mL−1 in pure culture and seawater, and 10 CFU g−1 in fish. While total bacterial counts by the multiplex assay were similar to those obtained by cultural methods, the levels of Vibrio detected by the multiplex assay were generally higher than by cultural methods of the same populations. Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp. Conclusion Our data demonstrate that this multiplex assay could be useful for the rapid detection and quantification of Vibrio spp. and total bacteria as a multipurpose tool for surveillance of fish and water quality as well as diagnostic method. © 2014 The Authors. Journal of the Science of Food and Agriculture published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:24752974

  17. A capillary-based multiplexed isothermal nucleic acid-based test for sexually transmitted diseases in patients.

    Science.gov (United States)

    Xu, Gaolian; Zhao, Hang; Cooper, Jonathan M; Reboud, Julien

    2016-10-06

    We demonstrate a multiplexed loop mediated isothermal amplification (LAMP) assay for infectious disease diagnostics, where the analytical process flow of target pathogens genomic DNA is performed manually by moving magnetic beads through a series of plugs in a capillary. Heat is provided by a water bath and the results are read by the naked eye, enabling applications in low resource settings.

  18. Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

    Directory of Open Access Journals (Sweden)

    Zhi Wan

    Full Text Available Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the "matrix effect" caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay. AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children's Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%. In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative

  19. A heuristic model linking yoga philosophy and self-reflection to examine underlying mechanisms of add-on yoga treatment in schizophrenia.

    Science.gov (United States)

    Rao, Naren; Menon, Sangeetha

    2016-06-01

    Preliminary evidence suggests efficacy of yoga as add-on treatment for schizophrenia, but the underlying mechanism by which yoga improves the symptoms of schizophrenia is not completely understood. Yoga improves self-reflection in healthy individuals, and self-reflection abnormalities are typically seen in schizophrenia. However, whether yoga treatment improves impairments in self-reflection typically seen in patients with schizophrenia is not examined. This paper discusses the potential mechanism of yoga in the treatment of schizophrenia and proposes a testable hypothesis for further empirical studies. It is proposed that self-reflection abnormalities in schizophrenia improve with yoga and the neurobiological changes associated with this can be examined using empirical behavioural measures and neuroimaging measures such as magnetic resonance imaging.

  20. Effects of add on PTH(1-84) substitution therapy in hypoparathyroidism: results from a double-blind randomised controlled study

    DEFF Research Database (Denmark)

    Sikjær, Tanja Tvistholm; Rejnmark, Lars; Mosekilde, Leif

    -phosphate levels within the physiological range. Compared with placebo, participants randomised to PTH (1-84) reduced their daily dose of calcium supplements by 68 % (pvitamin D dose by 53% (pneed of calcium supplements...... and 7 were not in need of active vitamin D. With the actual add on therapy there were no change in renal calcium excretion in response to treatment. Compared with placebo, bone mineral density (BMD) decreased significantly at the hip (-1.59 ± 0.57%), lumbar spine (-1.76 ± 1.03%) and whole body (-1...... bone packets. In conclusion, PTH substitution therapy is capable of maintaining normal p-calcium levels with a significantly reduced need for calcium supplements and active vitamin D. In contrast to the effect of PTH(1-84) treatment in patients with osteoporosis, causing an increase in BMD...

  1. Genetic barcoding with fluorescent proteins for multiplexed applications.

    Science.gov (United States)

    Smurthwaite, Cameron A; Williams, Wesley; Fetsko, Alexandra; Abbadessa, Darin; Stolp, Zachary D; Reed, Connor W; Dharmawan, Andre; Wolkowicz, Roland

    2015-04-14

    Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded 'indefinitely'. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

  2. Comparison of conventional inserts and an add-on electron MLC for chest wall irradiation of left-sided breast cancer

    International Nuclear Information System (INIS)

    Vatanen, Tero; Lahtinen, Tapani; Traneus, Erik

    2009-01-01

    Background. Collimation of irregularly shaped clinical electron beams is currently based on electron inserts made of low melting point alloys. The present investigation compares a conventional electron applicator with insert and add-on eMLC-based dose distributions in the postoperative chest wall irradiation of left-sided breast cancer. Material and methods. Voxel Monte Carlo++ (VMC++) calculated dose distributions related to electron fields were compared with 10 left-sided breast cancer patients after radical mastectomy. The prescription dose was 50 Gy at a build-up maximum. The same dose was prescribed for the ipsilateral axillary, parasternal and supraclavicular lymph nodes that were treated with photons and calculated with a pencil beam algorithm. The insert beams were shaped with 1.5 cm thick Wood's metal electron inserts in an electron applicator of a Varian 2100 C/D linac. Doses for the eMLC-shaped beams were calculated for an eMLC prototype with 2 cm thick and 5 mm wide steel leaves. The same collimator-to-surface distance (CSD) of 5.8 cm was used for both collimators. Results. The mean PTV dose was slightly higher for the eMLC plans (50.7 vs 49.5 Gy, p<0.001, respectively). The maximum doses assessed by D5% for the eMLC and insert were 60.9 and 59.1 Gy (p<0.001). The difference was due to the slightly higher doses near the field edges for the eMLC. The left lung V20 volumes were 34.5% and 34.0% (p<0.001). There was only a marginal difference in heart doses. Discussion: Despite a slight increase of maximum dose in PTV the add-on electron MLC for chest wall irradiation results in practically no differences in dose distributions compared with the present insert-based collimation

  3. Metabolic and other effects of pioglitazone as an add-on therapy to metformin in the treatment of polycystic ovary syndrome (PCOS).

    Science.gov (United States)

    Valsamakis, Georgios; Lois, Kostas; Kumar, Sudhesh; Mastorakos, George

    2013-01-01

    Insulin resistance is a key pathogenic defect of the clustered metabolic disturbances seen in polycystic ovary syndrome (PCOS). Metformin is an insulin sensitizer acting in the liver and the peripheral tissues that ameliorates the metabolic and reproductive defects in PCOS. In addition, pioglitazone is an insulin sensitizer used in diabetes mellitus type 2 (T2DM), improving insulin resistance (IR) in adipose tissue and muscles. In T2DM, these drugs are also used as a combined treatment due to their "add-on effect" on insulin resistance. Although the beneficial role of troglitazone (a member of the thiazolidinediones (TZDs) family) in PCOS has been shown in the past, currently only pioglitazone is available in the market. A few small randomized controlled trials have directly compared the effectiveness of pioglitazone in women with PCOS, while there are a limited number of small studies that support the beneficial metabolic add-on effect of pioglitazone on metformin-treated PCOS women as compared to metformin or pioglitazone monotherapy. These findings suggest a potentially promising role for combined pioglitazone/metformin treatment in the management of PCOS in metformin-resistant patients. In view of recent concerns regarding pioglitazone usage and its associated health risk, we aim to compare the pros and cons of each drug regarding their metabolic and other hormonal effects in women with PCOS and to explore the possible beneficial effect of combined therapy in certain cases, taking into consideration the teratogenic effect of pioglitazone. Finally, we discuss the need for a randomized controlled trial that will evaluate the metabolic and other hormonal effects of combined metformin/pioglitazone treatment in PCOS with selective treatment targets.

