Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

Science.gov (United States)

Llewellyn, Stacey; Inpankaew, Tawin; Nery, Susana Vaz; Gray, Darren J.; Verweij, Jaco J.; Clements, Archie C. A.; Gomes, Santina J.; Traub, Rebecca; McCarthy, James S.

2016-01-01

Background Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy. Methodology/Principal Findings Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections. Conclusions/Significance Multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and

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.

Multiplex Quantitative Histologic Analysis of Human Breast Cancer Cell Signaling and Cell Fate

National Research Council Canada - National Science Library

Lee, William M; Roysam, Badrinath

2008-01-01

.... We are developing a novel platform for immunohistological study of breast cancer specimens that will retrieve multiplex quantitative molecular information about tumor cells at a cytologic level...

Graphite nanocomposites sensor for multiplex detection of antioxidants in food.

Science.gov (United States)

Ng, Khan Loon; Tan, Guan Huat; Khor, Sook Mei

2017-12-15

Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ) are synthetic antioxidants used in the food industry. Herein, we describe the development of a novel graphite nanocomposite-based electrochemical sensor for the multiplex detection and measurement of BHA, BHT, and TBHQ levels in complex food samples using a linear sweep voltammetry technique. Moreover, our newly established analytical method exhibited good sensitivity, limit of detection, limit of quantitation, and selectivity. The accuracy and reliability of analytical results were challenged by method validation and comparison with the results of the liquid chromatography method, where a linear correlation of more than 0.99 was achieved. The addition of sodium dodecyl sulfate as supporting additive further enhanced the LSV response (anodic peak current, I pa ) of BHA and BHT by 2- and 20-times, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

Science.gov (United States)

Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

Microfluidic platform for multiplexed detection in single cells and methods thereof

Science.gov (United States)

Wu, Meiye; Singh, Anup K.

2018-05-01

The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.

Molecular diagnosis of urinary tract infections by semi-quantitative detection of uropathogens in a routine clinical hospital setting

NARCIS (Netherlands)

A. van der Zee (Anneke); L.D. Roorda (Lieuwe); G. Bosman (Gerda); J.M. Ossewaarde (Jacobus)

2016-01-01

textabstractBackground The objective of our study was the development of a semi-quantitative real-time PCR to detect uropathogens. Two multiplex PCR reactions were designed to detect Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus mirabilis, Enterococcus faecalis, and

Multiplex PCR detection of waterborne intestinal protozoa: microsporidia, Cyclospora, and Cryptosporidium.

Science.gov (United States)

Lee, Seung-Hyun; Joung, Migyo; Yoon, Sejoung; Choi, Kyoungjin; Park, Woo-Yoon; Yu, Jae-Ran

2010-12-01

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 10(1) to 10(2) oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

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.

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.

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

Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

Science.gov (United States)

Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

2008-09-01

In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR.

Science.gov (United States)

Malandraki, Ioanna; Beris, Despoina; Isaioglou, Ioannis; Olmos, Antonio; Varveri, Christina; Vassilakos, Nikon

2017-01-01

A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

Science.gov (United States)

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

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

Impact of Aerosol Dust on xMAP Multiplex Detection of Different Class Pathogens

Directory of Open Access Journals (Sweden)

Denis A. Kleymenov

2017-11-01

Full Text Available Environmental or city-scale bioaerosol surveillance can provide additional value for biodefense and public health. Efficient bioaerosol monitoring should rely on multiplex systems capable of detecting a wide range of biologically hazardous components potentially present in air (bacteria, viruses, toxins and allergens. xMAP technology from LuminexTM allows multiplex bead-based detection of antigens or nucleic acids, but its use for simultaneous detection of different classes of pathogens (bacteria, virus, toxin is questionable. Another problem is the detection of pathogens in complex matrices, e.g., in the presence of dust. In the this research, we developed the model xMAP multiplex test-system aiRDeTeX 1.0, which enables detection of influenza A virus, Adenovirus type 6 Salmonella typhimurium, and cholera toxin B subunit representing RNA virus, DNA virus, gram-negative bacteria and toxin respectively as model organisms of biologically hazardous components potentially present in or spreadable through the air. We have extensively studied the effect of matrix solution (PBS, distilled water, environmental dust and ultrasound treatment for monoplex and multiplex detection efficiency of individual targets. All targets were efficiently detectable in PBS and in the presence of dust. Ultrasound does not improve the detection except for bacterial LPS.

A simple approach for ultrasensitive detection of bisphenols by multiplexed surface-enhanced Raman scattering

Energy Technology Data Exchange (ETDEWEB)

De Bleye, C., E-mail: cdebleye@ulg.ac.be; Dumont, E.; Hubert, C.; Sacré, P.-Y.; Netchacovitch, L.; Chavez, P.-F.; Hubert, Ph.; Ziemons, E.

2015-08-12

Bisphenol A (BPA) is well known for its use in plastic manufacture and thermal paper production despite its risk of health toxicity as an endocrine disruptor in humans. Since the publication of new legislation regarding the use of BPA, manufacturers have begun to replace BPA with other phenolic molecules such as bisphenol F (BPF) and bisphenol B (BPB), but there are no guarantees regarding the health safety of these compounds at this time. In this context, a very simple, cheap and fast surface-enhanced Raman scattering (SERS) method was developed for the sensitive detection of these molecules in spiked tap water solutions. Silver nanoparticles were used as SERS substrates. An original strategy was employed to circumvent the issue of the affinity of bisphenols for metallic surfaces and the silver nanoparticles surface was functionalized using pyridine in order to improve again the sensitivity of the detection. Semi-quantitative detections were performed in tap water solutions at a concentrations range from 0.25 to 20 μg L{sup −1} for BPA and BPB and from 5 to 100 μg L{sup −1} for BPF. Moreover, a feasibility study for performing a multiplex-SERS detection of these molecules was also performed before successfully implementing the developed SERS method on real samples. - Highlights: • Development of a simple, fast and ultrasensitive SERS method to detect bisphenols. • Multiplexed-SERS detection of bisphenol A, bisphenol B and bisphenol F. • Implementation of the SERS developed method on real samples to detect bisphenols.

A simple approach for ultrasensitive detection of bisphenols by multiplexed surface-enhanced Raman scattering

International Nuclear Information System (INIS)

De Bleye, C.; Dumont, E.; Hubert, C.; Sacré, P.-Y.; Netchacovitch, L.; Chavez, P.-F.; Hubert, Ph.; Ziemons, E.

2015-01-01

Bisphenol A (BPA) is well known for its use in plastic manufacture and thermal paper production despite its risk of health toxicity as an endocrine disruptor in humans. Since the publication of new legislation regarding the use of BPA, manufacturers have begun to replace BPA with other phenolic molecules such as bisphenol F (BPF) and bisphenol B (BPB), but there are no guarantees regarding the health safety of these compounds at this time. In this context, a very simple, cheap and fast surface-enhanced Raman scattering (SERS) method was developed for the sensitive detection of these molecules in spiked tap water solutions. Silver nanoparticles were used as SERS substrates. An original strategy was employed to circumvent the issue of the affinity of bisphenols for metallic surfaces and the silver nanoparticles surface was functionalized using pyridine in order to improve again the sensitivity of the detection. Semi-quantitative detections were performed in tap water solutions at a concentrations range from 0.25 to 20 μg L −1 for BPA and BPB and from 5 to 100 μg L −1 for BPF. Moreover, a feasibility study for performing a multiplex-SERS detection of these molecules was also performed before successfully implementing the developed SERS method on real samples. - Highlights: • Development of a simple, fast and ultrasensitive SERS method to detect bisphenols. • Multiplexed-SERS detection of bisphenol A, bisphenol B and bisphenol F. • Implementation of the SERS developed method on real samples to detect bisphenols

Spectrally efficient polarization multiplexed direct-detection OFDM system without frequency gap.

Science.gov (United States)

Wei, Chia-Chien; Zeng, Wei-Siang; Lin, Chun-Ting

2016-01-25

We experimentally demonstrate a spectrally efficient direct-detection orthogonal frequency-division multiplexing (DD-OFDM) system. In addition to polarization-division multiplexing, removing the frequency gap further improves the spectral efficiency of the OFDM system. The frequency gap between a reference carrier and OFDM subcarriers avoids subcarrier-to-subcarrier beating interference (SSBI) in traditional DD-OFDM systems. Without dynamic polarization control, the resulting interference after square-law direct detection in the proposed gap-less system is polarization-dependent and composed of linear inter-carrier interference (ICI) and nonlinear SSBI. Thus, this work proposes an iterative multiple-input multiple-output detection scheme to remove the mixed polarization-dependent interference. Compared to the previous scheme, which only removes ICI, the proposed scheme can further eliminate SSBI to achieve the improvement of ∼ 7 dB in signal-to-noise ratio. Without the need for polarization control, we successfully utilize 7-GHz bandwidth to transmit a 39.5-Gbps polarization multiplexed OFDM signal over 100 km.

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

Science.gov (United States)

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

2016-08-01

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

Non-labeling multiplex surface enhanced Raman scattering (SERS) detection of volatile organic compounds (VOCs)

DEFF Research Database (Denmark)

Wong, Chi Lok; Dinish, U. S.; Schmidt, Michael Stenbæk

2014-01-01

chemical sensing layer for the enrichment of gas molecules on sensor surface. The leaning nano-pillar substrate also showed highly reproducible SERS signal in cyclic VOCs detection, which can reduce the detection cost in practical applications. Further, multiplex SERS detection on different combination...... device for multiplex, specific and highly sensitive detection of complex VOCs samples that can find potential applications in exhaled breath analysis, hazardous gas analysis, homeland security and environmental monitoring....

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.

Upconversion Nanoparticles-Encoded Hydrogel Microbeads-Based Multiplexed Protein Detection

Science.gov (United States)

Shikha, Swati; Zheng, Xiang; Zhang, Yong

2018-06-01

Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex's xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

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.

A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

Directory of Open Access Journals (Sweden)

Zhiyuan Wu

2014-01-01

Full Text Available A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph- negative myeloproliferative neoplasms (MPNs. The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS, quantitative PCR (qPCR and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.

A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

Science.gov (United States)

Zhang, Yunqing; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Zhang, Chen; Su, Bing

2014-01-01

A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. PMID:24729973

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

Science.gov (United States)

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Parallel segmented outlet flow high performance liquid chromatography with multiplexed detection

International Nuclear Information System (INIS)

Camenzuli, Michelle; Terry, Jessica M.; Shalliker, R. Andrew; Conlan, Xavier A.; Barnett, Neil W.; Francis, Paul S.

2013-01-01

Graphical abstract: -- Highlights: •Multiplexed detection for liquid chromatography. •‘Parallel segmented outlet flow’ distributes inner and outer portions of the analyte zone. •Three detectors were used simultaneously for the determination of opiate alkaloids. -- Abstract: We describe a new approach to multiplex detection for HPLC, exploiting parallel segmented outlet flow – a new column technology that provides pressure-regulated control of eluate flow through multiple outlet channels, which minimises the additional dead volume associated with conventional post-column flow splitting. Using three detectors: one UV-absorbance and two chemiluminescence systems (tris(2,2′-bipyridine)ruthenium(III) and permanganate), we examine the relative responses for six opium poppy (Papaver somniferum) alkaloids under conventional and multiplexed conditions, where approximately 30% of the eluate was distributed to each detector and the remaining solution directed to a collection vessel. The parallel segmented outlet flow mode of operation offers advantages in terms of solvent consumption, waste generation, total analysis time and solute band volume when applying multiple detectors to HPLC, but the manner in which each detection system is influenced by changes in solute concentration and solution flow rates must be carefully considered

Parallel segmented outlet flow high performance liquid chromatography with multiplexed detection

Energy Technology Data Exchange (ETDEWEB)

Camenzuli, Michelle [Australian Centre for Research on Separation Science (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), Sydney, NSW (Australia); Terry, Jessica M. [Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3216 (Australia); Shalliker, R. Andrew, E-mail: r.shalliker@uws.edu.au [Australian Centre for Research on Separation Science (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), Sydney, NSW (Australia); Conlan, Xavier A.; Barnett, Neil W. [Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3216 (Australia); Francis, Paul S., E-mail: paul.francis@deakin.edu.au [Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3216 (Australia)

2013-11-25

Graphical abstract: -- Highlights: •Multiplexed detection for liquid chromatography. •‘Parallel segmented outlet flow’ distributes inner and outer portions of the analyte zone. •Three detectors were used simultaneously for the determination of opiate alkaloids. -- Abstract: We describe a new approach to multiplex detection for HPLC, exploiting parallel segmented outlet flow – a new column technology that provides pressure-regulated control of eluate flow through multiple outlet channels, which minimises the additional dead volume associated with conventional post-column flow splitting. Using three detectors: one UV-absorbance and two chemiluminescence systems (tris(2,2′-bipyridine)ruthenium(III) and permanganate), we examine the relative responses for six opium poppy (Papaver somniferum) alkaloids under conventional and multiplexed conditions, where approximately 30% of the eluate was distributed to each detector and the remaining solution directed to a collection vessel. The parallel segmented outlet flow mode of operation offers advantages in terms of solvent consumption, waste generation, total analysis time and solute band volume when applying multiple detectors to HPLC, but the manner in which each detection system is influenced by changes in solute concentration and solution flow rates must be carefully considered.

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

Science.gov (United States)

Abdeldaim, Guma M K; Strålin, Kristoffer; Korsgaard, Jens; Blomberg, Jonas; Welinder-Olsson, Christina; Herrmann, Björn

2010-12-03

Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 10⁵ genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively.In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. The PCR provides increased

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis

Directory of Open Access Journals (Sweden)

Welinder-Olsson Christina

2010-12-01

Full Text Available Abstract Background Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR for detection of S. pneumoniae (9802 gene fragment, H. influenzae (omp P6 gene and N. meningitidis (ctrA gene. The method was evaluated on bronchoalveolar lavage (BAL samples from 156 adults with lower respiratory tract infection (LRTI and 31 controls, and on 87 cerebrospinal fluid (CSF samples from meningitis patients. Results The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both

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

Science.gov (United States)

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

2017-08-01

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

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.

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.

A multiplex microplatform for the detection of multiple DNA methylation events using gold-DNA affinity.

Science.gov (United States)

Sina, Abu Ali Ibn; Foster, Matthew Thomas; Korbie, Darren; Carrascosa, Laura G; Shiddiky, Muhammad J A; Gao, Jing; Dey, Shuvashis; Trau, Matt

2017-10-07

We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl -1 of DNA with no sequencing requirement.

Multiplexed capillary microfluidic immunoassay with smartphone data acquisition for parallel mycotoxin detection.

Science.gov (United States)

Machado, Jessica M D; Soares, Ruben R G; Chu, Virginia; Conde, João P

2018-01-15

The field of microfluidics holds great promise for the development of simple and portable lab-on-a-chip systems. The use of capillarity as a means of fluidic manipulation in lab-on-a-chip systems can potentially reduce the complexity of the instrumentation and allow the development of user-friendly devices for point-of-need analyses. In this work, a PDMS microchannel-based, colorimetric, autonomous capillary chip provides a multiplexed and semi-quantitative immunodetection assay. Results are acquired using a standard smartphone camera and analyzed with a simple gray scale quantification procedure. The performance of this device was tested for the simultaneous detection of the mycotoxins ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON) which are strictly regulated food contaminants with severe detrimental effects on human and animal health. The multiplexed assay was performed approximately within 10min and the achieved sensitivities of<40, 0.1-0.2 and<10ng/mL for OTA, AFB1 and DON, respectively, fall within the majority of currently enforced regulatory and/or recommended limits. Furthermore, to assess the potential of the device to analyze real samples, the immunoassay was successfully validated for these 3 mycotoxins in a corn-based feed sample after a simple sample preparation procedure. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Toward photostable multiplex analyte detection on a single mode planar optical waveguide

Energy Technology Data Exchange (ETDEWEB)

Mukundan, Harshini [Los Alamos National Laboratory; Xei, Hongshi [Los Alamos National Laboratory; Anderson, Aaron S [Los Alamos National Laboratory; Grace, Wynne K [Los Alamos National Laboratory; Martinez, Jennifer S [NON LANL; Swanson, Basil [Los Alamos National Laboratory

2009-01-01

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.