  4. Computerized cognitive training in children and adolescents with attention deficit/hyperactivity disorder as add-on treatment to stimulants: feasibility study and protocol description

    Directory of Open Access Journals (Sweden)

    Virginia de Oliveira Rosa

    Full Text Available Abstract Background Cognitive training has received increasing attention as a non-pharmacological approach for the treatment of attention deficit/hyperactivity disorder (ADHD in children and adolescents. Few studies have assessed cognitive training as add-on treatment to medication in randomized placebo controlled trials. The purpose of this preliminary study was to explore the feasibility of implementing a computerized cognitive training program for ADHD in our environment, describe its main characteristics and potential efficacy in a small pilot study. Methods Six ADHD patients aged 10-12-years old receiving stimulants and presenting residual symptoms were enrolled in a randomized clinical trial to either a standard cognitive training program or a controlled placebo condition for 12 weeks. The primary outcome was core ADHD symptoms measured using the Swanson, Nolan and Pelham Questionnaire (SNAP-IV scale. Results We faced higher resistance than expected to patient enrollment due to logistic issues to attend face-to-face sessions in the hospital and to fill the requirement of medication status and absence of some comorbidities. Both groups showed decrease in parent reported ADHD symptoms without statistical difference between them. In addition, improvements on neuropsychological tests were observed in both groups – mainly on trained tasks. Conclusions This protocol revealed the need for new strategies to better assess the effectiveness of cognitive training such as the need to implement the intervention in a school environment to have an assessment with more external validity. Given the small sample size of this pilot study, definitive conclusions on the effects of cognitive training as add-on treatment to stimulants would be premature.

  5. Granisetron as an add-on to risperidone for treatment of negative symptoms in patients with stable schizophrenia: randomized double-blind placebo-controlled study.

    Science.gov (United States)

    Khodaie-Ardakani, Mohammad-Reza; Seddighi, Sahar; Modabbernia, Amirhossein; Rezaei, Farzin; Salehi, Bahman; Ashrafi, Mandana; Shams-Alizadeh, Narges; Mohammad-Karimi, Maryam; Esfandiari, Gholam-Reza; Hajiaghaee, Reza; Akhondzadeh, Shahin

    2013-04-01

    Some 5-HT3 antagonists such as ondansetron have shown beneficial effects on negative symptoms of patients with schizophrenia. We aimed to evaluate the efficacy of granisetron (another 5-HT3 antagonist) add-on therapy in the treatment of negative symptoms of patients with stable schizophrenia. In a randomized, double-blind, and placebo-controlled study, forty stable patients with schizophrenia (DSM-IV-TR), were randomized to either granisetron (1 mg twice daily) or placebo (twice daily) in addition to risperidone up to 6 mg/day for eight weeks. The patients were assessed using positive and negative syndrome scale (PANSS) and extrapyramidal symptom rating scale (ESRS) at baseline, week 4 and 8. Hamilton depression rating scale (HDRS) was used to assess depression at baseline and week 8. Thirty-eight patients completed the trial. Granisetron group showed a significantly greater improvement on negative subscale than the placebo group at endpoint [t(38) = 6.046, mean difference (±95% CI) = 3.2(1.8-3.7), P granisetron groups did not differ in their reduction of positive and general psychopathology symptoms scores. HDRS scores and its changes did not differ between the two groups. The ESRS score at week 4 was significantly lower in the granisetron than the placebo group while the two groups showed similar ESRS score at week 8. Frequency of other side effects was similar between the two groups. In summary, granisetron add-on can safely and effectively reduce the primary negative symptoms of patients with schizophrenia. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Computerized cognitive training in children and adolescents with attention deficit/hyperactivity disorder as add-on treatment to stimulants: feasibility study and protocol description.

    Science.gov (United States)

    Rosa, Virginia de Oliveira; Schmitz, Marcelo; Moreira-Maia, Carlos Roberto; Wagner, Flavia; Londero, Igor; Bassotto, Caroline de Fraga; Moritz, Guilherme; de Souza, Caroline Dos Santos; Rohde, Luis Augusto Paim

    2017-01-01

    Cognitive training has received increasing attention as a non-pharmacological approach for the treatment of attention deficit/hyperactivity disorder (ADHD) in children and adolescents. Few studies have assessed cognitive training as add-on treatment to medication in randomized placebo controlled trials. The purpose of this preliminary study was to explore the feasibility of implementing a computerized cognitive training program for ADHD in our environment, describe its main characteristics and potential efficacy in a small pilot study. Six ADHD patients aged 10-12-years old receiving stimulants and presenting residual symptoms were enrolled in a randomized clinical trial to either a standard cognitive training program or a controlled placebo condition for 12 weeks. The primary outcome was core ADHD symptoms measured using the Swanson, Nolan and Pelham Questionnaire (SNAP-IV scale). We faced higher resistance than expected to patient enrollment due to logistic issues to attend face-to-face sessions in the hospital and to fill the requirement of medication status and absence of some comorbidities. Both groups showed decrease in parent reported ADHD symptoms without statistical difference between them. In addition, improvements on neuropsychological tests were observed in both groups - mainly on trained tasks. This protocol revealed the need for new strategies to better assess the effectiveness of cognitive training such as the need to implement the intervention in a school environment to have an assessment with more external validity. Given the small sample size of this pilot study, definitive conclusions on the effects of cognitive training as add-on treatment to stimulants would be premature.

  7. Phenobarbital or potassium bromide as an add-on antiepileptic drug for the management of canine idiopathic epilepsy refractory to imepitoin.

    Science.gov (United States)

    Royaux, E; Van Ham, L; Broeckx, B J G; Van Soens, I; Gielen, I; Deforce, D; Bhatti, S F M

    2017-02-01

    Imepitoin has recently been approved in Europe for the management of dogs with idiopathic epilepsy. Currently, there is no evidence-based information available on the efficacy of antiepileptic drugs used as additions to the therapeutic regimen in dogs with idiopathic epilepsy that are not well controlled with imepitoin. The goal of this study was to evaluate the efficacy of phenobarbital or potassium bromide (KBr) as add-on antiepileptic drugs for controlling dogs refractory to a maximum dose of imepitoin (30 mg/kg twice daily). The study was performed as a prospective, randomised, controlled clinical trial. The efficacy of phenobarbital and KBr was evaluated by comparing monthly seizure frequency (MSF), monthly seizure day frequency (MSDF), the presence of cluster seizures during a retrospective 2-month period with a prospective follow-up of 6 months, and the overall responder rate. Twenty-seven dogs were included in the study, 14 dogs in the phenobarbital group and 13 dogs in the KBr group. Both median MSF and MSDF decreased in the phenobarbital group (both P = 0.001) and in the KBr group (P = 0.004 and P = 0.003, respectively). Overall, the number of dogs with cluster seizures decreased (P = 0.0005). The responder rate was 79% vs. 69% in the phenobarbital and KBr groups, respectively. We conclude that phenobarbital or KBr add-on treatment decreases median MSF and MSDF in epileptic dogs refractory to a maximum dose of imepitoin. Combination therapy was generally well tolerated and resulted in an improvement in seizure management in the majority of the dogs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Cost-Utility Analysis of Heberprot-P as an Add-on Therapy to Good Wound Care for Patients in Slovakia with Advanced Diabetic Foot Ulcer

    Directory of Open Access Journals (Sweden)

    Tomas Tesar

    2017-12-01

    Full Text Available Objectives: To explore whether Heberprot-P (an epidermal growth factor is a cost-effective option for the treatment of advanced diabetic foot ulcer as an add-on therapy to good wound care (GWC in Slovakia from the perspective of health care payers.Methods: A Markov model was constructed to compare the costs and effects of Heberprot-P plus GWC to those of GWC alone from the perspective of health care payers. The 52-week clinical trial period was extended to 5- and 10-year time horizons. Transition probabilities were calculated based on a previous clinical trial of Heberprot, utility values were derived from the scientific literature, and cost vectors were collected from the General Health Insurance Fund database in Slovakia. A one-way deterministic sensitivity analysis was employed to explore the influence of uncertainty for each input parameter on the incremental cost-effectiveness ratio (ICER.Results: Based on the ICER threshold of €30,030 per quality-adjusted life year (QALY recommended by the Slovak Ministry of Health, Heberprot-P therapy plus GWC is not a cost-effective alternative to GWC alone over a 10-year time horizon. The ICER increases if a longer time horizon is applied, as the incremental costs are similar, but the aggregated utility gain from avoided amputation is lower. Based on the sensitivity analysis, the utility multiplier for the health state “no ulcer after small amputation” had the most impact on the ICER; however, the model was robust to changes in all input parameters.Conclusions: Heberprot-P, as an add-on therapy to GWC in the treatment of advanced diabetic foot ulcer, is not a cost-effective alternative to GWC alone. However, if the unit cost of Heberprot-P were to be reduced to <€273, its ICER would be <€30,030.

  9. Cost-effectiveness of omalizumab add-on to standard-of-care therapy in patients with uncontrolled severe allergic asthma in a Brazilian healthcare setting.

    Science.gov (United States)

    Suzuki, Cibele; Lopes da Silva, Nilceia; Kumar, Praveen; Pathak, Purnima; Ong, Siew Hwa

    2017-08-01

    Omalizumab add-on to standard-of-care therapy has proven to be efficacious in severe asthma patients for whom exacerbations cannot be controlled otherwise. Moreover, evidence from different healthcare settings suggests reduced healthcare resource utilization with omalizumab. Based on these findings, this study aimed to assess the cost-effectiveness of the addition of omalizumab to standard-of-care therapy in patients with uncontrolled severe allergic asthma in a Brazilian healthcare setting. A previously published Markov model was adapted using Brazil-specific unit costs to compare the costs and outcomes of the addition of omalizumab to standard-of-care therapy vs standard-of-care therapy alone. Model inputs were largely based on the eXpeRience study. Costs and health outcomes were calculated for lifetime-years and were annually discounted at 5%. Both one-way and probabilistic sensitivity analyses were performed. An additional cost of R$280,400 for 5.20 additional quality-adjusted life-years was estimated with the addition of omalizumab to standard-of-care therapy, resulting in an incremental cost-effectiveness ratio of R$53,890. One-way sensitivity analysis indicated that discount rates, standard-of-care therapy exacerbation rates, and exacerbation-related mortality rates had the largest impact on incremental cost-effectiveness ratios. Assumptions of lifetime treatment adherence and rate of future exacerbations, independent of previous events, might affect the findings. The lack of Brazilian patients in the eXpeRience study may affect the findings, although sample size and baseline characteristics suggest that the modeled population closely resembles Brazilian severe allergic asthma patients. Results indicate that omalizumab as an add-on therapy is more cost-effective than standard-of-care therapy alone for Brazilian patients with uncontrolled severe allergic asthma, based on the World Health Organization's cost-effectiveness threshold of up to 3-times the gross

  10. Silicon Chip-to-Chip Mode-Division Multiplexing

    DEFF Research Database (Denmark)

    Baumann, Jan Markus; Porto da Silva, Edson; Ding, Yunhong

    2018-01-01

    A chip-to-chip mode-division multiplexing connection is demonstrated using a pair of multiplexers/demultiplexers fabricated on the silicon-on-insulator platform. Successful mode multiplexing and demultiplexing is experimentally demonstrated, using the LP01, LP11a and LP11b modes.......A chip-to-chip mode-division multiplexing connection is demonstrated using a pair of multiplexers/demultiplexers fabricated on the silicon-on-insulator platform. Successful mode multiplexing and demultiplexing is experimentally demonstrated, using the LP01, LP11a and LP11b modes....

  11. Aptamer-based multiplexed proteomic technology for biomarker discovery.

    Science.gov (United States)

    Gold, Larry; Ayers, Deborah; Bertino, Jennifer; Bock, Christopher; Bock, Ashley; Brody, Edward N; Carter, Jeff; Dalby, Andrew B; Eaton, Bruce E; Fitzwater, Tim; Flather, Dylan; Forbes, Ashley; Foreman, Trudi; Fowler, Cate; Gawande, Bharat; Goss, Meredith; Gunn, Magda; Gupta, Shashi; Halladay, Dennis; Heil, Jim; Heilig, Joe; Hicke, Brian; Husar, Gregory; Janjic, Nebojsa; Jarvis, Thale; Jennings, Susan; Katilius, Evaldas; Keeney, Tracy R; Kim, Nancy; Koch, Tad H; Kraemer, Stephan; Kroiss, Luke; Le, Ngan; Levine, Daniel; Lindsey, Wes; Lollo, Bridget; Mayfield, Wes; Mehan, Mike; Mehler, Robert; Nelson, Sally K; Nelson, Michele; Nieuwlandt, Dan; Nikrad, Malti; Ochsner, Urs; Ostroff, Rachel M; Otis, Matt; Parker, Thomas; Pietrasiewicz, Steve; Resnicow, Daniel I; Rohloff, John; Sanders, Glenn; Sattin, Sarah; Schneider, Daniel; Singer, Britta; Stanton, Martin; Sterkel, Alana; Stewart, Alex; Stratford, Suzanne; Vaught, Jonathan D; Vrkljan, Mike; Walker, Jeffrey J; Watrobka, Mike; Waugh, Sheela; Weiss, Allison; Wilcox, Sheri K; Wolfson, Alexey; Wolk, Steven K; Zhang, Chi; Zichi, Dom

    2010-12-07

    The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

  12. Aptamer-based multiplexed proteomic technology for biomarker discovery.

    Directory of Open Access Journals (Sweden)

    Larry Gold

    2010-12-01

    Full Text Available The interrogation of proteomes ("proteomics" in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma. Our current assay measures 813 proteins with low limits of detection (1 pM median, 7 logs of overall dynamic range (~100 fM-1 µM, and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD. We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