Surface-enhanced Raman scattering based nonfluorescent probe for multiplex DNA detection.

Science.gov (United States)

Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

2007-06-01

To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive, and multiplex format, an alternative surface-enhanced Raman scattering based probe was designed and fabricated to covalently attach both DNA probing sequence and nonfluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the nonfluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA to its complementary targets was successfully accomplished with a long-term goal to use nonfluorescent RTags in a Raman-based DNA microarray platform.

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

Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis

Directory of Open Access Journals (Sweden)

Ki Young Yoo

2010-03-01

Full Text Available Purpose : The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs and conformation sensitive gel electrophoresis (CSGE to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption. Methods : A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA was conducted. Results : With direct sequencing, the mutations could be identified from 26 patients (96.3%, whereas for multiplex PCR- CSGE screened sequencing, the mutations could be detected in 23 (85.2%. One patient’s mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients. Conclusion : Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.

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.

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection.

Directory of Open Access Journals (Sweden)

Thadeous J Kacmarczyk

Full Text Available Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads. Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection

Science.gov (United States)

Kacmarczyk, Thadeous J.; Bourque, Caitlin; Zhang, Xihui; Jiang, Yanwen; Houvras, Yariv; Alonso, Alicia; Betel, Doron

2015-01-01

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal. PMID:26066343

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection.

Science.gov (United States)

Kacmarczyk, Thadeous J; Bourque, Caitlin; Zhang, Xihui; Jiang, Yanwen; Houvras, Yariv; Alonso, Alicia; Betel, Doron

2015-01-01

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.

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.

Screening DNA chip and event-specific multiplex PCR detection methods for biotech crops.

Science.gov (United States)

Lee, Seong-Hun

2014-11-01

There are about 80 biotech crop events that have been approved by safety assessment in Korea. They have been controlled by genetically modified organism (GMO) and living modified organism (LMO) labeling systems. The DNA-based detection method has been used as an efficient scientific management tool. Recently, the multiplex polymerase chain reaction (PCR) and DNA chip have been developed as simultaneous detection methods for several biotech crops' events. The event-specific multiplex PCR method was developed to detect five biotech maize events: MIR604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed from four endogenous genes of soybean, maize, cotton and canola respectively along with two regulatory elements and seven genes: P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac and cry3B. The specificity was confirmed and the sensitivity was 0.5% for four crops' 12 events: one soybean, six maize, three cotton and two canola events. The multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis of biotech crops as efficient detection methods by saving on workload and time. © 2014 Society of Chemical Industry. © 2014 Society of Chemical Industry.

RAPID DETECTION OF -THALASSEMIA MUTATIONS IN THAILAND USING MULTIPLEX ARMS

Directory of Open Access Journals (Sweden)

D. Shimbhu

2017-11-01

Full Text Available The number of mutations underlining b-thalassemia generate a wide variety of different clinical phenotypes. An understanding of the genotype is important for medical personnel in order to provide proper counseling to patients and their families. Characterization of these mutations should aid the planning of a prenatal diagnosis program for bthalassemia. The heterogeneity of the mutations makes it difficult and time consuming to identify the mutation in some individuals. We developed a single-tube multiplex amplification refractory mutation system (multiplex ARMS to identify common ethnic- specific b-thalassemia mutations. Confirmation of multiplex ARMS results was carried out using direct sequencing. Three thousand three hundred twenty two people from Phitsanulok province were screened for the b-thalassemia trait by quantitation of HbA2 with microcolumn chromatography and the genotypes of mutations were characterized using multiplex ARMS and direct sequencing. We found that the deletion at codons 41/42 (-TCTT was the most frequent (48%, codon 17 (A®T (30%, -28 (A®G (6% and IVS-I-1(G®T (6% were the second and third in frequency respectively. A -87 (C®A mutation (4%, IVS II-654 (C®T (2%, codons 71/72 (+A (2% and codon 35 (C®A mutations (2% were also found. These techniques were found to be a valuable tool for analysis of b-thalassemia mutations because they are accurate, simple, and speedy in operation. The application for the diagnosis of severe thalassemia in high-risk pregnancies is promising.

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.

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.

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....

Detection of sexually transmitted infection and human papillomavirus in negative cytology by multiplex-PCR

Directory of Open Access Journals (Sweden)

Chung Hyun-Jae

2010-09-01

Full Text Available Abstract Background The aim of this study was to determine the prevalence of human papillomavirus (HPV and 15 species that cause sexually transmitted infections (STIs in negative cytology. In addition, we compared the diagnostic performance of multiplex polymerase chain reaction (PCR with widely available techniques used to detect HPV. Methods We recruited 235 women of reproductive age who had negative cytology findings in a liquid-based cervical smear. STIs were identified by multiplex PCR, and HPV genotypes by multiplex PCR, hybrid capture 2, and DNA microaray; discordant results were analyzed by direct sequencing. Results Approximately 96.6% of patients with negative cytology results were positive for pathogens that cause STIs. The pathogens most frequently detected were Gardnerella vaginalis, Ureaplasma urealyticum. The incidence of HPV in negative cytology was 23.3%. Low-risk HPV infection was significantly correlated with Chalmaydia trachomatis, and high-risk HPV infection was significantly correlated with Group β streptococcus. The analytical sensitivities of the multiplex PCR and DNA microarray were higher than 80%, and the analytical specificity was nearly 100% for all tests. Conclusions Multiplex PCR yielded results that most of patients with negative cytology were positive for pathogens that cause STIs, and were more similar to that of DNA microarray, than that of hybrid capture 2 in terms of analytical sensitivity and prediction value of HPV infection.

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

Gold-silver-alloy nanoprobes for one-pot multiplex DNA detection

Energy Technology Data Exchange (ETDEWEB)

Doria, G; Larguinho, M; Dias, J T; Baptista, P V [Centro de Investigacao em Genetica Molecular Humana (CIGMH), Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Pereira, E [Rede de Quimica e Tecnologia (REQUIMTE), Departamento de Quimica, Faculdade de Ciencias, Universidade do Porto, 4169-007 Porto (Portugal); Franco, R, E-mail: pmvb@fct.unl.pt [Rede de Quimica e Tecnologia (REQUIMTE), Departamento de Quimica, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal)

2010-06-25

A specific colorimetric DNA detection method based on oligonucleotide functionalized gold-silver-alloy nanoparticles (AuAg-alloy-nanoprobes) is presented. The AuAg-alloy-nanoprobes were then used for the specific detection of a DNA sequence from TP53-a gene involved in cancer development. The AuAg-alloy-nanoprobes were then used in combination with Au-nanoprobes for a one-pot dual-colour detection strategy that allowed for the simultaneous differential detection of two distinct target sequences. This system poses an unprecedented opportunity to explore the combined use of metal nanoparticles with different composition towards the development of a multiplex one-pot colorimetric assay for DNA detection.

Gold-silver-alloy nanoprobes for one-pot multiplex DNA detection

International Nuclear Information System (INIS)

Doria, G; Larguinho, M; Dias, J T; Baptista, P V; Pereira, E; Franco, R

2010-01-01

A specific colorimetric DNA detection method based on oligonucleotide functionalized gold-silver-alloy nanoparticles (AuAg-alloy-nanoprobes) is presented. The AuAg-alloy-nanoprobes were then used for the specific detection of a DNA sequence from TP53-a gene involved in cancer development. The AuAg-alloy-nanoprobes were then used in combination with Au-nanoprobes for a one-pot dual-colour detection strategy that allowed for the simultaneous differential detection of two distinct target sequences. This system poses an unprecedented opportunity to explore the combined use of metal nanoparticles with different composition towards the development of a multiplex one-pot colorimetric assay for DNA detection.

Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions.

Science.gov (United States)

Compston, Lara Isobel; Sarkobie, Francis; Li, Chengyao; Candotti, Daniel; Opare-Sem, Ohene; Allain, Jean-Pierre

2008-07-01

In common with latent viruses such as herpesviruses, parvovirus B19, HBV and GBV-C are contained successfully by the immune response and persist in the host. When immune control breaks down, reactivation of both latent and persistent viruses occurs. Two multiplex assays were developed (B19, HBV, HHV-8), (EBV, CMV, VZV) for blood screening, and tested on blood donor samples from Ghana to determine baseline prevalence of viraemia in immunocompetent persons. Single-virus real-time quantitative PCR (qPCR) assays were optimised for viral load determination of positive initial screening. The qPCR method utilised was absolute quantification with external standards. Multiplex and single-virus qPCR assays had similar sensitivity, except for the B19 assay in which sensitivity was 100-fold lower. Assays were optimised for reproducibility and repeatability, with R(2) of 0.9 being obtained for most assays. With the exception of B19 and CMV, assays had 100% detection limit ranging between 10(1) and 10(2) copies, IU or arbitrary units under single-virus and multiplex assay conditions. The prevalence of viraemia was 1.6% HBV (0.8% DNA+/HBsAg-, 0.8% DNA+/HBsAg+), 0.8% parvovirus B19, and 3.3% GBV-C viraemia in the plasma fraction. The prevalence of four herpesviruses was 1.0% HHV-8, 0.85% CMV, and 8.3% EBV, and no detectable VZV viraemia.

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...

Multiplex detection of plant pathogens through the luminex magplex bead system

NARCIS (Netherlands)

Vlugt, van der R.A.A.; Raaij, van H.M.G.; Weerdt, de M.; Bergervoet, J.H.W.

2015-01-01

Here we describe a versatile multiplex method for both the serological and molecular detection of plant pathogens. The Luminex MagPlex bead system uses small paramagnetic microspheres (“beads”), either coated with specific antibodies or oligonucleotides, which capture respectively viruses and/or

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.

Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance

Directory of Open Access Journals (Sweden)

Ralevski Filip

2009-12-01

Full Text Available Abstract Background Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. Materials and methods A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR, and two rapid diagnostic immuno-chromatographic tests (ICT in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. Results QPCR is the most analytically sensitive method (sensitivity 99.41%, followed by CARESTART (sensitivity 88.24%, and BINAXNOW (sensitivity 86.47% for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R2 = 0.9746 in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/μl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more

Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR

Science.gov (United States)

Li, Jun-Wen; Wang, Xin-Wei; Yuan, Chang-Qing; Zheng, Jin-Lai; Jin, Min; Song, Nong; Shi, Xiu-Quan; Chao, Fu-Huan

2002-01-01

AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-, 671-, 1084-, and 1128 bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU for Coxsackie virus, 60 PFU for Echovirus and 105 TCID50 for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID50 for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness. PMID:12174381

Multiplexed detection of cardiac biomarkers in serum with nanowire arrays using readout ASIC.

Science.gov (United States)

Zhang, Guo-Jun; Chai, Kevin Tshun Chuan; Luo, Henry Zhan Hong; Huang, Joon Min; Tay, Ignatius Guang Kai; Lim, Andy Eu-Jin; Je, Minkyu

2012-05-15

Early detection of cardiac biomarkers for diagnosis of heart attack is the key to saving lives. Conventional method of detection like the enzyme-linked immunosorbent assay (ELISA) is time consuming and low in sensitivity. Here, we present a label-free detection system consisting of an array of silicon nanowire sensors and an interface readout application specific integrated circuit (ASIC). This system provides a rapid solution that is highly sensitive and is able to perform direct simultaneous-multiplexed detection of cardiac biomarkers in serum. Nanowire sensor arrays were demonstrated to have the required selectivity and sensitivity to perform multiplexed detection of 100 fg/ml troponin T, creatine kinase MM, and creatine kinase MB in serum. A good correlation between measurements from a probe station and the readout ASIC was obtained. Our detection system is expected to address the existing limitations in cardiac health management that are currently imposed by the conventional testing platform, and opens up possibilities in the development of a miniaturized device for point-of-care diagnostic applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Community Detection for Multiplex Social Networks Based on Relational Bayesian Networks

DEFF Research Database (Denmark)

Jiang, Jiuchuan; Jaeger, Manfred

2014-01-01

Many techniques have been proposed for community detection in social networks. Most of these techniques are only designed for networks defined by a single relation. However, many real networks are multiplex networks that contain multiple types of relations and different attributes on the nodes...

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.

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

Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC.

Directory of Open Access Journals (Sweden)

Zachary F Phillips

Full Text Available We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification-an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC, is a single-shot variant of Differential Phase Contrast (DPC, which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps for various in vitro cell samples and c. elegans in a micro-fluidic channel.

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.

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.

Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

Science.gov (United States)

Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

2015-01-01

An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

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.

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

Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

Science.gov (United States)

Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

2014-05-01

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR.

Science.gov (United States)

Lee, Shin-Young; Kim, Mi-Ju; Kim, Hyun-Joong; Jeong, KwangCheol Casey; Kim, Hae-Yeong

2018-02-28

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

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.

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR.

Science.gov (United States)

Kuamsab, Napaporn; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai

2012-06-10

Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.

Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods†

Science.gov (United States)

Jean, Julie; D'Souza, Doris H.; Jaykus, Lee-Ann

2004-01-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10−1 reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 100 to 102 reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods. PMID:15528524

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

Science.gov (United States)

Jean, Julie; D'Souza, Doris H; Jaykus, Lee-Ann

2004-11-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients.

Science.gov (United States)

Taira, Cleison Ledesma; Okay, Thelma Suely; Delgado, Artur Figueiredo; Ceccon, Maria Esther Jurfest Rivero; de Almeida, Margarete Teresa Gottardo; Del Negro, Gilda Maria Barbaro

2014-07-21

Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients. DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures. The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients. Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.

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.

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.

Detection of virulence genes in Uropathogenic E. coli (UPEC strains by Multiplex-PCR method

Directory of Open Access Journals (Sweden)

Javad Mohammadi

2017-06-01

Full Text Available Background & Objectives: Urinary tract infection caused by E. coli is one of the most common illnesses in all age groups worldwide. Presence of virulence genes is a key factor in bacterial pathogens in uroepithelial cells. The present study was performed to detect iha, iroN, ompT genes in the Uropathogenic E.coli isolates from clinical samples using multiplex-PCR method in Kerman. Materials & Methods: In this descriptive cross-sectional study, 200 samples of patients with urinary tract infections in Kerman hospitals were collected. After biochemical and microbiological tests, all strains were tested with regard to the presence of iha, iroN, and ompT genes using multiplex-PCR method. Results: The results of Multiplex-PCR showed that all specimens had one, two, or three virulence genes simultaneously. The highest and lowest frequency distribution of genes was related to iha (56.7% and iroN (20% respectively. Conclusion: According to the prevalence of urinary tract infection in the community and distribution of resistance and virulence factors, the fast and accurate detection of the strains and virulence genes is necessary

A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species.

Science.gov (United States)

Radhika, M; Saugata, Majumder; Murali, H S; Batra, H V

2014-01-01

Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC). The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

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.

Development of an In-House Multiplex Nested RT-PCR Method for Detecting Acute HIV-1 Infection in High Risk Populations.

Science.gov (United States)

Liu, Zhiying; Li, Wei; Xu, Meng; Sheng, Bo; Yang, Zixuan; Jiao, Yanmei; Zhang, Tong; Mou, Danlei; Chen, Dexi; Wu, Hao

2015-01-01

The detection of acute HIV infection (AHI) among high risk populations can help reduce secondary transmission of HIV. The nucleic acid testing (NAT) can shorten the test window period by up to 7-12 days. In this study, we describe an in-house NAT based on the multiplex nested RT-PCR method to detect the HIV RNA. We also evaluated it in a high risk cohort in Beijing. Four primer pairs were designed and evaluated for the detection of different HIV-1 subtypes in group M. Multiplex RT-PCR and nested PCR were performed. The sensitivity, specialty, primers compatibility among HIV subtypes were evaluated simultaneously. In an MSM cohort in Beijing during a 3-year period, a total of 11,808 blood samples that were negative by ELISA or indeterminate by Western blot were analyzed by this multiplex nested RT-PCR with pooling strategy. The multiplex nested RT-PCR was successfully applied for the detection of at least six HIV-1 subtypes. The sensitivity was 40 copies/ml and the specificity was 100%. A total of 29 people were tested HIV-1 positive with acute infection in a MSM cohort of Beijing during a 3 years period. This multiplex nested RT-PCR provides a useful tool for the rapid detection of acute HIV-1 infection. When used in combination with the 3(rd) generation ELISA, it can improve the detection rate of HIV infection, especially in the source limited regions.

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

Science.gov (United States)

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

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.

Droplet-based microscale colorimetric biosensor for multiplexed DNA analysis via a graphene nanoprobe

International Nuclear Information System (INIS)

Xiang Xia; Luo Ming; Shi Liyang; Ji Xinghu; He Zhike

2012-01-01

Graphical abstract: With a microvalve manipulate technique combined with droplet platform, a microscale fluorescence-based colorimetric sensor for multiplexed DNA analysis is developed via a graphene nanoprobe. Highlights: ► A quantitative detection for multiplexed DNA is first realized on droplet platform. ► The DNA detection is relied on a simple fluorescence-based colorimetric method. ► GO is served as a quencher for two different DNA fluorescent probes. ► This present work provides a rapid, sensitive, visual and convenient detection tool for droplet biosensor. - Abstract: The development of simple and inexpensive DNA detection strategy is very significant for droplet-based microfluidic system. Here, a droplet-based biosensor for multiplexed DNA analysis is developed with a common imaging device by using fluorescence-based colorimetric method and a graphene nanoprobe. With the aid of droplet manipulation technique, droplet size adjustment, droplet fusion and droplet trap are realized accurately and precisely. Due to the high quenching efficiency of graphene oxide (GO), in the absence of target DNAs, the droplet containing two single-stranded DNA probes and GO shows dark color, in which the DNA probes are labeled carboxy fluorescein (FAM) and 6-carboxy-X-rhodamine (ROX), respectively. The droplet changes from dark to bright color when the DNA probes form double helix with the specific target DNAs leading to the dyes far away from GO. This colorimetric droplet biosensor exhibits a quantitative capability for simultaneous detection of two different target DNAs with the detection limits of 9.46 and 9.67 × 10 −8 M, respectively. It is also demonstrated that this biosensor platform can become a promising detection tool in high throughput applications with low consumption of reagents. Moreover, the incorporation of graphene nanoprobe and droplet technique can drive the biosensor field one more step to some extent.

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.

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.

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

Real-time quantitative phase reconstruction in off-axis digital holography using multiplexing.

Science.gov (United States)

Girshovitz, Pinhas; Shaked, Natan T

2014-04-15

We present a new approach for obtaining significant speedup in the digital processing of extracting unwrapped phase profiles from off-axis digital holograms. The new technique digitally multiplexes two orthogonal off-axis holograms, where the digital reconstruction, including spatial filtering and two-dimensional phase unwrapping on a decreased number of pixels, can be performed on both holograms together, without redundant operations. Using this technique, we were able to reconstruct, for the first time to our knowledge, unwrapped phase profiles from off-axis holograms with 1 megapixel in more than 30 frames per second using a standard single-core personal computer on a MATLAB platform, without using graphic-processing-unit programming or parallel computing. This new technique is important for real-time quantitative visualization and measurements of highly dynamic samples and is applicable for a wide range of applications, including rapid biological cell imaging and real-time nondestructive testing. After comparing the speedups obtained by the new technique for holograms of various sizes, we present experimental results of real-time quantitative phase visualization of cells flowing rapidly through a microchannel.

Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

Directory of Open Access Journals (Sweden)

Kelly Grace Magalhães

2004-06-01

Full Text Available From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.

A multiplex PCR method for detection of Aspergillus spp. and Mycobacterium tuberculosis in BAL specimens.

Science.gov (United States)

Amini, F; Kachuei, R; Noorbakhsh, F; Imani Fooladi, A A

2015-06-01

The aim of this study was the detection of Aspergillus species and Mycobacterium tuberculosis together in bronchoalveolar lavage (BAL) using of multiplex PCR. In this study, from September 2012 until June 2013, 100 bronchoalveolar lavage (BAL) specimens were collected from patients suspected of tuberculosis (TB). After the direct and culture test, multiplex PCR were utilized in order to diagnose Aspergillus species and M. tuberculosis. Phenol-chloroform manual method was used in order to extract DNA from these microorganisms. Aspergillus specific primers, M. tuberculosis designed primers and beta actin primers were used for multiplex PCR. In this study, by multiplex PCR method, Aspergillus species were identified in 12 samples (12%), positive samples in direct and culture test were respectively 11% and 10%. Sensitivity and specificity of this method in comparison to direct test were respectively 100% and 98.8%, also sensitivity and specificity of this method in comparison to culture test were respectively 100% and 97.7%. In this assay, M. tuberculosis was identified in 8 samples (8%). Mycobacterium-positive samples in molecular method, direct and culture test were respectively 6%, 5% and 7%. Sensitivity and specificity of PCR method in comparison to direct test were 80% and 97.8% also sensitivity and specificity of this method in comparison to culture test was 71.4% and 98.9%. In the present study, multiplex PCR method had higher sensitivity than direct and culture test in order to identify and detect Aspergillus, also this method had lower sensitivity for identification of M. tuberculosis, suggesting that the method of DNA extraction was not suitable. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

Science.gov (United States)

Li, Wenbin; Abad, Jorge A; French-Monar, Ronald D; Rascoe, John; Wen, Aimin; Gudmestad, Neil C; Secor, Gary A; Lee, Ing-Ming; Duan, Yongping; Levy, Laurene

2009-07-01

The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated

Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites

Energy Technology Data Exchange (ETDEWEB)

Labroo, Pratima; Cui, Yue, E-mail: yue.cui@usu.edu

2014-02-01

Graphical abstract: - Highlights: • We report graphene-ink biosensor arrays on a microfluidic paper for metabolites. • The device is able to detect multiple metabolites sensitively and rapidly. • The device fabrication process is simple and inexpensive. - Abstract: The development of a miniaturized and low-cost platform for the highly sensitive, selective and rapid detection of multiplexed metabolites is of great interest for healthcare, pharmaceuticals, food science, and environmental monitoring. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with extraordinary electrical sensing capability. Microfluidic paper with printing technique is a low cost matrix. Here, we demonstrated the development of graphene-ink based biosensor arrays on a microfluidic paper for the multiplexed detection of different metabolites, such as glucose, lactate, xanthine and cholesterol. Our results show that the graphene biosensor arrays can detect multiple metabolites on a microfluidic paper sensitively, rapidly and simultaneously. The device exhibits a fast measuring time of less than 2 min, a low detection limit of 0.3 μM, and a dynamic detection range of 0.3–15 μM. The process is simple and inexpensive to operate and requires a low consumption of sample volume. We anticipate that these results could open exciting opportunities for a variety of applications.

Detection of Shiga toxins genes by Multiplex PCR in clinical samples

Directory of Open Access Journals (Sweden)

2013-09-01

Full Text Available Background: Different methods have been used for detection of shiga toxins; such as,  cell culture, ELISA, and RFPLA. However, all of these methods suffer from high cost, time-consumption and relatively low sensitivity. In this study we used Multiplex PCR method for detection of genes encoding shiga toxins. Material and Methods: In this study, 63 clinical samples were obtained from positive cultures of Shigella and E. coli O157, from Bahman 1391 until Ordibehesht 1392 in Mazandaran province. Initial confirmation of shiga toxins producing bacteria was performed by biochemical and serological methods. After DNA extraction, detection of stx1 and stx2 genes was accomplished by multiplex PCR.  For confirmation of the PCR amplicon, DNA sequencing was used. Antibiotic sensitivity tests were performed by disk diffusion method. Results:  Among the positive strains, 13 strains contained stx2 genes, 4 strains contained Stx/Stx1 genes and 4 strains harbored both Stx/Stx1 and Stx2. The DNA extracted from other Gram-negative bacteria was not protected by the relevant parts of these toxins. Sequencing of the amplified fragments indicated the correct toxin sequences.  The sensitivity for identification of Stx/Stx1 gene was 1.56 pg/ µl and for Stx2 was 1.08 pg/µl. The toxin positive strains were all sensitive to Cefixime, Gentamicin, Amikacin, Ceftriaxone, and Nitrofurantoin. Conclusion: This method is fast and accurate for detection of bacteria producing shiga toxin and can be used to identify different types of shiga toxin.

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

Directory of Open Access Journals (Sweden)

Qiuying Huang

2011-04-01

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

Dual-excitation upconverting nanoparticle and quantum dot aptasensor for multiplexed food pathogen detection.

Science.gov (United States)

Kurt, Hasan; Yüce, Meral; Hussain, Babar; Budak, Hikmet

2016-07-15

In this report, a dual-excitation sensing method was developed using aptamer-functionalized quantum dots and upconverting nanoparticles, exhibiting Stokes and anti-Stokes type excitation profiles, respectively. Conjugation of the aptamer-functionalized luminescent nanoparticles with the magnetic beads, comprising short DNA sequences that were partially complementary to the aptamer sequences, enabled facile separation of the analyte-free conjugates for fluorescent measurement. UV-Visible spectroscopy, Circular Dichroism spectroscopy, Dynamic Light Scattering and Polyacrylamide Gel Electrophoresis techniques were used to characterize the aptamer probes developed. The target-specific luminescent conjugates were applied for multiplex detection of model food pathogens, Salmonella typhimurium, and Staphylococcus aureus, in which the fluorescent emission spectra were obtained under UV excitation at 325nm for quantum dots and NIR excitation at 980nm for upconverting nanoparticles, respectively. The dual-excitation strategy was aimed to minimize cross-talk between the luminescent signals for multiplexed detection, and yielded limit of detection values of 16 and 28cfumL(-1) for Staphylococcus aureus, and Salmonella typhimurium, respectively. By employing a greater number of quantum dots and upconverting nanoparticles with non-overlapping fluorescent emissions, the proposed methodology might be exploited further to detect several analytes, simultaneously. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a multiplex lateral flow strip test for foot-and-mouth disease virus detection using monoclonal antibodies.

Science.gov (United States)

Yang, Ming; Caterer, Nigel R; Xu, Wanhong; Goolia, Melissa

2015-09-01

Foot-and-mouth disease (FMD) is one of the world's most highly contagious animal diseases with tremendous economic consequences. A rapid and specific test for FMD diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. This project developed a multiplex lateral flow immunochromatographic strip test (multiplex-LFI) for the rapid detection and serotyping of FMD viruses. The monoclonal antibodies (mAbs) against serotypes O, A, and Asia 1 were used as capture mAbs. The mAbs were conjugated with fluorescein, rhodamine or biotin for serotype O, A and Asia 1, respectively. The detection mAbs which consisted of a serotype-independent mAb in combination with one serotype A-specific mAb and one Asia 1-specific mAb, were each colloidal gold-conjugated. The strips used in this study contained one control line and three test lines, which corresponded to one of the three serotypes, O, A or Asia 1. The newly developed multiplex-LFI strip test specifically identified serotype O (n=46), A (n=45) and Asia 1 (n=17) in all tested field isolates. The sensitivity of this strip test was comparable to the double antibody sandwich ELISA for serotypes O and A, but lower than the ELISA for serotype Asia 1. The multiplex-LFI strip test identified all tissue suspensions from animals that were experimentally inoculated with serotypes O, A or Asia 1. FMD viruses were detected in 38% and 50% of the swab samples from the lesion areas of experimentally inoculated sheep for serotypes O and A, respectively. The capability of the multiplex-LFI strip tests to produce rapid results with high specificity for FMD viruses of multiple serotypes makes this test a valuable tool to detect FMD viruses at outbreak sites. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

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.

A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control.

Science.gov (United States)

Cui, Xingye; Hu, Jie; Choi, Jane Ru; Huang, Yalin; Wang, Xuemin; Lu, Tian Jian; Xu, Feng

2016-09-07

A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed "U shape" reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 μg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

78 FR 16513 - Application of Advances in Nucleic Acid and Protein Based Detection Methods to Multiplex...

Science.gov (United States)

2013-03-15

... Methods to Multiplex Detection of Transfusion- Transmissible Agents and Blood Cell Antigens in Blood... Transfusion-Transmissible Agents and Blood Cell Antigens in Blood Donations; Public Workshop AGENCY: Food and... technological advances in gene based and protein based pathogen and blood cell antigen detection methods and to...

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.

A PDMS/paper/glass hybrid microfluidic biochip integrated with aptamer-functionalized graphene oxide nano-biosensors for one-step multiplexed pathogen detection

OpenAIRE

Zuo, Peng; Li, XiuJun; Dominguez, Delfina C.; Ye, Bang-Ce

2013-01-01

Infectious pathogens often cause serious public health concerns throughout the world. There is an increasing demand for simple, rapid and sensitive approaches for multiplexed pathogen detection. In this paper we have developed a polydimethylsiloxane (PDMS)/paper/glass hybrid microfluidic system integrated with aptamer-functionalized graphene oxide (GO) nano-biosensors for simple, one-step, multiplexed pathogen detection. The paper substrate used in this hybrid microfluidic system facilitated ...

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

Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine

Directory of Open Access Journals (Sweden)

Elena Mettifogo

2015-01-01

Full Text Available Mycoplasma gallisepticum (MG and Mycoplasma synoviae (MS are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%, and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks.

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.

Systematic optimization of multiplex zymography protocol to detect active cathepsins K, L, S, and V in healthy and diseased tissue: compromise among limits of detection, reduced time, and resources.

Science.gov (United States)

Dumas, Jerald E; Platt, Manu O

2013-07-01

Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis, and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as clear bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 h to 10 min, incubation assay time from overnight to 4 h, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by Smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings.

Systematic optimization of multiplex zymography protocol to detect active cathepsins K, L, S, and V in healthy and diseased tissue: compromise between limits of detection, reduced time, and resources

Science.gov (United States)

Dumas, Jerald E.; Platt, Manu O.

2013-01-01

Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as cleared bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 hours to 10 minutes, incubation assay time from overnight to 4 hours, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings. PMID:23532386

Multiplexed microRNA detection using lanthanide-labeled DNA probes and laser ablation inductively coupled plasma mass spectrometry

DEFF Research Database (Denmark)

de Bang, Thomas Christian; Shah, Pratik; Cho, Seok Keun

2014-01-01

coupled plasma mass spectrometry (LA-ICPMS). Three miRNAs from Arabidopsis thaliana were analyzed simultaneously with high specificity, and the sensitivity of the method was comparable to radioactive detection (low femtomol range). The perspective of the developed method is highly multiplexed......In the past decade, microRNAs (miRNAs) have drawn increasing attention due to their role in regulation of gene expression. Especially, their potential as biomarkers in disease diagnostics has motivated miRNA research, including the development of simple, accurate, and sensitive detection methods....... The narrow size range of miRNAs (20-24 nucleotides) combined with the chemical properties of conventional reporter tags has hampered the development of multiplexed miRNA assays. In this study, we have used lanthanide-labeled DNA probes for the detection of miRNAs on membranes using laser ablation inductively...