  13. 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

    potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

  14. Inter-laboratory assessment of a prototype multiplex kit for determination of recent HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Kelly A Curtis

    Full Text Available BACKGROUND: Accurate and reliable laboratory-based assays are needed for estimating HIV-1 incidence from cross-sectional samples. We recently described the development of a customized, HIV-1-specific Bio-Plex assay that allows for the measurement of HIV-specific antibody levels and avidity to multiple analytes for improved HIV-1 incidence estimates. METHODS: To assess intra- and inter-laboratory assay performance, prototype multiplex kits were developed and evaluated by three distinct laboratories. Longitudinal seroconversion specimens were tested in parallel by each laboratory and kit performance was compared to that of an in-house assay. Additionally, the ability of the kit to distinguish recent from long-term HIV-1 infection, as compared to the in-house assay, was determined by comparing the reactivity of known recent (infected 12 months drug naïve specimens. RESULTS: Although the range of reactivity for each analyte varied between the prototype kit and in-house assay, a measurable distinction in reactivity between recent and long-term specimens was observed with both assays in all three laboratories. Additionally, kit performance was consistent between all three laboratories. The intra-assay coefficient of variation (CV, between sample replicates for all laboratories, ranged from 0.5% to 6.1%. The inter-laboratory CVs ranged from 8.5% to 21.3% for gp160-avidity index (a and gp120-normalized mean fluorescent intensity (MFI value (n, respectively. CONCLUSION: We demonstrate the feasibility of producing a multiplex kit for measuring HIV antibody levels and avidity, with the potential for improved incidence estimates based on multi-analyte algorithms. The availability of a commercial kit will facilitate the transfer of technology among diverse laboratories for widespread assay use.

  15. 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.

  16. Blood pressure control with amlodipine add-on therapy in patients with hypertension and diabetes: results of the Amlodipine Diabetic Hypertension Efficacy Response Evaluation Trial.

    Science.gov (United States)

    Kloner, Robert A; Neutel, Joel; Roth, Eli M; Weiss, Robert; Weinberger, Myron H; Thakker, Kamlesh M; Schwartz, Brian; Shi, Harry; Gregg, Anne-Marie

    2008-11-01

    Attainment of blood pressure (BP) goals in patients with diabetes is critical both to reduce the risk of cardiovascular events and to delay the progression of renal disease. While therapeutic guidelines advise initial therapy with an angiotensin-converting enzyme inhibitor or angiotensin receptor blocker, monotherapy with these agents may not be sufficient to attain target BP. The ADHT (Amlodipine Diabetic Hypertension Efficacy Response Evaluation Trial) evaluated the efficacy and safety of adding amlodipine to the treatment regimen of patients with hypertension and diabetes who were already receiving either quinapril or losartan as monotherapy. ADHT was a double-blind, double-dummy, 22-week trial conducted in the US. After a washout period of 7-13 days, patients (aged 30-75 y) with hypertension and diabetes were randomized to receive quinapril 20 mg/day plus placebo or losartan 50 mg/day plus placebo for 4 weeks, titrated to 40 mg or 100 mg (if required), respectively, for an additional 4 weeks to achieve their BP goals (<130/80 mm Hg). At week 8, either amlodipine 5 mg/day or placebo was added for an additional 12 weeks, with titration to 10 mg at week 14 if the BP goal was not achieved. Efficacy of add-on therapy was evaluated in 411 patients (amlodipine 211, placebo 200). BP goal was reached by 27.5% of patients when amlodipine was added to quinapril or losartan monotherapy, compared with 12.5% when placebo was added (OR 2.73; 95% CI 1.61 to 4.64; p < 0.001). When added to quinapril or losartan monotherapy, amlodipine reduced BP by 8.1/5.4 mm Hg, compared with a 1.6/0.7 mm Hg decrease with add-on placebo (p < 0.001). Amlodipine, quinapril, and losartan were well tolerated. Amlodipine is safe and effective when added to quinapril or losartan monotherapy to help lower BP toward therapeutic targets in patients with hypertension and diabetes.

  17. Management implications for the perioperative surgical home related to inpatient case cancellations and add-on case scheduling on the day of surgery.

    Science.gov (United States)

    Epstein, Richard H; Dexter, Franklin

    2015-07-01

    The American Society of Anesthesiologists has embraced the concept of the Perioperative Surgical Home as a means through which anesthesiologists can add value to the health systems in which they practice. One key listed element of the Perioperative Surgical Home is to support "scheduling initiatives to reduce cancellations and increase efficiency." In this study, we explored the potential benefits of the Perioperative Surgical Home with respect to inpatient cancellations and add-on case scheduling. We evaluated 6 hypotheses related to the timing of inpatient cancellations and preoperative anesthesia evaluations. Inpatient cancellations were studied during 26 consecutive 4-week intervals between July 2012 and June 2014 at a tertiary care academic hospital. All timestamps related to scheduling, rescheduling, and cancellation activities were retrieved from the operating room (OR) case scheduling system. Timestamps when patients were seen by anesthesia residents were obtained from the preoperative evaluation system database. Batch mean methods were used to calculate means and SE. For cases cancelled, we determined whether, for "most" (>50%) cancellations, a subsequent procedure (of any type) was performed on the patient within 7 days of the cancellation. Comparisons with most and other fractions were assessed using the 1 group, 1-sided Student t test. We evaluated whether a few procedures were highly represented among the cancelled cases via the Herfindahl (Simpson's) index, comparing it with Data from 24,735 scheduled inpatient cases were assessed. Cases cancelled after 7 AM on the day before or at any time on the scheduled day of surgery accounted for 22.6% ± 0.5% (SE) of the scheduled minutes all scheduled cases, and 26.8% ± 0.4% of the case volume (i.e., number of cases). Most (83.1% ± 0.6%, P 50%, P = 0.12), implying that the indication for the cancelled procedure no longer existed or the patient/family decided not to proceed with surgery. When only

  18. Social contagions on correlated multiplex networks

    Science.gov (United States)

    Wang, Wei; Cai, Meng; Zheng, Muhua

    2018-06-01

    The existence of interlayer degree correlations has been disclosed by abundant multiplex network analysis. However, how they impose on the dynamics of social contagions are remain largely unknown. In this paper, we propose a non-Markovian social contagion model in multiplex networks with inter-layer degree correlations to delineate the behavior spreading, and develop an edge-based compartmental (EBC) theory to describe the model. We find that multiplex networks promote the final behavior adoption size. Remarkably, it can be observed that the growth pattern of the final behavior adoption size, versus the behavioral information transmission probability, changes from discontinuous to continuous once decreasing the behavior adoption threshold in one layer. We finally unravel that the inter-layer degree correlations play a role on the final behavior adoption size but have no effects on the growth pattern, which is coincidence with our prediction by using the suggested theory.

  19. Multiplexed image storage by electromagnetically induced transparency in a solid

    Science.gov (United States)

    Heinze, G.; Rentzsch, N.; Halfmann, T.