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

International Nuclear Information System (INIS)

Lattanzio, Veronica M.T.; Nivarlet, Noan; Lippolis, Vincenzo; Gatta, Stefania Della; Huet, Anne-Catherine; Delahaut, Philippe; Granier, Benoit; Visconti, Angelo

2012-01-01

Highlights: ► We developed a rapid method based on a multiplex dipstick immunoassay. ► The assay allowed the determination of major Fusarium toxins in wheat, oats, maize. ► We obtained cut off levels close to EU regulatory levels. - Abstract: A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin–BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73–109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 μg kg −1 , respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg kg −1 , respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC–MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

Functionalization of optical nanotip arrays with an electrochemical microcantilever for multiplexed DNA detection.

Science.gov (United States)

Descamps, Emeline; Duroure, Nathalie; Deiss, Frédérique; Leichlé, Thierry; Adam, Catherine; Mailley, Pascal; Aït-Ikhlef, Ali; Livache, Thierry; Nicu, Liviu; Sojic, Neso

2013-08-07

Optical nanotip arrays fabricated on etched fiber bundles were functionalized with DNA spots. Such unconventional substrates (3D and non-planar) are difficult to pattern with standard microfabrication techniques but, using an electrochemical cantilever, up to 400 spots were electrodeposited on the nanostructured optical surface in 5 min. This approach allows each spot to be addressed individually and multiplexed fluorescence detection is demonstrated. Finally, remote fluorescence detection was performed by imaging through the optical fiber bundle itself after hybridisation with the complementary sequence.

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.

Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

Energy Technology Data Exchange (ETDEWEB)

Tholouli, Eleni [Department of Haematology, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL (United Kingdom); MacDermott, Sarah [The Medical School, The University of Manchester, Oxford Road, M13 9PT Manchester (United Kingdom); Hoyland, Judith [School of Biomedicine, Faculty of Medical and Human Sciences, The University of Manchester, Oxford Road, M13 9PT Manchester (United Kingdom); Yin, John Liu [Department of Haematology, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL (United Kingdom); Byers, Richard, E-mail: richard.byers@cmft.nhs.uk [School of Cancer and Enabling Sciences, Faculty of Medical and Human Sciences, The University of Manchester, Stopford Building, Oxford Road, M13 9PT Manchester (United Kingdom)

2012-08-24

Highlights: Black-Right-Pointing-Pointer Development of a quantitative high throughput in situ expression profiling method. Black-Right-Pointing-Pointer Application to a tissue microarray of 242 AML bone marrow samples. Black-Right-Pointing-Pointer Identification of HOXA4, HOXA9, Meis1 and DNMT3A as prognostic markers in AML. -- Abstract: Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection in archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p = 0.009), whilst high expression of HOXA9 (p < 0.0001), Meis1 (p = 0.005) and DNMT3A (p = 0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.

Simultaneous detection of peanut and hazelnut allergens in food matrices using multiplex PCR method

Directory of Open Access Journals (Sweden)

Eva Renčová

2014-01-01

Full Text Available Multiplex PCR analysis for the detection of two targeting segments of genes coding major food protein allergens as peanut (Arachis hypogaea Ara h 1 gene and hazelnut (Corylus avellana Cor a 1 gene was developed. Two sets of primers were designed and tested to their specificity on a broad range of ingredients. The identity of amplicons (Ara h 1- 180 bp, Cor a 1 – 258 bp by sequencing and alignment of sequences with sequences deposited in Genbank was confirmed. When testing the specificity of designed primer pairs on a spectrum of food ingredients, no cross reactions were detected. A potential inhibition of PCR reaction was eliminated using the universal plant primers of chloroplast gene 124 bp for the plant matrices confirmation. The intrinsic detection limit was 10 pg·ml-1 and the practical detection limit was 0.001% w/w (10 mg·kg-1 for both peanuts and hazelnuts. The method was applied to the investigation of 60 commercial food samples. The developed multiplex PCR method is cheap, specific and sensitive enough and can be used as a simple, one day procedure for the checking of undeclared peanut and hazelnut major allergens in food.

Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection.

Science.gov (United States)

Morgenstern, Christian; Cabric, Sabrina; Perka, Carsten; Trampuz, Andrej; Renz, Nora

2018-02-01

Analysis of joint aspirate is the standard preoperative investigation for diagnosis of periprosthetic joint infection (PJI). We compared the diagnostic performance of culture and multiplex polymerase chain reaction (PCR) of synovial fluid for diagnosis of PJI. Patients in whom aspiration of the prosthetic hip or knee joint was performed before revision arthroplasty were prospectively included. The performance of synovial fluid culture and multiplex PCR was compared by McNemar's chi-squared test. A total of 142 patients were included, 82 with knee and 60 with hip prosthesis. PJI was diagnosed in 77 patients (54%) and aseptic failure in 65 patients (46%). The sensitivity of synovial fluid culture and PCR was 52% and 60%, respectively, showing concordant results in 116 patients (82%). In patients with PJI, PCR missed 6 high-virulent pathogens (S. aureus, streptococci, E. faecalis, E. coli) which grew in synovial fluid culture, whereas synovial fluid culture missed 12 pathogens detected by multiplex PCR, predominantly low-virulent pathogens (Cutibacterium acnes and coagulase-negative staphylococci). In patients with aseptic failure, PCR detected 6 low-virulent organisms (predominantly C. acnes). While the overall performance of synovial fluid PCR was comparable to culture, PCR was superior for detection of low-virulent bacteria such as Cutibacterium spp. and coagulase-negative staphylococci. In addition, synovial fluid culture required several days for growth, whereas multiplex PCR provided results within 5hours in an automated manner. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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.

Metal-doped inorganic nanoparticles for multiplex detection of biomarkers by a sandwich-type ICP-MS immunoassay.

Science.gov (United States)

Ko, Jung Aa; Lim, H B

2016-09-28

Metal-doped inorganic nanoparticles were synthesized for the multiplex detection of biomarkers by a sandwich-type inductively coupled plasma mass spectrometry (ICP-MS) immunoassay. The synthesized Cs-doped multicore magnetic nanoparticles (MMNPs) were used not only for magnetic extraction of targets but also for ratiometric measurement in ICP-MS. In addition, three different metal/dye-doped silica nanoparticles (SNPs) were synthesized as probes for multiplex detection: Y/RhBITC (rhodamine B isothiocyanate)-doped SNPs for CRP (cardiovascular disease), Cd/RhBITC-doped SNPs for AFP (tumor), and Au/5(6)-XRITC (X-rhodamine-5-(and-6)-isothiocyanate)-doped SNPs for NSE (heart disease). For quantification, the doped metals of SNPs were measured by ICP-MS and then the signal ratio to Cs of MMNPs was plotted with respect to the concentration of targets by a ratiometry. Limits of detection (LOD) of 0.35 ng/mL to 77 ng mL(-1) and recoveries of 83%-125% were obtained for serum samples spiked with the biomarkers. Since no sample treatment was necessary prior to the extraction, the proposed method provided short analysis time and convenience for the multiplex determination of biomarkers, which will be valuable for clinical application. Copyright © 2016 Elsevier B.V. All rights reserved.

Establishment and application of a multiplex genetic mutation-detection method of lung cancer based on MassARRAY platform

International Nuclear Information System (INIS)

Tian, Hong-Xia; Zhang, Xu-Chao; Wang, Zhen; Chen, Jian-Guang; Chen, Shi-Liang; Guo, Wei-Bang; Wu, Yi-Long

2016-01-01

Objective: This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on MassARRAY mass spectrometry platform. Methods: We reviewed the related literature and data on lung cancer treatments. We also identified 99 mutation hot spots in 13 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. A total of 297 primers, composed of 99 paired forward and reverse amplification primers and 99 matched extension primers, were designed using Assay Design software. The detection method was established by analyzing eight cell lines and six lung cancer specimens. The proposed method was then validated through comparisons by using a LungCarta TM kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. Results: The proposed method was able to detect multiplex genetic mutations in lung cancer cell lines. This finding was consistent with the observations on previously reported mutations. The proposed method can also detect such mutations in clinical lung cancer specimens. This result was consistent with the observations with LungCarta TM kit. However, an FGFR2 mutation was detected only through the proposed method. The measured sensitivity and specificity were 100% and 96.3%, respectively. Conclusions: The proposed MassARRAY technology-based multiplex method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method can be applied to detect mutations in other cancer tissues

Multi-beam synchronous measurement based on PSD phase detection using frequency-domain multiplexing

Science.gov (United States)

Duan, Ying; Qin, Lan; Xue, Lian; Xi, Feng; Mao, Jiubing

2013-10-01

According to the principle of centroid measurement, position-sensitive detectors (PSD) are commonly used for micro displacement detection. However, single-beam detection method cannot satisfy such tasks as multi-dimension position measurement, three dimension vision reconstruction, and robot precision positioning, which require synchronous measurement of multiple light beams. Consequently, we designed PSD phase detection method using frequency-domain multiplexing for synchronous detection of multiple modulated light beams. Compared to previous PSD amplitude detection method, the phase detection method using FDM has advantages of simplified measuring system, low cost, high capability of resistance to light interference as well as improved resolution. The feasibility of multi-beam synchronous measurement based on PSD phase detection using FDM was validated by multi-beam measuring experiments. The maximum non-linearity error of the multi-beam synchronous measurement is 6.62%.

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

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs.

Directory of Open Access Journals (Sweden)

José Navarrete-Perea

2017-09-01

Full Text Available In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts can infest several tissues including the central nervous system (CNS and the skeletal muscles (SM. The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs.

Science.gov (United States)

Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P; Laclette, Juan P

2017-09-01

In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.

SERS-based inverse molecular sentinel (iMS) nanoprobes for multiplexed detection of microRNA cancer biomarkers in biological samples

Science.gov (United States)

Crawford, Bridget M.; Wang, Hsin-Neng; Fales, Andrew M.; Bowie, Michelle L.; Seewaldt, Victoria L.; Vo-Dinh, Tuan

2017-02-01

The development of sensitive and selective biosensing techniques is of great interest for clinical diagnostics. Here, we describe the development and application of a surface enhanced Raman scattering (SERS) sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, for the detection of nucleic acid biomarkers in biological samples. This iMS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform for a homogenous assay for multiplexed detection of nucleic acid biomarkers, including DNA, RNA and microRNA (miRNA). The "OFF-to-ON" signal switch is based on a non-enzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. Here, we demonstrate the development of iMS nanoprobes for the detection of DNA sequences as well as a modified design of the nanoprobe for the detection of short (22-nt) microRNA sequences. The application of iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of nucleic acid biomarkers, including short miRNAs molecules.

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.

Turbulence mitigation scheme based on multiple-user detection in an orbital-angular-momentum multiplexed system

Science.gov (United States)

Zou, Li; Wang, Le; Zhao, Sheng-Mei; Chen, Han-Wu

2016-11-01

Atmospheric turbulence (AT) induced crosstalk can significantly impair the performance of a free-space optical (FSO) communication link using orbital angular momentum (OAM) multiplexing. In this paper, we propose a multiple-user detection (MUD) turbulence mitigation scheme in an OAM-multiplexed FSO communication link. First, we present a MUD equivalent communication model for an OAM-multiplexed FSO communication link under AT. In the equivalent model, each input bit stream represents one user’s information. The deformed OAM spatial modes caused by AT, instead of the pure OAM spatial modes, are used as information carriers, and the overlapping between the deformed OAM spatial modes are computed as the correlation coefficients between the users. Then, we present a turbulence mitigation scheme based on MUD idea to enhance AT tolerance of the OAM-multiplexed FSO communication link. In the proposed scheme, the crosstalk caused by AT is used as a useful component to deduce users’ information. The numerical results show that the performance of the OAM-multiplexed communication link has greatly improved by the proposed scheme. When the turbulence strength is 1 × 10-15 m-2/3, the transmission distance is 1000 m and the channel signal-to-noise ratio (SNR) is 26 dB, the bit-error-rate (BER) performance of four spatial multiplexed OAM modes lm = +1,+2,+3,+4 are all close to 10-5, and there is a 2-3 fold increase in the BER performance in comparison with those results without the proposed scheme. In addition, the proposed scheme is more effective for an OAM-multiplexed FSO communication link with a larger OAM mode topological charge interval. The proposed scheme is a promising direction for compensating the interference caused by AT in the OAM-multiplexed FSO communication link. Project supported by the National Natural Science Foundation of China (Grant Nos. 61271238 and 61475075), the Open Research Fund of Key Lab of Broadband Wireless Communication and Sensor Network

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.

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.

Quantitative secondary electron detection

Science.gov (United States)

Agrawal, Jyoti; Joy, David C.; Nayak, Subuhadarshi

2018-05-08

Quantitative Secondary Electron Detection (QSED) using the array of solid state devices (SSD) based electron-counters enable critical dimension metrology measurements in materials such as semiconductors, nanomaterials, and biological samples (FIG. 3). Methods and devices effect a quantitative detection of secondary electrons with the array of solid state detectors comprising a number of solid state detectors. An array senses the number of secondary electrons with a plurality of solid state detectors, counting the number of secondary electrons with a time to digital converter circuit in counter mode.

A Multiplex PCR for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in Clinical Specimens

National Research Council Canada - National Science Library

McDonough, E. A; Barrozo, C. P; Russell, K. L; Metzgar, D

2005-01-01

A multiplex PCR was developed that is capable of detecting four of the most important bacterial agents of atypical pneumophia, Mycaplasma pneumoniae, Chlamydophia pneumoniae, Legionella pneumophila...

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�

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.

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

Energy Technology Data Exchange (ETDEWEB)

Lattanzio, Veronica M.T., E-mail: veronica.lattanzio@ispa.cnr.it [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy); Nivarlet, Noan [UNISENSOR S.A., Zoning industriel du Dossay, Rue du Dossay no 3, B-4020 Liege (Belgium); Lippolis, Vincenzo; Gatta, Stefania Della [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy); Huet, Anne-Catherine; Delahaut, Philippe [Centre d' Economie Rurale (CER Groupe), Rue du Point du Jour no 8, B-6900 Marloie (Belgium); Granier, Benoit [UNISENSOR S.A., Zoning industriel du Dossay, Rue du Dossay no 3, B-4020 Liege (Belgium); Visconti, Angelo [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy)

2012-03-09

Highlights: Black-Right-Pointing-Pointer We developed a rapid method based on a multiplex dipstick immunoassay. Black-Right-Pointing-Pointer The assay allowed the determination of major Fusarium toxins in wheat, oats, maize. Black-Right-Pointing-Pointer We obtained cut off levels close to EU regulatory levels. - Abstract: A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 {mu}g kg{sup -1}, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 {mu}g kg{sup -1}, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins

Multiplexed Colorimetric Solid-Phase Extraction

Science.gov (United States)

Gazda, Daniel B.; Fritz, James S.; Porter, Marc D.

2009-01-01

Multiplexed colorimetric solid-phase extraction (MC-SPE) is an extension of colorimetric solid-phase extraction (C-SPE) an analytical platform that combines colorimetric reagents, solid phase extraction, and diffuse reflectance spectroscopy to quantify trace analytes in water. In CSPE, analytes are extracted and complexed on the surface of an extraction membrane impregnated with a colorimetric reagent. The analytes are then quantified directly on the membrane surface using a handheld diffuse reflectance spectrophotometer. Importantly, the use of solid-phase extraction membranes as the matrix for impregnation of the colorimetric reagents creates a concentration factor that enables the detection of low concentrations of analytes in small sample volumes. In extending C-SPE to a multiplexed format, a filter holder that incorporates discrete analysis channels and a jig that facilitates the concurrent operation of multiple sample syringes have been designed, enabling the simultaneous determination of multiple analytes. Separate, single analyte membranes, placed in a readout cartridge create unique, analyte-specific addresses at the exit of each channel. Following sample exposure, the diffuse reflectance spectrum of each address is collected serially and the Kubelka-Munk function is used to quantify each water quality parameter via calibration curves. In a demonstration, MC-SPE was used to measure the pH of a sample and quantitate Ag(I) and Ni(II).

A Multiplex PCR for Simultaneous Detection of Three Zoonotic Parasites Ancylostoma ceylanicum, A. caninum, and Giardia lamblia Assemblage A

Directory of Open Access Journals (Sweden)

Wei Hu

2015-01-01

Full Text Available Ancylostoma ceylanicum, A. caninum, and Giardia lamblia assemblage A are common intestinal parasites of dogs and cats; they can also infect humans, causing parasitic zoonoses. In this study, a multiplex PCR method was developed for simultaneous identification and detection of those three zoonotic parasites. Three pairs of specific primers were designed based on ITS sequence of A. ceylanicum and A. caninum and TPI gene of G. lamblia available in the GenBank. The multiplex PCR reaction system was established by optimizing the reaction condition, and a series of tests on the sensitivity, specificity, and clinical application were also conducted. Results showed that three target fragments were amplified specifically; the detection limit was 10 eggs for both A. ceylanicum and A. caninum, 72 pg DNA for G. lamblia. Of 112 clinical fecal samples, 34.8% and 17.8% samples were positive for A. caninum and A. ceylanicum, respectively, while only 2.7% samples were positive for G. lamblia assemblage A. It is concluded that the established multiplex PCR assay is a convenient, rapid, cost-effective, and high-efficiency method for molecular detection and epidemiological investigation of three zoonotic parasites.

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.

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

Science.gov (United States)

Vasquez, Joshua J; Hussien, Rajaa; Aguilar-Rodriguez, Brandon; Junger, Henrik; Dobi, Dejan; Henrich, Timothy J; Thanh, Cassandra; Gibson, Erica; Hogan, Louise E; McCune, Joseph; Hunt, Peter W; Stoddart, Cheryl A; Laszik, Zoltan G

2018-02-01

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

Development of an integrated microsystem for the multiplexed detection of breast cancer markers in serum using electrochemical immunosensors

Science.gov (United States)

Fragoso, Alex; Laboria, Noemi; Botero, Mary Luz; Bejarano, Diego; Latta, Daniel; Hansen-Hagge, Thomas E.; Kemmner, Wolfgang; Katakis, Ioanis; Gärtner, Claudia; Drese, Klaus; O'Sullivan, Ciara K.

2010-02-01

A microsystem integrating electrochemical biosensoric detection for the simultaneous multiplexed detection of protein markers of breast cancer is reported. The immobilization of antibodies against each of carcinoembryonic antigen (CEA), prostate specific antigen (PSA) and cancer antigen 15-3 (CA15-3) was achieved via crosslinking to a bipodal dithiol chemisorbed on gold electrodes. This bipodal dithiol had the double function of eliminating non-specific binding and optimal spacing of the anchor antibodies for maximum accessibility to the target proteins. Storage conditions were optimized, demonstrating a long-term stability of the reporter conjugates jointly stored within a single reservoir in the microsystem. The final system has been optimized in terms of incubation times, temperatures and simultaneous, multiplexed detection of the protein markers was achieved in less than 10 minutes with less than ng/mL detection limits. The microsystem has been validated using real patient serum samples and excellent correlation with ELISA results obtained.

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.

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.

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.

Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

Science.gov (United States)

Davidson, Irit; Raibshtein, I; Al-Touri, A

2013-06-01

The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

Comparison of three human papillomavirus DNA detection methods: Next generation sequencing, multiplex-PCR and nested-PCR followed by Sanger based sequencing.

Science.gov (United States)

da Fonseca, Allex Jardim; Galvão, Renata Silva; Miranda, Angelica Espinosa; Ferreira, Luiz Carlos de Lima; Chen, Zigui

2016-05-01

To compare the diagnostic performance for HPV infection using three laboratorial techniques. Ninty-five cervicovaginal samples were randomly selected; each was tested for HPV DNA and genotypes using 3 methods in parallel: Multiplex-PCR, the Nested PCR followed by Sanger sequencing, and the Next_Gen Sequencing (NGS) with two assays (NGS-A1, NGS-A2). The study was approved by the Brazilian National IRB (CONEP protocol 16,800). The prevalence of HPV by the NGS assays was higher than that using the Multiplex-PCR (64.2% vs. 45.2%, respectively; P = 0.001) and the Nested-PCR (64.2% vs. 49.5%, respectively; P = 0.003). NGS also showed better performance in detecting high-risk HPV (HR-HPV) and HPV16. There was a weak interobservers agreement between the results of Multiplex-PCR and Nested-PCR in relation to NGS for the diagnosis of HPV infection, and a moderate correlation for HR-HPV detection. Both NGS assays showed a strong correlation for detection of HPVs (k = 0.86), HR-HPVs (k = 0.91), HPV16 (k = 0.92) and HPV18 (k = 0.91). NGS is more sensitive than the traditional Sanger sequencing and the Multiplex PCR to genotype HPVs, with promising ability to detect multiple infections, and may have the potential to establish an alternative method for the diagnosis and genotyping of HPV. © 2015 Wiley Periodicals, Inc.

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.

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

Design of a Modular DNA Triangular-Prism Sensor Enabling Ratiometric and Multiplexed Biomolecule Detection on a Single Microbead.

Science.gov (United States)

Liu, Yu; Chen, Qiaoshu; Liu, Jianbo; Yang, Xiaohai; Guo, Qiuping; Li, Li; Liu, Wei; Wang, Kemin

2017-03-21

DNA nanostructures have emerged as powerful and versatile building blocks for the construction of programmable nanoscale structures and functional sensors for biomarker detection, disease diagnostics, and therapy. Here we integrated multiple sensing modules into a single DNA three-dimensional (3D) nanoarchitecture with a triangular-prism (TP) structure for ratiometric and multiplexed biomolecule detection on a single microbead. In our design, the complementary hybridization of three clip sequences formed TP nanoassemblies in which the six single-strand regions in the top and bottom faces act as binding sites for different sensing modules, including an anchor module, reference sequence module, and capture sequence module. The multifunctional modular TP nanostructures were thus exploited for ratiometric and multiplexed biomolecule detection on microbeads. Microbead imaging demonstrated that, after ratiometric self-calibration analysis, the imaging deviations resulting from uneven fluorescence intensity distribution and differing probe concentrations were greatly reduced. The rigid nanostructure also conferred the TP as a framework for geometric positioning of different capture sequences. The inclusion of multiple targets led to the formation of sandwich hybridization structures that gave a readily detectable optical response at different fluorescence channels and distinct fingerprint-like pattern arrays. This approach allowed us to discriminate multiplexed biomolecule targets in a simple and efficient fashion. In this module-designed strategy, the diversity of the controlled DNA assembly coupled with the geometrically well-defined rigid nanostructures of the TP assembly provides a flexible and reliable biosensing approach that shows great promise for biomedical applications.

Optimizing diffusion in multiplexes by maximizing layer dissimilarity

Science.gov (United States)

Serrano, Alfredo B.; Gómez-Gardeñes, Jesús; Andrade, Roberto F. S.

2017-05-01

Diffusion in a multiplex depends on the specific link distribution between the nodes in each layer, but also on the set of the intralayer and interlayer diffusion coefficients. In this work we investigate, in a quantitative way, the efficiency of multiplex diffusion as a function of the topological similarity among multiplex layers. This similarity is measured by the distance between layers, taken among the pairs of layers. Results are presented for a simple two-layer multiplex, where one of the layers is held fixed, while the other one can be rewired in a controlled way in order to increase or decrease the interlayer distance. The results indicate that, for fixed values of all intra- and interlayer diffusion coefficients, a large interlayer distance generally enhances the global multiplex diffusion, providing a topological mechanism to control the global diffusive process. For some sets of networks, we develop an algorithm to identify the most sensitive nodes in the rewirable layer, so that changes in a small set of connections produce a drastic enhancement of the global diffusion of the whole multiplex system.

A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

Science.gov (United States)

Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

2016-05-01

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

Simultaneous aptasensor for multiplex pathogenic bacteria detection based on multicolor upconversion nanoparticles labels.

Science.gov (United States)

Wu, Shijia; Duan, Nuo; Shi, Zhao; Fang, Congcong; Wang, Zhouping

2014-03-18

A highly sensitive and specific multiplex method for the simultaneous detection of three pathogenic bacteria was fabricated using multicolor upconversion nanoparticles (UCNPs) as luminescence labels coupled with aptamers as the molecular recognition elements. Multicolor UCNPs were synthesized via doping with various rare-earth ions to obtain well-separated emission peaks. The aptamer sequences were selected using the systematic evolution of ligands by exponential enrichment (SELEX) strategy for Staphylococcus aureus, Vibrio parahemolyticus, and Salmonella typhimurium. When applied in this method, aptamers can be used for the specific recognition of the bacteria from complex mixtures, including those found in real food matrixes. Aptamers and multicolor UCNPs were employed to selectively capture and simultaneously quantify the three target bacteria on the basis of the independent peaks. Under optimal conditions, the correlation between the concentration of three bacteria and the luminescence signal was found to be linear from 50-10(6) cfu mL(-1). Improved by the magnetic separation and concentration effect of Fe3O4 magnetic nanoparticles, the limits of detection of the developed method were found to be 25, 10, and 15 cfu mL(-1) for S. aureus, V. parahemolyticus, and S. typhimurium, respectively. The capability of the bioassay in real food samples was also investigated, and the results were consistent with experimental results obtained from plate-counting methods. This proposed method for the detection of various pathogenic bacteria based on multicolor UCNPs has great potential in the application of food safety and multiplex nanosensors.

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.

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...

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.

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

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

Detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR.

Science.gov (United States)

Maas, L; Dorigo-Zetsma, J W; de Groot, C J; Bouter, S; Plötz, F B; van Ewijk, B E

2014-06-01

The performance of a multiplex real-time PCR for the detection of Blastocystis, Dientamoeba fragilis, Giardia lamblia, Cryptosporidium species and Entamoeba species in faecal samples was evaluated in an observational prospective study. Paediatric patients (0-18 years) presenting with gastrointestinal symptoms and suspected of having enteroparasitic disease were included. A questionnaire on gastrointestinal symptoms and the chosen treatment was completed at the start of the study and after 6 weeks. Of 163 paediatric patients (mean age, 7.8 years), 114 (70%) had a PCR-positive faecal sample. D. fragilis was detected most frequently, in 101 patients, followed by Blastocystis in 49. In faecal samples of 47 patients, more than one protozoan was detected, mainly the combination of D. fragilis and Blastocystis. Reported gastrointestinal symptoms were abdominal pain (78%), nausea (30%), and altered bowel habits (28%). Eighty-nine of the PCR-positive patients were treated with antibiotics. A significant reduction in abdominal pain was observed both in treated and in untreated patients. This study demonstrated that multiplex real-time PCR detects a high percentage of intestinal protozoa in paediatric patients with gastrointestinal symptoms. However, interpretation and determination of the clinical relevance of a positive PCR result in this population are still difficult. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

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.

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.

Multiplex polymerase chain reaction for the detection of high-risk-human papillomavirus types in formalin-fixed paraffin-embedded cervical tissues

Directory of Open Access Journals (Sweden)

Mini P Singh

2017-01-01

Full Text Available Detecting high-risk-human papillomavirus (HPV types has become an integral part of the cervical cancer screening programmes. This study aimed to develop a multiplex polymerase chain reaction (PCR for identification of HPV types 16 and 18 along with the beta globin gene in formalin-fixed and paraffin-embedded cervical biopsy specimens. A total of 59 samples from patients with cervical abnormalities were tested. HPV 16 positivity was 50% in cervical cancers and 52.9% in cervical intraepithelial neoplasia. Our multiplex PCR protocol can be used as a simple and cost-effective tool for high-risk-HPV detection in cervical cancer screening programmes.

Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

Directory of Open Access Journals (Sweden)

Zhang PF

2015-09-01

Full Text Available Pengfei Zhang,1,* Yan Bao,1,* Mohamed Shehata Draz,2,3,* Huiqi Lu,1 Chang Liu,1 Huanxing Han11Center for Translational Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Faculty of Science, Tanta University, Tanta, Egypt*These authors contributed equally to this workAbstract: Convenient and rapid immunofiltration assays (IFAs enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP. CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.Keywords: C-reactive proteins, point-of-care test, Glutathione capped QDs, PEGylation

High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy.

Science.gov (United States)

Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

2011-03-10

Historical records suggest that multiple burial sites from the 14th-16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

Directory of Open Access Journals (Sweden)

Takao Ito

Full Text Available Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

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.

Multicolor fluorescent biosensor for multiplexed detection of DNA.

Science.gov (United States)

Hu, Rong; Liu, Tao; Zhang, Xiao-Bing; Huan, Shuang-Yan; Wu, Cuichen; Fu, Ting; Tan, Weihong

2014-05-20

Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

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.

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

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.

Multiplex Cathepsin Zymography to Detect Amounts of Active Cathepsins K, L, S, and V.

Science.gov (United States)

Platt, Manu O

2017-01-01

Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracellular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for specific identification which tremendously reduces cost. This chapter will demonstrate the utility and interpretation of this method with mammalian cells and tissue to quantify amounts of active cathepsins K, L, S, and V without complicating signals of the procathepsin. Multiplex cathepsin zymography has many advantages: (1) it separates cathepsins K, L, S, and V by electrophoretic migration distance, (2) allows visual confirmation of cathepsin identity, (3) does not detect procathepsins, and (4) can be quantified with densitometry.

A real-time multi-gases detection and concentration measurements based-on time-division multiplexed-lasers

Science.gov (United States)

Yazdandoust, Fatemeh; Tatenguem Fankem, Hervé; Milde, Tobias; Jimenez, Alvaro; Sacher, Joachim

2018-02-01

We report the development of a platform, based-on a Field-Programmable Gate Arrays (FPGAs) and suitable for Time-Division-Multiplexed DFB lasers. The designed platform is subsequently combined with a spectroscopy setup, for detection and quantification of species in a gas mixture. The experimental results show a detection limit of 460 ppm, an uncertainty of 0.1% and a computation time of less than 1000 clock cycles. The proposed system offers a high level of flexibility and is applicable to arbitrary types of gas-mixtures.

The Role of Multiplex Polymerase Chain Reaction in Detecting Etiological Causes of Bacterial Prostatitis Associated Benign Prostatic Hyperplasia

Directory of Open Access Journals (Sweden)

Bramastha Rosadi

2015-01-01

Full Text Available Background: Benign Prostatic Hyperplasia (BPH has been correlated with chronic prostatitis according recent study. Chronic pelvic pain is the chief complain of BPH followed by prostatitis. The gold standard of the etiological diagnosis is urine culture, but the negativity rate is still high. Multiplex polymerase chain reaction (PCR as a diagnostic tool in search of etiological causes could identify microorganism on DNA level. This research aims to find out the role of multiplex polymerase chain reaction as diagnostic tools on prostatitis patients. Material and Method: A total of 12 samples collected during the TURP procedure in Sanglah General Hospital Denpasar – Bali from February until May 2015. All of the samples has been diagnosed prostatitis clinically and perform urine culture test. The prostate specimen taken was sent to the Pathological anatomy for histopathology diagnostic and underwent multiplex PCR for etiologic diagnostic. Result: 12 samples have been declared as prostatitis based on histopathology examination, and then were analyzed using multiplex PCR. 10 samples were positive (6 were E. coli, 2 were C. trachomatis, the rest were N. gonorrhea and P. aeruginosa. The urine culture revealed 9 positive, within the result 6 were E. coli, and the others were P. aeruginosa, M. morganii and A. haemolyticus. Conclusion: In prostatitis patient, the etiological diagnostic was important. Multiplex PCR as diagnostic tools could detect the microorganism on a negative urine culture. The combination of the urine culture test and multiplex PCR revealed a better result on etiologic diagnosis which leads to a better management of the disease. 