    2012-11-01

    We report on frequency- and angle-multiplexed image storage by electromagnetically induced transparency (EIT) in a Pr3+:Y2SiO5 crystal. Frequency multiplexing by EIT relies on simultaneous storage of light pulses in atomic coherences, driven in different frequency ensembles of the inhomogeneously broadened solid medium. Angular multiplexing by EIT relies on phase matching of the driving laser beams, which permits simultaneous storage of light pulses propagating under different angles into the crystal. We apply the multiplexing techniques to increase the storage capacity of the EIT-driven optical memory, in particular to implement multiplexed storage of larger two-dimensional amounts of data (images). We demonstrate selective storage and readout of images by frequency-multiplexed EIT and angular-multiplexed EIT, as well as the potential to combine both multiplexing approaches towards further enhanced storage capacities.

  20. Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

    International Nuclear Information System (INIS)

    Snellenberg, Suzanne; Strooper, Lise MA De; Hesselink, Albertus T; Meijer, Chris JLM; Snijders, Peter JF; Heideman, Daniëlle AM; Steenbergen, Renske DM

    2012-01-01

    Quantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design. Primers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings. Upon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R 2 =0.985), MAL (R 2 =0.986) and hsa-miR-124-2 (R 2 =0.944). Multiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions

  1. Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

    Science.gov (United States)

    Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

    2012-06-01

    Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

  2. Performance modeling, loss networks, and statistical multiplexing

    CERN Document Server

    Mazumdar, Ravi

    2009-01-01

    This monograph presents a concise mathematical approach for modeling and analyzing the performance of communication networks with the aim of understanding the phenomenon of statistical multiplexing. The novelty of the monograph is the fresh approach and insights provided by a sample-path methodology for queueing models that highlights the important ideas of Palm distributions associated with traffic models and their role in performance measures. Also presented are recent ideas of large buffer, and many sources asymptotics that play an important role in understanding statistical multiplexing. I

  3. Digital holograms for laser mode multiplexing

    CSIR Research Space (South Africa)

    Mhlanga, T

    2014-10-02

    Full Text Available multiplexing Thandeka Mhlangaa, b, Abderrahmen Trichilic, Angela Dudleya, Darryl Naidooa, b, Mourad Zghalc and Andrew Forbesa, b aCSIR National Laser Centre, P.O. Box 395, Pretoria 0001, South Africa bSchool of Physics, University of KwaZulu-Natal, Private Bag... problems. In this context, we demonstrate a method of multiplexing laser modes using spatial light modulators (SLMs). In our proposed technique, we use Laguerre Gaussian (LG) modes, which form a complete basis set; hence multi-mode masks can be created...

  4. Simple Multiplexing Hand-Held Control Unit

    Science.gov (United States)

    Hannaford, Blake

    1989-01-01

    Multiplexer consists of series of resistors, each shunted by single-pole, single-throw switch. User operates switches by pressing buttons or squeezing triggers. Prototype includes three switches operated successfully in over 200 hours of system operations. Number of switches accommodated determined by signal-to-noise ratio of current source, noise induced in control unit and cable, and number of bits in output of analog-to-digital converter. Because many computer-contolled robots have extra analog-to-digital channels, such multiplexer added at little extra cost.

  5. Cooperative spreading processes in multiplex networks.

    Science.gov (United States)

    Wei, Xiang; Chen, Shihua; Wu, Xiaoqun; Ning, Di; Lu, Jun-An

    2016-06-01

    This study is concerned with the dynamic behaviors of epidemic spreading in multiplex networks. A model composed of two interacting complex networks is proposed to describe cooperative spreading processes, wherein the virus spreading in one layer can penetrate into the other to promote the spreading process. The global epidemic threshold of the model is smaller than the epidemic thresholds of the corresponding isolated networks. Thus, global epidemic onset arises in the interacting networks even though an epidemic onset does not arise in each isolated network. Simulations verify the analysis results and indicate that cooperative spreading processes in multiplex networks enhance the final infection fraction.

  6. Longitudinal Multiplexed Measurement of Quantitative Proteomic Signatures in Mouse Lymphoma Models Using Magneto-Nanosensors.

    Science.gov (United States)

    Lee, Jung-Rok; Appelmann, Iris; Miething, Cornelius; Shultz, Tyler O; Ruderman, Daniel; Kim, Dokyoon; Mallick, Parag; Lowe, Scott W; Wang, Shan X

    2018-01-01

    Cancer proteomics is the manifestation of relevant biological processes in cancer development. Thus, it reflects the activities of tumor cells, host-tumor interactions, and systemic responses to cancer therapy. To understand the causal effects of tumorigenesis or therapeutic intervention, longitudinal studies are greatly needed. However, most of the conventional mouse experiments are unlikely to accommodate frequent collection of serum samples with a large enough volume for multiple protein assays towards single-object analysis. Here, we present a technique based on magneto-nanosensors to longitudinally monitor the protein profiles in individual mice of lymphoma models using a small volume of a sample for multiplex assays. Methods: Drug-sensitive and -resistant cancer cell lines were used to develop the mouse models that render different outcomes upon the drug treatment. Two groups of mice were inoculated with each cell line, and treated with either cyclophosphamide or vehicle solution. Serum samples taken longitudinally from each mouse in the groups were measured with 6-plex magneto-nanosensor cytokine assays. To find the origin of IL-6, experiments were performed using IL-6 knock-out mice. Results: The differences in serum IL-6 and GCSF levels between the drug-treated and untreated groups were revealed by the magneto-nanosensor measurement on individual mice. Using the multiplex assays and mouse models, we found that IL-6 is secreted by the host in the presence of tumor cells upon the drug treatment. Conclusion: The multiplex magneto-nanosensor assays enable longitudinal proteomic studies on mouse tumor models to understand tumor development and therapy mechanisms more precisely within a single biological object.

  7. Advanced combinational microfluidic multiplexer for fuel cell reactors

    International Nuclear Information System (INIS)

    Lee, D W; Kim, Y; Cho, Y-H; Doh, I

    2013-01-01

    An advanced combinational microfluidic multiplexer capable to address multiple fluidic channels for fuel cell reactors is proposed. Using only 4 control lines and two different levels of control pressures, the proposed multiplexer addresses up to 19 fluidic channels, at least two times larger than the previous microfluidic multiplexers. The present multiplexer providing high control efficiency and simple structure for channel addressing would be used in the application areas of the integrated microfluidic systems such as fuel cell reactors and dynamic pressure generators

  8. Twelve-Week Treatment With Liraglutide as Add-on to Insulin in Normal-Weight Patients With Poorly Controlled Type 1 Diabetes

    DEFF Research Database (Denmark)

    Frandsen, Christian S; Dejgaard, Thomas F; Holst, Jens J

    2015-01-01

    OBJECTIVE: This study investigated the efficacy and safety of once-daily liraglutide 1.2 mg versus placebo as add-on to insulin treatment in normal-weight patients with poorly controlled type 1 diabetes. RESEARCH DESIGN AND METHODS: In a randomized (1:1), double-blind, placebo-controlled design, 40...... patients with type 1 diabetes (HbA1c ≥8% [64 mmol/mol]) received once-daily liraglutide 1.2 mg or placebo for 12 weeks. Continuous glucose monitoring was performed before and at the end of treatment. The primary end point was change in HbA1c. Secondary end points included change in insulin dose, weight...... was more frequently associated with gastrointestinal adverse effects. The incidence of hypoglycemia did not differ between groups. CONCLUSIONS: Liraglutide significantly reduces body weight and insulin requirements but has no additional effect on HbA1c in normal-weight patients with type 1 diabetes...