High Throughput, Multiplexed Pathogen Detection Authenticates Plague Waves in Medieval Venice, Italy

Science.gov (United States)

Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

2011-01-01

Background Historical records suggest that multiple burial sites from the 14th–16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. Methodology/Principal Findings High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. Conclusions These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century. PMID:21423736

SiDIVS: Simple Detection of Inductive Vehicle Signatures with a Multiplex Resonant Sensor

Directory of Open Access Journals (Sweden)

José J. Lamas-Seco

2016-08-01

Full Text Available This work provides a system capable of obtaining simultaneous inductive signatures of vehicles traveling on a roadway with minimal cost. Based on Time-Division Multiplexing (TDM with multiple oscillators, one for each inductive loop, the proposed system detects the presence of vehicles by means of a shift in the oscillation period of the selected loop and registers the signature of the detected vehicles by measuring the duration of a fixed number of oscillator pulses. In order to test the system in an actual environment, we implement a prototype that we denote as SiDIVS (Simple Detection of Inductive Vehicle Signatures and acquire different vehicle inductive signatures under real scenarios. We also test the robustness of the detector by simulating the effect of noise on the signature acquisition.

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.

Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray.

Directory of Open Access Journals (Sweden)

Bettina Stieber

Full Text Available S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.

DNA Differential Diagnosis of Taeniasis and Cysticercosis by Multiplex PCR

Science.gov (United States)

Yamasaki, Hiroshi; Allan, James C.; Sato, Marcello Otake; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Qiu, Dongchuan; Mamuti, Wulamu; Craig, Philip S.; Ito, Akira

2004-01-01

Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections. PMID:14766815

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

Science.gov (United States)

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

2008-05-01

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

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers.

Science.gov (United States)

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C; Chorianopoulos, Nikos

2015-10-22

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers

Directory of Open Access Journals (Sweden)

Alex Galanis

2015-10-01

Full Text Available Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD Sequenced Characterized Amplified Region (SCAR analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum.

Science.gov (United States)

Anniballi, Fabrizio; Auricchio, Bruna; Delibato, Elisabetta; Antonacci, Monia; De Medici, Dario; Fenicia, Lucia

2012-01-27

Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities. Copyright © 2011 Elsevier B.V. All rights reserved.

Methods for detection of GMOs in food and feed.

Science.gov (United States)

Marmiroli, Nelson; Maestri, Elena; Gullì, Mariolina; Malcevschi, Alessio; Peano, Clelia; Bordoni, Roberta; De Bellis, Gianluca

2008-10-01

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.

Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction.

Science.gov (United States)

Saade, M; Aparicio, F; Sánchez-Navarro, J A; Herranz, M C; Myrta, A; Di Terlizzi, B; Pallás, V

2000-12-01

ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Long-term desorption of trichloroethylene from flint clay using multiplexed optical detection

International Nuclear Information System (INIS)

Stager, M.P.; Perram, G.P.

1999-01-01

The long-term desorption of trichloroethylene (TCE) from powdered flint clay was examined using a multiplexed, phase sensitive infrared technique which provided a gas phase detection limit of 0.0045 torr for continuous monitoring of the desorption process for at least 3 days. The vapor phase TCE concentrations as a function of desorption time exhibit a significant deviation from Langmuir kinetics. The desorption process is adequately described by bonding sites with a gamma distribution for the desorption rate coefficients. The mean desorption rate for powdered flint clay at 25°C is k d = 0.50 ± 0.02 h −1 . (author)

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

Directory of Open Access Journals (Sweden)

Ahmad N. A. A

2017-01-01

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

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

Science.gov (United States)

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

2017-11-01

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

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.

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...

Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

Science.gov (United States)

Frei, Andreas P; Bava, Felice-Alessio; Zunder, Eli R; Hsieh, Elena W Y; Chen, Shih-Yu; Nolan, Garry P; Gherardini, Pier Federico

2016-03-01

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

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.

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

DEFF Research Database (Denmark)

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

2017-01-01

, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair...

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

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

Non-Linear Detection for Joint Space-Frequency Block Coding and Spatial Multiplexing in OFDM-MIMO Systems

DEFF Research Database (Denmark)

Rahman, Imadur Mohamed; Marchetti, Nicola; Fitzek, Frank

2005-01-01

(SIC) receiver where the detection is done on subcarrier by sub-carrier basis based on both Zero Forcing (ZF) and Minimum Mean Square Error (MMSE) nulling criterion for the system. In terms of Frame Error Rate (FER), MMSE based SIC receiver performs better than all other receivers compared......In this work, we have analyzed a joint spatial diversity and multiplexing transmission structure for MIMO-OFDM system, where Orthogonal Space-Frequency Block Coding (OSFBC) is used across all spatial multiplexing branches. We have derived a BLAST-like non-linear Successive Interference Cancellation...... in this paper. We have found that a linear two-stage receiver for the proposed system [1] performs very close to the non-linear receiver studied in this work. Finally, we compared the system performance in spatially correlated scenario. It is found that higher amount of spatial correlation at the transmitter...

Multiplexed detection of mycotoxins in foods with a regenerable array.

Science.gov (United States)

Ngundi, Miriam M; Shriver-Lake, Lisa C; Moore, Martin H; Ligler, Frances S; Taitt, Chris R

2006-12-01

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Comparison between Conventional OCDMA and Subcarrier Multiplexing SAC OCDMA System Based on Single Photodiode Detection

OpenAIRE

Ahmad N. A. A; Junita M. N; Aljunid Syed Alwi; Che Beson Mohd Rashidi; Endut Rosdisham

2017-01-01

This paper demonstrates the comparison between conventional OCDMA system and subcarrier multiplexing (SCM) SAC-OCDMA system by applying Recursive Combinatorial (RC) code based on single photodiode detection (SPD). SPD is used in the receiver part to reduce the effect of multiple access interference (MAI) which contributes as a dominant noise in incoherent SAC-OCDMA systems. From this analysis, the performance of SCM OCDMA network could be improved by using lower data rates and higher received...

Multiplexing of spatial modes in the mid-IR region

Science.gov (United States)

Gailele, Lucas; Maweza, Loyiso; Dudley, Angela; Ndagano, Bienvenu; Rosales-Guzman, Carmelo; Forbes, Andrew

2017-02-01

Traditional optical communication systems optimize multiplexing in polarization and wavelength both trans- mitted in fiber and free-space to attain high bandwidth data communication. Yet despite these technologies, we are expected to reach a bandwidth ceiling in the near future. Communications using orbital angular momentum (OAM) carrying modes offers infinite dimensional states, providing means to increase link capacity by multiplexing spatially overlapping modes in both the azimuthal and radial degrees of freedom. OAM modes are multiplexed and de-multiplexed by the use of spatial light modulators (SLM). Implementation of complex amplitude modulation is employed on laser beams phase and amplitude to generate Laguerre-Gaussian (LG) modes. Modal decomposition is employed to detect these modes due to their orthogonality as they propagate in space. We demonstrate data transfer by sending images as a proof-of concept in a lab-based scheme. We demonstrate the creation and detection of OAM modes in the mid-IR region as a precursor to a mid-IR free-space communication link.

A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control

International Nuclear Information System (INIS)

Cui, Xingye; Hu, Jie; Choi, Jane Ru; Huang, Yalin; Wang, Xuemin; Lu, Tian Jian; Xu, Feng

2016-01-01

A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed “U shape” reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 μg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications. - Highlights: • The meter chip is a standalone point-of-care diagnostic tool with visible readouts of quantification results. • A fast and low cost fabrication protocol (~3 min and ~$0.2 per chip) of meter chip was proposed. • The chip may hold the potential for rapid scaning of bovine mastitis in cattle farms for food safety control.

A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control

Energy Technology Data Exchange (ETDEWEB)

Cui, Xingye; Hu, Jie; Choi, Jane Ru; Huang, Yalin; Wang, Xuemin [The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi' an Jiaotong University, Xi' an, 710049 (China); Bioinspired Engineering and Biomechanics Center (BEBC), Xi' an Jiaotong University, Xi' an, 710049 (China); Lu, Tian Jian, E-mail: tjlu@mail.xjtu.edu.cn [Bioinspired Engineering and Biomechanics Center (BEBC), Xi' an Jiaotong University, Xi' an, 710049 (China); Xu, Feng, E-mail: fengxu@mail.xjtu.edu.cn [The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi' an Jiaotong University, Xi' an, 710049 (China); Bioinspired Engineering and Biomechanics Center (BEBC), Xi' an Jiaotong University, Xi' an, 710049 (China)

2016-09-07

A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed “U shape” reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 μg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications. - Highlights: • The meter chip is a standalone point-of-care diagnostic tool with visible readouts of quantification results. • A fast and low cost fabrication protocol (~3 min and ~$0.2 per chip) of meter chip was proposed. • The chip may hold the potential for rapid scaning of bovine mastitis in cattle farms for food safety control.

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

Science.gov (United States)

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

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

Fluorescence-Raman Dual Modal Endoscopic System for Multiplexed Molecular Diagnostics

Science.gov (United States)

Jeong, Sinyoung; Kim, Yong-Il; Kang, Homan; Kim, Gunsung; Cha, Myeong Geun; Chang, Hyejin; Jung, Kyung Oh; Kim, Young-Hwa; Jun, Bong-Hyun; Hwang, Do Won; Lee, Yun-Sang; Youn, Hyewon; Lee, Yoon-Sik; Kang, Keon Wook; Lee, Dong Soo; Jeong, Dae Hong

2015-03-01

Optical endoscopic imaging, which was recently equipped with bioluminescence, fluorescence, and Raman scattering, allows minimally invasive real-time detection of pathologies on the surface of hollow organs. To characterize pathologic lesions in a multiplexed way, we developed a dual modal fluorescence-Raman endomicroscopic system (FRES), which used fluorescence and surface-enhanced Raman scattering nanoprobes (F-SERS dots). Real-time, in vivo, and multiple target detection of a specific cancer was successful, based on the fast imaging capability of fluorescence signals and the multiplex capability of simultaneously detected SERS signals using an optical fiber bundle for intraoperative endoscopic system. Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) on the breast cancer xenografts in a mouse orthotopic model were successfully detected in a multiplexed way, illustrating the potential of FRES as a molecular diagnostic instrument that enables real-time tumor characterization of receptors during routine endoscopic procedures.

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

Science.gov (United States)

Hernández, Marta; Rodríguez-Lázaro, David; Esteve, Teresa; Prat, Salomé; Pla, Maria

2003-12-15

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

Science.gov (United States)

Majumder, S; Baranwal, V K

2014-06-01

Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiplex and high-throughput DNA detection using surface plasmon mediated fluorescence

Science.gov (United States)

Mei, Zhong

The overall objective of this research project was to develop a user-friendly and sensitive biosensor for nucleic acid aptamers with multiplexing and high-throughput capability. The sensing was based on the fluorescence signals emitted by the fluorophores coupling with plamonic nanoparticle (gold nanorod) deposited on a patterned substrate. Gold nanorods (GNRs) were synthesized using a binary mixture of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NaOL) in seed mediated growth method. Polytetrafluoroethylene (PTFE) printed glass slides were selectively coated with a gold thin-film to define hydrophilic areas for GNR deposition. Due to the wettablity contrast, GNR solution dropped on the slide was induced to assemble exclusively in the hydrophilic spots. By controlling temperature and humidity of the evaporation process, vertically-standing GNR arrays were achieved on the pattered slide. Fluorescence was conjugated to GNR surface via DNA double strand with tunable length. Theoretical simulation predicted a flat layer ( 30 nm thick) of uniform "hot spots" presented on the GNR tips, which could modify the nearby fluorescence. Experimentally, the vertical GNR arrays yielded metallic enhanced fluorescence (MEF) effect, which was dependent on the spectrum overlap and GNR-fluorophore distance. Specifically, the maximum enhancement of Quasar 670 and Alexa 750 was observed when it was coupled with GNR664 (plasmonic wavelength 664 nm) and GNR778 respectively at a distance of 16 nm, while the carboxyfluorescein (FAM) was at maximal intensity when attached to gold nanosphere520. This offers an opportunity for multiplexed DNA sensing. Based on this, we developed a novel GNR mediated fluorescence biosensor for DNA detection. Fluorescence labeled haipin-DNA probes were introduced to designated spots of GNR array with the matching LSPR wavelengths on the substrate. The fluorescence was quenched originally because of Forster resonance energy transfer (FRET) effect

Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

Science.gov (United States)

Ranjan, R K; Singh, Dinesh; Baranwal, V K

2016-11-01

Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors

Directory of Open Access Journals (Sweden)

Fadel Sayes

2018-04-01

Full Text Available Summary: The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II. : Sayes et al. develop an approach to express distinct fluorescent reporters that is based on the recognition of specific Mycobacterium tuberculosis MHC class II epitopes by highly discriminative T cell hybridomas. This multiplexed technology allows the study of secretion, subcellular location, and regulation patterns of these instrumental protein members. Keywords: mycobacterium tuberculosis, type VII secretion systems, intracellular bacteria, T-cell hybridomas, mycobacterial virulence factors, bacterial antigen presentation, lentiviral vectors, reporter T cells, in vivo antigen presentation, protein localization

Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System.

Science.gov (United States)

Rocchetti, Talita T; Silbert, Suzane; Gostnell, Alicia; Kubasek, Carly; Campos Pignatari, Antonio C; Widen, Raymond

2017-03-01

A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Lesion detection and quantitation of positron emission mammography

International Nuclear Information System (INIS)

Qi, Jinyi; Huesman, Ronald H.

2001-01-01

A Positron Emission Mammography (PEM) scanner dedicated to breast imaging is being developed at our laboratory. We have developed a list mode likelihood reconstruction algorithm for this scanner. Here we theoretically study the lesion detection and quantitation. The lesion detectability is studied theoretically using computer observers. We found that for the zero-order quadratic prior, the region of interest observer can achieve the performance of the prewhitening observer with a properly selected smoothing parameter. We also study the lesion quantitation using the test statistic of the region of interest observer. The theoretical expressions for the bias, variance, and ensemble mean squared error of the quantitation are derived. Computer simulations show that the theoretical predictions are in good agreement with the Monte Carlo results for both lesion detection and quantitation

Detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-PCR.

Directory of Open Access Journals (Sweden)

Kerstin Wernike

Full Text Available Porcine reproductive and respiratory syndrome (PRRS causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV are classified into the two distinct genotypes "North American (NA, type 2" and "European (EU, type 1". In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV, characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per µl for the type 1-assay and 20 copies per µl for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes as well as the direct detection of HP-PRRSV.

Multiplex autoantibody detection for autoimmune liver diseases and autoimmune gastritis.

Science.gov (United States)

Vanderlocht, Joris; van der Cruys, Mart; Stals, Frans; Bakker-Jonges, Liesbeth; Damoiseaux, Jan

2017-09-01

Autoantibody detection for autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and autoimmune gastritis (AIG) is traditionally performed by IIF on a combination of tissues. Multiplex line/dot blots (LIA/DIA) offer multiple advantages, i.e. automation, objective reading, no interfering reactivities, no coincidental findings. In the current study we evaluated automated DIA (D-Tek) for detecting autoantibodies related to autoimmune diseases of the gastrointestinal tract. We tested samples of the Dutch EQC program and compared the results with the consensus of the participating labs. For the autoimmune liver diseases and AIG, respectively, 64 and 36 samples were tested. For anti-mitochondrial and anti-smooth muscle antibodies a concordance rate of 97% and 88% was observed, respectively. The concordance rate for anti-parietal cell antibodies was 92% when samples without EQC consensus (n=15) were excluded. For antibodies against intrinsic factor a concordance of 96% was observed. For all these antibodies discrepancies were identified that relate to the different test characteristics and the preponderance of IIF utilizing labs in the EQC program. In conclusion, we observed good agreement of the tested DIA blots with the consensus results of the Dutch EQC program. Taken together with the logistic advantages these blots are a good alternative for autoantibody detection in the respective diseases. A large prospective multicenter study is warranted to position these novel tests further in the whole spectrum of assays for the detection of these antibodies in a routine autoimmune laboratory. Copyright © 2017 Elsevier B.V. All rights reserved.

A PDMS/paper/glass hybrid microfluidic biochip integrated with aptamer-functionalized graphene oxide nano-biosensors for one-step multiplexed pathogen detection.

Science.gov (United States)

Zuo, Peng; Li, XiuJun; Dominguez, Delfina C; Ye, Bang-Ce

2013-10-07

Infectious pathogens often cause serious public health concerns throughout the world. There is an increasing demand for simple, rapid and sensitive approaches for multiplexed pathogen detection. In this paper we have developed a polydimethylsiloxane (PDMS)/paper/glass hybrid microfluidic system integrated with aptamer-functionalized graphene oxide (GO) nano-biosensors for simple, one-step, multiplexed pathogen detection. The paper substrate used in this hybrid microfluidic system facilitated the integration of aptamer biosensors on the microfluidic biochip, and avoided complicated surface treatment and aptamer probe immobilization in a PDMS or glass-only microfluidic system. Lactobacillus acidophilus was used as a bacterium model to develop the microfluidic platform with a detection limit of 11.0 cfu mL(-1). We have also successfully extended this method to the simultaneous detection of two infectious pathogens - Staphylococcus aureus and Salmonella enterica. This method is simple and fast. The one-step 'turn on' pathogen assay in a ready-to-use microfluidic device only takes ~10 min to complete on the biochip. Furthermore, this microfluidic device has great potential in rapid detection of a wide variety of different other bacterial and viral pathogens.

A multiplex PCR-based method for the detection and early identification of wood rotting fungi in standing trees.

Science.gov (United States)

Guglielmo, F; Bergemann, S E; Gonthier, P; Nicolotti, G; Garbelotto, M

2007-11-01

The goal of this research was the development of a PCR-based assay to identify important decay fungi from wood of hardwood tree species in northern temperate regions. Eleven taxon-specific primers were designed for PCR amplification of either nuclear or mitochondrial ribosomal DNA regions of Armillaria spp., Ganoderma spp., Hericium spp., Hypoxylon thouarsianum var. thouarsianum, Inonotus/Phellinus-group, Laetiporus spp., Perenniporia fraxinea, Pleurotus spp., Schizophyllum spp., Stereum spp. and Trametes spp. Multiplex PCR reactions were developed and optimized to detect fungal DNA and identify each taxon with a sensitivity of at least 1 pg of target DNA in the template. This assay correctly identified the agents of decay in 82% of tested wood samples. The development and optimization of multiplex PCRs allowed for reliable identification of wood rotting fungi directly from wood. Early detection of wood decay fungi is crucial for assessment of tree stability in urban landscapes. Furthermore, this method may prove useful for prediction of the severity and the evolution of decay in standing trees.

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.

Group-specific multiplex PCR detection systems for the identification of flying insect prey.

Directory of Open Access Journals (Sweden)

Daniela Sint

Full Text Available The applicability of species-specific primers to study feeding interactions is restricted to those ecosystems where the targeted prey species occur. Therefore, group-specific primer pairs, targeting higher taxonomic levels, are often desired to investigate interactions in a range of habitats that do not share the same species but the same groups of prey. Such primers are also valuable to study the diet of generalist predators when next generation sequencing approaches cannot be applied beneficially. Moreover, due to the large range of prey consumed by generalists, it is impossible to investigate the breadth of their diet with species-specific primers, even if multiplexing them. However, only few group-specific primers are available to date and important groups of prey such as flying insects have rarely been targeted. Our aim was to fill this gap and develop group-specific primers suitable to detect and identify the DNA of common taxa of flying insects. The primers were combined in two multiplex PCR systems, which allow a time- and cost-effective screening of samples for DNA of the dipteran subsection Calyptratae (including Anthomyiidae, Calliphoridae, Muscidae, other common dipteran families (Phoridae, Syrphidae, Bibionidae, Chironomidae, Sciaridae, Tipulidae, three orders of flying insects (Hymenoptera, Lepidoptera, Plecoptera and coniferous aphids within the genus Cinara. The two PCR assays were highly specific and sensitive and their suitability to detect prey was confirmed by testing field-collected dietary samples from arthropods and vertebrates. The PCR assays presented here allow targeting prey at higher taxonomic levels such as family or order and therefore improve our ability to assess (trophic interactions with flying insects in terrestrial and aquatic habitats.

A Portable Immunoassay Platform for Multiplexed Detection of Biotoxins in Clinical and Environmental Samples

Energy Technology Data Exchange (ETDEWEB)

Koh, Chung-Yan [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Piccini, Matthew Ernest [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Cepheid, Sunnyvale, CA (United States); Schaff, Ulrich Y. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandstone Diagnostics, Livermore, CA (United States); Stanker, Larry H. [US Dept. of Agriculture, Albany, CA (United States). Western Regional Research Center, Foodborne Contaminants Research Unit; Cheng, Luisa W. [US Dept. of Agriculture, Albany, CA (United States). Western Regional Research Center, Foodborne Contaminants Research Unit; Ravichandran, Easwaran [Univ. of Massachusetts, Dartmouth, MA (United States); Singh, Bal-Ram [Univ. of Massachusetts, Dartmouth, MA (United States); Sommer, Greg J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandstone Diagnostics, Livermore, CA (United States); Singh, Anup K. [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

2015-01-01

Multiple cases of attempted bioterrorism events using biotoxins have highlighted the urgent need for tools capable of rapid screening of suspect samples in the field (e.g., mailroom and public events). We present a portable microfluidic device capable of analyzing environmental (e.g., white powder), food (e.g., milk) and clinical (e.g., blood) samples for multiplexed detection of biotoxins. The device is rapid (<15-30 min sample-to-answer), sensitive (< 0.08 pg/mL detection limit for botulinum toxin), multiplexed (up to 64 parallel assays) and capable of analyzing small volume samples (< 20 μL total sample input). The immunoassay approach (SpinDx) is based on binding of toxins in a sample to antibody-laden capture particles followed by sedimentation of particles through a density-media in a microfluidic disk and quantification using a laser-induced fluorescence detector. A direct, blinded comparison with a gold standard ELISA revealed a 5-fold more sensitive detection limit for botulinum toxin while requiring 250-fold less sample volume and a 30 minute assay time with a near unity correlation. A key advantage of the technique is its compatibility with a variety of sample matrices with no additional sample preparation required. Ultrasensitive quantification has been demonstrated from direct analysis of multiple clinical, environmental and food samples, including white powder, whole blood, saliva, salad dressing, whole milk, peanut butter, half and half, honey, and canned meat. We believe that this device can met an urgent need in screening both potentially exposed people as well as suspicious samples in mail-rooms, airports, public sporting venues and emergency rooms. The general-purpose immunodiagnostics device can also find applications in screening of infectious and systemic diseases or serve as a lab device for conducting rapid immunoassays.

The Design of a Quantitative Western Blot Experiment

Directory of Open Access Journals (Sweden)

Sean C. Taylor

2014-01-01

Full Text Available Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013 and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

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.

Multiplex PCR detection of Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica directly from dried stool samples from Guinea-Bissauan children with diarrhoea

DEFF Research Database (Denmark)

Mero, Sointu; Kirveskari, Juha; Antikainen, Jenni

2017-01-01

and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. Methods: A real-time multiplex PCR method with internal inhibition control...... microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. Conclusions: Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof...

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.

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.

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

Science.gov (United States)

Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

2018-04-03

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

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.

Multiplex PCR for the detection and identification of dairy bacteriophages in milk.

Science.gov (United States)

del Rio, B; Binetti, A G; Martín, M C; Fernández, M; Magadán, A H; Alvarez, M A

2007-02-01

Bacteriophage infections of starter lactic acid bacteria are a serious risk in the dairy industry. Phage infection can lead to slow lactic acid production or even the total failure of fermentation. The associated economic losses can be substantial. Rapid and sensitive methods are therefore required to detect and identify phages at all stages of the manufacture of fermented dairy products. This study describes a simple and rapid multiplex PCR method that, in a single reaction, detects the presence of bacteriophages infecting Streptococcus thermophilus and Lactobacillus delbrueckii, plus three genetically distinct 'species' of Lactococcus lactis phages commonly found in dairy plants (P335, 936 and c2). Available bacteriophage genome sequences were examined and the conserved regions used to design five pairs of primers, one for each of the above bacteriophage species. These primers were designed to generate specific fragments of different size depending on the species. Since this method can detect the above phages in untreated milk and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms or for use in those that involve phage-deactivating conditions.

Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor

Directory of Open Access Journals (Sweden)

Ge Y

2017-04-01

Full Text Available Yiyue Ge,1 Qiang Zhou,2 Kangchen Zhao,1 Ying Chi,1 Bin Liu,3 Xiaoyan Min,4 Zhiyang Shi,1 Bingjie Zou,2 Lunbiao Cui1 1Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health, Jiangsu Provincial Center for Disease Control and Prevention, 2Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, 3Department of Biomedical Engineering, Nanjing Medical University, 4Department of Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China Abstract: Influenza virus infections represent a worldwide public health and economic problem due to the significant morbidity and mortality caused by seasonal epidemics and pandemics. Sensitive and convenient methodologies for detection of influenza viruses are essential for further disease control. Loop-mediated isothermal amplification (LAMP is the most commonly used method of nucleic acid isothermal amplification. However, with regard to multiplex LAMP, differentiating the ladder-like LAMP products derived from multiple targets is still challenging today. The requirement of specialized instruments has further hindered the on-site application of multiplex LAMP. We have developed an integrated assay coupling multiplex reverse transcription LAMP with cascade invasive reaction using nanoparticles (mRT-LAMP-CIRN as a sensor for the detection of three subtypes of influenza viruses: A/H1N1pdm09, A/H3 and influenza B. The analytic sensitivities of the mRT-LAMP-CIRN assay were 101 copies of RNA for both A/H1N1pdm09 and A/H3, and 102 copies of RNA for influenza B. This assay demonstrated highly specific detection of target viruses and could differentiate them from other genetically or clinically related viruses. Clinical specimen analysis showed the mRT-LAMP-CIRN assay had an overall sensitivity and specificity of 98.3% and 100%, respectively. In summary, the mRT-LAMP-CIRN assay is

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.

A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

Science.gov (United States)

Saingamsook, Jassada; Saeung, Atiporn; Yanola, Jintana; Lumjuan, Nongkran; Walton, Catherine; Somboon, Pradya

2017-10-10

Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale. A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR). The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory. Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.

Directory of Open Access Journals (Sweden)

Sandrine Prost

Full Text Available The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family. Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705, Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches.

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.

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.

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....

Detection of HPV and co-infecting pathogens in healthy Italian women by multiplex real-time PCR.

Science.gov (United States)

Camporiondo, Maria Pia; Farchi, Francesca; Ciccozzi, Massimo; Denaro, Aurelia; Gallone, Domenica; Maracchioni, Fabio; Favalli, Cartesio; Ciotti, Marco

2016-01-01

Several pathogens can be transmitted sexually and are an important cause of morbidity among sexually active women. The aim of the study was to detect the presence of human papillomavirus (HPV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP) in a group of 309 healthy women enrolled at the San Camillo - Forlanini hospital of Rome by using two multiplex real-time PCR assays based on TOCE® technology. The women's ages ranged from 34 to 60 years, median 49 [IQR 45-54]. Of the 309 women tested, HPV DNA was detected in 77/309 (24.9%) patients. Of these, 44 (14.2%) harboured a single infection while 33 (10.7%) were infected by multiple genotypes. Prevalence of HPV infection was highest among females aged 40-50 years (15.2%). Of the other pathogens sought, CT, MG and NG were not detected while positive results were found for MH (12/309, 3.9%), TV (4/309, 1.3%), UP (89/309, 28.8%) and UU (14/309, 4.5%). Co-infections were as follows: 5 MH/HPV, 4 TV/HPV, 34 UP/HPV and 9 UU/HPV. In HPV-positive women, the probability of being infected by UP and UU was 2.5 (p=0.00045) and 6 fold higher (p=0.0016) than in HPV-negative women. The study supports the use of multiplex real-time PCR assays in a routine diagnostic setting. The high sensitivity and specificity of these assays along with the simultaneous detection of the most common sexually transmitted pathogens confers an advantage with respect to more obsolete methods reducing costs and time to diagnosis.

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.

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.

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.

Immunomagnetic nanoparticle based quantitative PCR for rapid detection of Salmonella

International Nuclear Information System (INIS)

Bakthavathsalam, Padmavathy; Rajendran, Vinoth Kumar; Saran, Uttara; Chatterjee, Suvro; Ali, Baquir Mohammed Jaffar

2013-01-01

We have developed a rapid and sensitive method for immunomagnetic separation (IMS) of Salmonella along with their real time detection via PCR. Silica-coated magnetic nanoparticles were functionalized with carboxy groups to which anti-Salmonella antibody raised against heat-inactivated whole cells of Salmonella were covalently attached. The immuno-captured target cells were detected in beverages like milk and lemon juice by multiplex PCR and real time PCR with a detection limit of 10 4 cfu.mL −1 and 10 3 cfu.mL −1 , respectively. We demonstrate that IMS can be used for selective concentration of target bacteria from beverages for subsequent use in PCR detection. PCR also enables differentiation of Salmonella typhi and Salmonella paratyphi A using a set of four specific primers. In addition, IMS—PCR can be used as a screening tool in the food and beverage industry for the detection of Salmonella within 3–4 h which compares favorably to the time of several days that is needed in case of conventional detection based on culture and biochemical methods. (author)

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.

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.

Multiplex quantification of 16S rDNA of predominant bacteria group within human fecal samples by polymerase chain reaction--ligase detection reaction (PCR-LDR).

Science.gov (United States)

Li, Kai; Chen, Bei; Zhou, Yuxun; Huang, Rui; Liang, Yinming; Wang, Qinxi; Xiao, Zhenxian; Xiao, Junhua

2009-03-01

A new method, based on ligase detection reaction (LDR), was developed for quantitative detection of multiplex PCR amplicons of 16S rRNA genes present in complex mixtures (specifically feces). LDR has been widely used in single nucleotide polymorphism (SNP) assay but never applied for quantification of multiplex PCR products. This method employs one pair of DNA probes, one of which is labeled with fluorescence for signal capture, complementary to the target sequence. For multiple target sequence analysis, probes were modified with different lengths of polyT at the 5' end and 3' end. Using a DNA sequencer, these ligated probes were separated and identified by size and dye color. Then, relative abundance of target DNA were normalized and quantified based on the fluorescence intensities and exterior size standards. 16S rRNA gene of three preponderant bacteria groups in human feces: Clostridium coccoides, Bacteroides and related genera, and Clostridium leptum group, were amplified and cloned into plasmid DNA so as to make standard curves. After PCR-LDR analysis, a strong linear relationship was found between the florescence intensity and the diluted plasmid DNA concentrations. Furthermore, based on this method, 100 human fecal samples were quantified for the relative abundance of the three bacterial groups. Relative abundance of C. coccoides was significantly higher in elderly people in comparison with young adults, without gender differences. Relative abundance of Bacteroides and related genera and C. leptum group were significantly higher in young and middle aged than in the elderly. Regarding the whole set of sample, C. coccoides showed the highest relative abundance, followed by decreasing groups Bacteroides and related genera, and C. leptum. These results imply that PCR-LDR can be feasible and flexible applied to large scale epidemiological studies.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

Science.gov (United States)

Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2018-05-01

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

Science.gov (United States)

Bradford, Jolene A; Buller, Gayle; Suter, Michael; Ignatius, Michael; Beechem, Joseph M

2004-10-01

Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon

A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.