  9. Effectiveness of vildagliptin as add-on to metformin monotherapy among uncontrolled type 2 diabetes mellitus patients in a real-world setting.

    Science.gov (United States)

    Melzer Cohen, Cheli; Davis, Carla; Shalev, Varda; Chodick, Gabriel

    2018-01-01

    Vildagliptin is a dipeptidyl peptidase-4 inhibitor commonly used as a dual oral agent with metformin, thiazolidinediones, or sulfonylurea for the treatment of type 2 diabetes mellitus (T2DM). The efficacy of dual therapy with vildagliptin and metformin has been established in randomized controlled trials, but there is little evidence from observational studies. The aims of the present study were to evaluate the effectiveness of vildagliptin as an add-on therapy to metformin in reducing HbA1c and its affects on body weight and blood lipids in a real-life setting. Included in the present retrospective cohort were T2DM patients (n = 345) who were uncontrolled on metformin monotherapy and intensified treatment with vildagliptin. The efficacy of at least 90 days of dual therapy with vildagliptin and metformin in reducing HbA1c levels, as well as changes in blood lipids and body weight, were evaluated. After 180 days (range 90-365 days) from the index date with a mean daily dose of 92 mg vildagliptin, HbA1c was significantly (P vildagliptin in uncontrolled patients on metformin monotherapy is associated with a significant improvement in the control of HbA1c. © 2017 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  10. Add-On Aliskiren Elicits Stronger Renoprotection Than High-Dose Valsartan in Type 2 Diabetic KKAy Mice That Do Not Respond to Low-Dose Valsartan

    Science.gov (United States)

    Lei, Bai; Nakano, Daisuke; Fan, Yu-Yan; Kitada, Kento; Hitomi, Hirofumi; Kobori, Hiroyuki; Mori, Hirohito; Masaki, Tsutomu; Nishiyama, Akira

    2012-01-01

    We hypothesized that aliskiren provides renoprotection in diabetic animals that did not receive sufficient renoprotection by AT1-receptor antagonist treatment. Type 2 diabetic KKAy mice were treated with group 1: vehicle or group 2: valsartan (15 mg/kg per day) from 12 to 16 weeks of age. The mice were subsequently divided into 4 groups and treated with the following combinations of drugs for another 6 weeks: 1: group 1 kept receiving vehicle, 2: group 2 continuously received 15 mg/kg per day of valsartan (Val-Val15), 3: group 2 received 50 mg/kg per day of valsartan (Val-Val50), 4: group 2 continuously received 15 mg/kg per day of valsartan with 25 mg/kg per day of aliskiren (Val-Val+Ali). Aliskiren exerted significant anti-albuminuric effects, whereas valsartan failed to ameliorate the albuminuria in the first four weeks. Surprisingly, the increasing dosage of valsartan in the Val-Val50 group showed non-significant tendencies to attenuate the albuminuria compared with vehicle infusion. Val-Val+Ali significantly suppressed the development of albuminuria and podocyte injury. Val-Val50 and Val-Val+Ali showed similar suppression of angiotensin II contents in the kidney of KKAy mice. In conclusion, the anti-albuminuric effect that was observed in the type 2 diabetic mice showing no anti-albuminuric effect by valsartan can be attributed to the add-on aliskiren. PMID:22673148

  11. "Effect of pranayama and meditation as an add-on therapy in rehabilitation of patients with Guillain-Barré syndrome--a randomized control pilot study".

    Science.gov (United States)

    Sendhilkumar, Ragupathy; Gupta, Anupam; Nagarathna, Raghuram; Taly, Arun B

    2013-01-01

    To study the add-on effects of pranayama and meditation in rehabilitation of patients with Guillain-Barré syndrome (GBS). This randomized control pilot study was conducted in neurological rehabilitation unit of university tertiary research hospital. Twenty-two GBS patients, who consented for the study and satisfied selection criteria, were randomly assigned to yoga and control groups. Ten patients in each group completed the study. The yoga group received 15 sessions in total over a period of 3 weeks (1 h/session), one session per day on 5 days per week that consisted of relaxation, Pranayama (breathing practices) and Guided meditation in addition to conventional rehabilitation therapeutics. The control group received usual rehabilitation care. All the patients were assessed using Pittsburgh Sleep Quality Index, Numeric pain rating scale, Hospital anxiety and Depression scale and Barthel index score. Mann-Whitney U test and Wilcoxon's signed rank test were used for statistical analysis. Quality of sleep improved significantly with reduction of PSQI score in the yoga group (p = 0.04). There was reduction of pain scores, anxiety and depression in both the groups without statistical significance between groups (pain p > 0.05, anxiety p > 0.05 and depression p > 0.05). Overall functional status improved in both groups without significant difference (p > 0.05). Significant improvement was observed in quality of sleep with yogic relaxation, pranayama, and meditation in GBS patients.

  12. Stereotaxic percutaneous core biopsy versus surgical biopsy of nonpalpable breast lesions using a standard mammographic table with an add-on device

    International Nuclear Information System (INIS)

    Levin, M.F.; Papoff, W.J.; Doan, L.; Eliasziw, M.

    2001-01-01

    To determine the accuracy of using a regular mammographic table with an add-on device for biopsy of nonpalpable breast lesions in women in a community hospital setting. During a 3-year period, 70 consenting women (39-80 years of age) with a nonpalpable mammographically suspicious lesion on routine screening mammography underwent 14-gauge automated percutaneous core biopsies, immediate needle localization and lumpectomy. The needle and surgical biopsy results were independently classified into 1 of 5 categories: cancer, fibroadenomal fibrocystic change, normal or other. The procedure was well tolerated, and all core specimens yielded adequate tissue for pathologic evaluation. There were 3 episodes of vasovagal reaction. There was complete agreement in histologic findings in 64 cases (91%), including 22 of 24 cancers (92%). The overall agreement for categorizing lesions was 91% (k 0.88), and there was 97% agreement (k = 0.94) for the classification of cancer versus benign lesion. The results are similar to those of studies performed with dedicated prone equipment. Stereotaxic core biopsies can be done safely and accurately in a community hospital setting with relatively inexpensive nondedicated mammographic equipment. (author)

  13. Stereotaxic percutaneous core biopsy versus surgical biopsy of nonpalpable breast lesions using a standard mammographic table with an add-on device

    Energy Technology Data Exchange (ETDEWEB)

    Levin, M.F.; Papoff, W.J.; Doan, L.; Eliasziw, M. [St. Thomas-Elgin General Hospital, St. Thomas, Ontario (Canada)

    2001-02-01

    To determine the accuracy of using a regular mammographic table with an add-on device for biopsy of nonpalpable breast lesions in women in a community hospital setting. During a 3-year period, 70 consenting women (39-80 years of age) with a nonpalpable mammographically suspicious lesion on routine screening mammography underwent 14-gauge automated percutaneous core biopsies, immediate needle localization and lumpectomy. The needle and surgical biopsy results were independently classified into 1 of 5 categories: cancer, fibroadenomal fibrocystic change, normal or other. The procedure was well tolerated, and all core specimens yielded adequate tissue for pathologic evaluation. There were 3 episodes of vasovagal reaction. There was complete agreement in histologic findings in 64 cases (91%), including 22 of 24 cancers (92%). The overall agreement for categorizing lesions was 91% (k 0.88), and there was 97% agreement (k = 0.94) for the classification of cancer versus benign lesion. The results are similar to those of studies performed with dedicated prone equipment. Stereotaxic core biopsies can be done safely and accurately in a community hospital setting with relatively inexpensive nondedicated mammographic equipment. (author)

  14. Comparison of the Effects of Continuous Subcutaneous Insulin Infusion and Add-On Therapy with Sitagliptin in Patients with Newly Diagnosed Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Heng Wan

    2016-01-01

    Full Text Available To identify a new regimen to optimize treatment for patients with newly diagnosed type 2 diabetes (T2DM by short-term continuous subcutaneous insulin infusion (CSII alone. Methods. 60 patients with newly diagnosed T2DM were randomized into two groups (n=30 each and treated for 2 weeks with CSII alone (CSII group or with CSII plus sitagliptin (CSII + Sig group. The glycemic variability of the patients was measured using a continuous glucose monitoring system (CGMS for the last 72 hours. A standard meal test was performed before and after the interventions, and the levels of glycated albumin, fasting glucose, fasting C-peptide, postprandial 2 h blood glucose, and postprandial 2 h C-peptide were examined. Results. Compared with the CSII group, the indicators of glycemic variability, such as the mean amplitude of glycemic excursion (MAGE and the standard deviation of blood glucose (SDBG, were decreased significantly in the CSII + Sig group. The changes before and after treatment in the C-peptide reactivity index (ΔCPI and the secretory unit of islet in transplantation index (ΔSUIT indicated a significant improvement in the CSII + Sig group. Conclusions. Add-on therapy with sitagliptin may be an optimized treatment for patients with newly diagnosed T2DM compared with short-term CSII alone.

  15. Add-on-Statin Extended Release Nicotinic Acid/Laropiprant but Not the Switch to High-Dose Rosuvastatin Lowers Blood Pressure: An Open-Label Randomized Study

    Directory of Open Access Journals (Sweden)

    Anastazia Kei

    2011-01-01

    Full Text Available Introduction. Nicotinic acid (NA and statins have been associated with reductions in blood pressure (BP. Patients and Methods. We recruited 68 normotensive and hypertensive dyslipidemic patients who were treated with a conventional statin dose and had not achieved lipid targets. Patients were randomized to switch to high-dose rosuvastatin (40 mg/day or to add-on current statin treatment with extended release (ER NA/laropiprant (1000/20 mg/day for the first 4 weeks followed by 2000/40 mg/day for the next 8 weeks for 3 months. Results. Switching to rosuvastatin 40 mg/day was not associated with significant BP alterations. In contrast, the addition of ER-NA/laropiprant to current statin treatment resulted in a 7% reduction of systolic BP (from 134±12 to 125±10 mmHg, <.001 versus baseline and =.01 versus rosuvastatin group and a 5% reduction of diastolic BP (from 81±9 to 77±6 mmHg, =.009 versus baseline and =.01 versus rosuvastatin group. These reductions were significant only in the subgroup of hypertensives and were independent of the hypolipidemic effects of ER-NA/laropiprant. Conclusions. Contrary to the switch to high-dose rosuvastatin, the addition of ER-NA/laropiprant to statin treatment was associated with significant reductions in both systolic and diastolic BP.

  16. Aplicación de las pruebas de PCR convencional simple y múltiple para la identificación de aislamientos de Leptospira spp. en Colombia Application of conventional and multiplex PCR assays for identification of isolates of Leptospira spp. in Colombia

    Directory of Open Access Journals (Sweden)

    Natali Moreno

    2010-12-01

    and multiplex PCR methods (using primers directed against lipl32 and secY/flaB genes, respectively. To assess the capacity of PCR methods to identify pathogenic and saprophytic species of Leptospira ssp. Material and methods. 22 international reference strains and 12 colombian isolates were used. DNA was extracted with a commercial kit (Wizard. Specificity and sensitivity of both PCR methods were evaluated. Results. The maximum dilution of DNA samples allowing the detection of Leptospira ssp was determined to be 1:10000 for the PCR lipL32 and 1:100/1:1000 for the multiplex PCR secY/flaB. Both PCR didn’t detect DNA from microorganisms unrelated to Leptospira ssp. The lipL32 PCR specifically amplified a 423bp fragment from all pathogenic Leptospira reference strains, while the secY/flaB PCR amplified both 285bp (secY and 793bp (flaB fragments from 18 reference strains. The lipL32 PCR detected 7/12 colombian isolates, while secY/flaB PCR detected both secY and flaB genes from 6/12 isolates. Conclusions. Best results were obtained with the lipL32 PCR, which displayed a better sensitivity and a better capacity to detect different strains than the multiplex PCR. The secY primers showed a poor specificity to pathogenic species and a poor sensitivity. Thus, lipL32 primers show high potential for molecular diagnosis of Leptospira spp in clinical and environmental samples.

  17. Ultrasensitive aptamer-based multiplexed electrochemical detection by coupling distinguishable signal tags with catalytic recycling of DNase I.

    Science.gov (United States)

    Tang, Dianping; Tang, Juan; Li, Qunfang; Su, Biling; Chen, Guonan

    2011-10-01

    This work reports an aptamer-based, disposable, and multiplexed sensing platform for simultaneous electrochemical determination of small molecules, employing adenosine triphosphate (ATP) and cocaine as the model target analytes. The multiplexed sensing strategy is based on target-induced release of distinguishable redox tag-conjugated aptamers from a magnetic graphene platform. The electronic signal of the aptasensors could be further amplified by coupling DNase I with catalytic recycling of self-produced reactants. The assay was based on the change in the current at the various peak potentials in the presence of the corresponding signal tags. Experimental results revealed that the multiplexed electrochemical aptasensor enabled the simultaneous monitoring of ATP and cocaine in a single run with wide working ranges and low detection limits (LODs: 0.1 pM for ATP and 1.5 pM for cocaine). This concept offers promise for rapid, simple, and cost-effective analysis of biological samples.

  18. Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

    Science.gov (United States)

    Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

    2015-02-21

    This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

  19. Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

    Science.gov (United States)

    Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

    2012-01-01

    Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

  20. Rapid identification of HPV 16 and 18 by multiplex nested PCR-immunochromatographic test.

    Science.gov (United States)

    Kuo, Yung-Bin; Li, Yi-Shuan; Chan, Err-Cheng

    2015-02-01

    Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application. Copyright © 2014. Published by Elsevier B.V.

  1. Evaluation of Optical Detection Platforms for Multiplexed Detection of Proteins and the Need for Point-of-Care Biosensors for Clinical Use

    Directory of Open Access Journals (Sweden)

    Samantha Spindel

    2014-11-01

    Full Text Available This review investigates optical sensor platforms for protein multiplexing, the ability to analyze multiple analytes simultaneously. Multiplexing is becoming increasingly important for clinical needs because disease and therapeutic response often involve the interplay between a variety of complex biological networks encompassing multiple, rather than single, proteins. Multiplexing is generally achieved through one of two routes, either through spatial separation on a surface (different wells or spots or with the use of unique identifiers/labels (such as spectral separation—different colored dyes, or unique beads—size or color. The strengths and weaknesses of conventional platforms such as immunoassays and new platforms involving protein arrays and lab-on-a-chip technology, including commercially-available devices, are discussed. Three major public health concerns are identified whereby detecting medically-relevant markers using Point-of-Care (POC multiplex assays could potentially allow for a more efficient diagnosis and treatment of diseases.

  2. Comparison of Multiplex Suspension Array Large-Panel Kits for Profiling Cytokines and Chemokines in Rheumatoid Arthritis Patients

    Science.gov (United States)

    Khan, Imran H.; Krishnan, V.V.; Ziman, Melanie; Janatpour, Kim; Wun, Ted; Luciw, Paul A.; Tuscano, Joseph

    2015-01-01

    Background Multiplex analysis allows measurements of a large number of analytes simultaneously in each sample. Based on the Luminex multiplex technology (xMAP), kits for measuring multiple cytokines and chemokines (immunomodulators) are commercially available and are useful in investigations on inflammatory diseases. This study evaluated four multiplex kits (Bio-Plex, LINCOplex, Fluorokine, and Beadlyte) that contained 27, 29, 20 and 22 analytes each, respectively, for the analysis of immunomodulators in plasma of rheumatoid arthritis (RA) patients who underwent treatment with antibody against CD20 (rituximab), a B-cell reductive therapy. Methods Multiplex kits were tested on serial plasma samples obtained from six RA patients at baseline and multiple time points (3, 6, and 9 months) post-treatment with rituximab. The RA patients included in this study had previously failed therapy with disease modifying anti-arthritis drugs (DMARD) and treatment with anti-TNFα antibody (infliximab). Results Computer modeling and hierarchical cluster analysis of the multiplex data allowed a comparison of the performance of multiplex assay kits and revealed profiles of immunomodulators in the RA patients. Conclusions In plasma of RA patients who appeared to have benefited from rituximab treatment the profile of significantly elevated immunomodulators by at least two of the three kits (BioPlex, LINCOplex, Beadlyte), is as follows: IL-12p70, Eotaxin, IL-4, TNFα, Il-9, IL-1β, IFNγ, IL-10, IL-6, and IL-13. Immunomodulator profiling by multiplex analysis may provide useful plasma biomarkers for monitoring response to B-cell reductive therapy in RA patients. PMID:18823005

  3. Moving through a multiplex holographic scene

    Science.gov (United States)

    Mrongovius, Martina

    2013-02-01

    This paper explores how movement can be used as a compositional element in installations of multiplex holograms. My holographic images are created from montages of hand-held video and photo-sequences. These spatially dynamic compositions are visually complex but anchored to landmarks and hints of the capturing process - such as the appearance of the photographer's shadow - to establish a sense of connection to the holographic scene. Moving around in front of the hologram, the viewer animates the holographic scene. A perception of motion then results from the viewer's bodily awareness of physical motion and the visual reading of dynamics within the scene or movement of perspective through a virtual suggestion of space. By linking and transforming the physical motion of the viewer with the visual animation, the viewer's bodily awareness - including proprioception, balance and orientation - play into the holographic composition. How multiplex holography can be a tool for exploring coupled, cross-referenced and transformed perceptions of movement is demonstrated with a number of holographic image installations. Through this process I expanded my creative composition practice to consider how dynamic and spatial scenes can be conveyed through the fragmented view of a multiplex hologram. This body of work was developed through an installation art practice and was the basis of my recently completed doctoral thesis: 'The Emergent Holographic Scene — compositions of movement and affect using multiplex holographic images'.

  4. Prototype data terminal-multiplexer/demultiplexer

    Science.gov (United States)

    Leck, D. E.; Goodwin, J. E.

    1972-01-01

    The design and operation of a quad redundant data terminal and a multiplexer/demultiplexer (MDU) is described. The most unique feature is the design of the quad redundant data terminal. This is one of the few designs where the unit is fail/op, fail/op, fail/safe. Laboratory tests confirm that the unit will operate satisfactorily with the failure of three out of four channels. Although the design utilizes state-of-the-art technology, the waveform error checks, the voting techniques, and the parity bit checks are believed to be used in unique configurations. Correct word selection routines are also novel. The MDU design, while not redundant, utilizes, the latest state-of-the-art advantages of light coupler and interested amplifiers. Much of the technology employed was an evolution of prior NASA contracts related to the Addressable Time Division Data System. A good example of the earlier technology development was the development of a low level analog multiplexer, a high level analog multiplexer, and a digital multiplexer. A list of all drawings is included for reference and all schematic, block and timing diagrams are incorporated.

  5. Silicon Photonic Integrated Circuit Mode Multiplexer

    DEFF Research Database (Denmark)

    Ding, Yunhong; Ou, Haiyan; Xu, Jing

    2013-01-01

    We propose and demonstrate a novel silicon photonic integrated circuit enabling multiplexing of orthogonal modes in a few-mode fiber (FMF). By selectively launching light to four vertical grating couplers, all six orthogonal spatial and polarization modes supported by the FMF are successfully...

  6. Coherence-Multiplexed Optical RF Feeder Networks

    NARCIS (Netherlands)

    Meijerink, Arjan; Taniman, R.O.; van Etten, Wim

    2007-01-01

    An optical RF feeding system for wireless access is proposed, in which the radio access points are distinguished by means of coherence multiplexing (CM). CM is a rather unknown and potentially inexpensive optical code division multiple access technique, which is particularly suitable for relatively

  7. Multiple routes transmitted epidemics on multiplex networks

    International Nuclear Information System (INIS)

    Zhao, Dawei; Li, Lixiang; Peng, Haipeng; Luo, Qun; Yang, Yixian

    2014-01-01

    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.

  8. Beyond Multiplexing Gain in Large MIMO Systems

    DEFF Research Database (Denmark)

    Cakmak, Burak; Müller, Ralf R.; Fleury, Bernard Henri

    growth (multiplexing gain). Even when the channel entries are i.i.d. the deviation from the linear growth is significant. We also find an additive property of the deviation for a concatenated MIMO system. Finally, we quantify the deviation of the large SNR capacity from the exact capacity and find...

  9. 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.