Science.gov (United States)

Xu, Youchun; Yan, He; Zhang, Yan; Jiang, Kewei; Lu, Ying; Ren, Yonghong; Wang, Hui; Wang, Shan; Xing, Wanli

2015-07-07

Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing.

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

Science.gov (United States)

Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

2013-08-01

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

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.

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.

Detection and Antigenic Profiling of Undeclared Peanut in Imported Garlic Using an xMAP Multiplex Immunoassay for Food Allergens.

Science.gov (United States)

Pedersen, Ronnie O; Peters, Tim; Panda, Rakhi; Wehling, Paul; Garber, Eric A E

2017-07-01

A shipment of imported garlic powder was suspected of containing peanut. Samples (subs) collected from the shipment displayed considerable variability in peanut antigenicity when analyzed by enzyme-linked immunosorbent assay (ELISA). This raised questions regarding whether peanut was actually present, the amount present, and the basis for the variability in antigenic content. Analyses that used an xMAP multiplex assay for the detection of peanut and additional food allergens generated responses that were characteristic of peanut. Specifically, the relative intensities of two different peanut-specific antibodies coupled to beads (peanut-37 and -38) and the antigen profiles were identical to garlic controls spiked with peanut. In addition, the xMAP data did not indicate the presence of other allergens. Quantitative analyses indicated an approximately fivefold variation in peanut concentration among different subs. In contrast, within a sub, the apparent peanut concentration appeared constant. Particle size analyses of the garlic powder subs indicated a single distribution profile, with a peak at 380 μm. ELISA analysis of sieve-fractionated garlic powder from one of the subs indicated that slightly less than half of the detectable peanut was smaller than 212 μm, with the remainder almost evenly split between 212 and 300 μm and >300 μm. Modeling to predict possible oral exposure levels of peanut other than those directly measured requires additional research on the physicochemical properties of peanut and garlic, along with information on the production of the garlic powder.

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

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

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.

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.

The new challenges of multiplex networks: Measures and models

Science.gov (United States)

Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

2017-02-01

What do societies, the Internet, and the human brain have in common? They are all examples of complex relational systems, whose emerging behaviours are largely determined by the non-trivial networks of interactions among their constituents, namely individuals, computers, or neurons, rather than only by the properties of the units themselves. In the last two decades, network scientists have proposed models of increasing complexity to better understand real-world systems. Only recently we have realised that multiplexity, i.e. the coexistence of several types of interactions among the constituents of a complex system, is responsible for substantial qualitative and quantitative differences in the type and variety of behaviours that a complex system can exhibit. As a consequence, multilayer and multiplex networks have become a hot topic in complexity science. Here we provide an overview of some of the measures proposed so far to characterise the structure of multiplex networks, and a selection of models aiming at reproducing those structural properties and quantifying their statistical significance. Focusing on a subset of relevant topics, this brief review is a quite comprehensive introduction to the most basic tools for the analysis of multiplex networks observed in the real-world. The wide applicability of multiplex networks as a framework to model complex systems in different fields, from biology to social sciences, and the colloquial tone of the paper will make it an interesting read for researchers working on both theoretical and experimental analysis of networked systems.

Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction.

Science.gov (United States)

Zhang, Xiaoyan; Peng, Yanmei; Wang, Ying; Zhang, Zongying; Li, Dawei; Yu, Jialin; Han, Chenggui

2016-11-22

Brassica yellows virus (BrYV), proposed to be a new polerovirus species, three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) have been described. This study was to develop a simple, rapid, sensitive, cost-effective method for simultaneous detection and differentiation of three genotypes of BrYV. In this study, a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection and differentiation of the three genotypes of BrYV. The three genotypes of BrYV and Tunip yellows virus (TuYV) could be differentiated simultaneously using six optimized specific oligonucleotide primers, including one universal primer for detecting BrYV, three BrYV genotype-specific primers, and a pair of primers for specific detection of TuYV. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. The mRT-PCR products were 278 bp for BrYV-A, 674 bp for BrYV-B, 505 bp for BrYV-C, and 205 bp for TuYV. Amplification of three target genotypes was optimized by increasing the PCR annealing temperatures to 62 °C. One to three fragments specific for the virus genotypes were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. No specific products could be amplified from cDNAs of other viruses which could infect crucifer crops. Detection limits of the plasmids for multiplex PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively. The mRT-PCR was applied successfully for detection of three BrYV genotypes from field samples collected in China. The simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.

DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS

Directory of Open Access Journals (Sweden)

M. A. Gordukova

2015-01-01

Full Text Available Primary immunodeficiencies (PID such as severe combined immunodeficiency (SCID and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to their death or disability. Our purpose was developing of simple, inexpensive, high throughput technique based on the quantitative determination of TREC and KREC molecules by real-time PCR, and its validation in a group of children with a verified diagnosis of SCID and X-linked agammaglobulinemia.In this study, we developed and validated multiplex real-time PCR for the TREC’s and KREC’s quantitative analysis. We have shown that linear range of Ct changes depending on the concentrations of targets with a correlation coefficient R2 not worse than 0.98 was observed at concentrations from 109 to 5 × 104 copies per ml. The lowest amount of targets reliably detected in a reaction volume was 10 TREC’s copies, 5 KREC ‘s copies and 5 copies of internal control (IL17RA. We determined the age-depended reference values of TRECs and KRECs in whole blood in 29 boys and 27 girls with normal immunological parameters. The normal cut-offs for TRECs and KRECs were defined in dry blood spots depending on the method of extraction.The proposed method showed 100% diagnostic sensitivity and specificity in the studied group. The method can be proposed as a screening tool for the diagnosis of SCID and X-linked agammaglobulinemia both in whole blood and in the dry blood spots. The further investigation is required with larger number of samples. 

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.

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.

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.

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

Science.gov (United States)

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

2017-12-01

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

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

International Nuclear Information System (INIS)

Nurul Najian, A.B.; Engku Nur Syafirah, E.A.R.; Ismail, Nabilah; Mohamed, Maizan; Yean, Chan Yean

2016-01-01

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10 −1 genomic equivalent ml −1 . An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device. - Highlights: • We develop multiplex LAMP label-based lateral flow dipstick biosensor for detection of pathogenic Leptospira. • We design primers for multiplex LAMP targeting the conserved LipL32 gene of pathogenic Leptospira and LAMP internal

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

Energy Technology Data Exchange (ETDEWEB)

Nurul Najian, A.B.; Engku Nur Syafirah, E.A.R.; Ismail, Nabilah [Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Mohamed, Maizan [Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, City Campus, Pengkalan Chepa, Locked Bag 36, 16100 Kota Bharu, Kelantan (Malaysia); Yean, Chan Yean, E-mail: yeancyn@yahoo.com [Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

2016-01-15

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10{sup −1} genomic equivalent ml{sup −1}. An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device. - Highlights: • We develop multiplex LAMP label-based lateral flow dipstick biosensor for detection of pathogenic Leptospira. • We design primers for multiplex LAMP targeting the conserved LipL32 gene of pathogenic Leptospira and LAMP

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

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.

Lab-on-capillary: a rapid, simple and quantitative genetic analysis platform integrating nucleic acid extraction, amplification and detection.

Science.gov (United States)

Fu, Yu; Zhou, Xiaoming; Xing, Da

2017-12-05

In this work, we describe for the first time a genetic diagnosis platform employing a polydiallyldimethylammonium chloride (PDDA)-modified capillary and a liquid-based thermalization system for rapid, simple and quantitative DNA analysis with minimal user interaction. Positively charged PDDA is modified on the inner surface of the silicon dioxide capillary by using an electrostatic self-assembly approach that allows the negatively charged DNA to be separated from the lysate in less than 20 seconds. The capillary loaded with the PCR mix is incorporated in the thermalization system, which can achieve on-site real-time PCR. This system is based on the circulation of pre-heated liquids in the chamber, allowing for high-speed thermalization of the capillary and fast amplification. Multiple targets can be simultaneously analysed with multiplex spatial melting. Starting with live Escherichia coli (E. coli) cells in milk, as a realistic sample, the current method can achieve DNA extraction, amplification, and detection within 40 min.

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...

Targeted deposition of antibodies on a multiplex CMOS microarray and optimization of a sensitive immunoassay using electrochemical detection.

Directory of Open Access Journals (Sweden)

John Cooper

2010-03-01

Full Text Available The CombiMatrix ElectraSense microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs specifically on electrodes using complementary DNA sequences conjugated to the Abs.An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense microarray based upon targeted deposition of polypyrrole (Ppy and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different.Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.

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.

Development of a Multiplexed Microsphere PCR for Culture-Free Detection and Gram-Typing of Bacteria in Human Blood Samples.

Science.gov (United States)

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

2018-05-11

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

Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

Science.gov (United States)

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.

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.

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.

Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

DEFF Research Database (Denmark)

Llewellyn, Stacey; Inpankaew, Tawin; Nery, Susana Vaz

2016-01-01

Background Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission...... of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity...... of intestinal parasite infections, and compare them to standard microscopy. Methodology/Principal Findings Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two...

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

DEFF Research Database (Denmark)

Rebelo, Ana Rita; Bortolaia, Valeria; Kjeldgaard, Jette S.

2018-01-01

Background and aim: Plasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriace...

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

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.

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.

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

Detection of Human Bocavirus DNA by Multiplex PCR Analysis: Postmortem Case Report

Directory of Open Access Journals (Sweden)

Nihan Ziyade

2015-06-01

Full Text Available Background: Human bocavirus (HBoV is a virus belonging to the Parvoviridae family, which has been newly discovered to be associated with respiratory tract infections in children. There are many reports worldwide on the endemicity of this virus. Since it is relatively new, it is not routinely detected in clinical laboratory investigations. Case Report: We demonstrated that HBoV infection caused the death of a 5-month-old girl with a history of high fever and wheezing. Human bocavirus (HBoV 1/2/3/4 was found in a nasopharyngeal swab, paraffin-embedded lung tissue and stool samples by multiplex PCR methods using postmortem microbiological analysis. Conclusion: This case suggests that lower respiratory tract infections due to HBoV may cause severe and life-threatening diseases. Postmortem microbiology is useful in both clinical and forensic autopsies, and allows a suspected infection to be confirmed. To our knowledge, this report is the first document of a HBoV postmortem case in Turkey.

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

International Nuclear Information System (INIS)

Wang, Xuan; Mu, Zhongde; Shangguan, Fengqi; Liu, Ran; Pu, Yuepu; Yin, Lihong

2014-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-05-01

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

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise.

Science.gov (United States)

Sanchez, J J; Børsting, C; Balogh, K; Berger, B; Bogus, M; Butler, J M; Carracedo, A; Court, D Syndercombe; Dixon, L A; Filipović, B; Fondevila, M; Gill, P; Harrison, C D; Hohoff, C; Huel, R; Ludes, B; Parson, W; Parsons, T J; Petkovski, E; Phillips, C; Schmitter, H; Schneider, P M; Vallone, P M; Morling, N

2008-06-01

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.

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.

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.

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.

Typing of Y chromosome SNPs with multiplex PCR methods

DEFF Research Database (Denmark)

Sanchez Sanchez, Juan Jose; Børsting, Claus; Morling, Niels

2005-01-01

We describe a method for the simultaneous typing of Y-chromosome single nucleotide polymorphism (SNP) markers by means of multiplex polymerase chain reaction (PCR) strategies that allow the detection of 35 Y chromosome SNPs on 25 amplicons from 100 to 200 pg of chromosomal deoxyribonucleic acid...... factors for the creation of larger SNP typing PCR multiplexes include careful selection of primers for the primary amplification and the SBE reaction, use of DNA primers with homogenous composition, and balancing the primer concentrations for both the amplification and the SBE reactions....

Mode Division Multiplexing Exploring Hollow-Core Photonic Bandgap Fibers

DEFF Research Database (Denmark)

Xu, Jing; Lyngso, Jens Kristian; Leick, Lasse

2013-01-01

We review our recent exploratory investigations on mode division multiplexing using hollow-core photonic bandgap fibers (HC-PBGFs). Compared with traditional multimode fibers, HC-PBGFs have several attractive features such as ultra-low nonlinearities, low-loss transmission window around 2 µm etc....... After having discussed the potential and challenges of using HC-PBGFs as transmission fibers for mode multiplexing applications, we will report a number of recent proof-of-concept results obtained in our group using direct detection receivers. The first one is the transmission of two 10.7 Gbit/s non...

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...

Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

OpenAIRE

Parida Manmohan; Shrivastava Ambuj; Santhosh SR; Dash Paban; Saxena Parag; Rao PV

2008-01-01

Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Jap...

Molecular detection of β-lactamase and integron genes in clinical strains of Klebsiella pneumoniae by multiplex polymerase chain reaction

Directory of Open Access Journals (Sweden)

Mansour Sedighi

Full Text Available Abstract INTRODUCTION: Infections caused by β-lactamase-producing gram-negative bacteria, such as Klebsiella pneumoniae, are increasing globally with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the prevalence of antibiotic resistance genes (β-lactamase and integron genes using multiplex PCR. METHODS One-hundred K. pneumoniae isolates were collected from different clinical samples. Antibiotic susceptibility testing was performed with thirteen different antibiotics. Multiplex-PCR was used to detect β-lactamase (bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC and integron genes (int I, int II, and int III. RESULTS: The highest and lowest rate of resistance was exhibited against amikacin (93% and imipenem (8%, respectively. The frequency of β-lactamase-positive K. pneumoniae was 37%, and the prevalence of the bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC genes was 38%, 24%, 19%, 12%, 6%, 11%, 33%, 0%, 28%, and 23%, respectively. Of the 100 isolates, eight (8% were positive for class I integrons; however, class II and III integrons were not detected in any of the strains. CONCLUSIONS: These results indicate co-carriage of a number of β-lactamase genes and antibiotic resistance integrons on the same plasmids harboring multi-drug resistance genes. It seems that these properties help to decrease treatment complications due to resistant bacterial infections by rapid detection, infection-control programs and prevention of transmission of drug resistance.

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.

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.

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.

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.

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....

Evolution of cooperation under social pressure in multiplex networks.

Science.gov (United States)

Pereda, María

2016-09-01

In this work, we aim to contribute to the understanding of human prosocial behavior by studying the influence that a particular form of social pressure, "being watched," has on the evolution of cooperative behavior. We study how cooperation emerges in multiplex complex topologies by analyzing a particular bidirectionally coupled dynamics on top of a two-layer multiplex network (duplex). The coupled dynamics appears between the prisoner's dilemma game in a network and a threshold cascade model in the other. The threshold model is intended to abstract the behavior of a network of vigilant nodes that impose the pressure of being observed altering hence the temptation to defect of the dilemma. Cooperation or defection in the game also affects the state of a node of being vigilant. We analyze these processes on different duplex networks structures and assess the influence of the topology, average degree and correlated multiplexity, on the outcome of cooperation. Interestingly, we find that the social pressure of vigilance may impact cooperation positively or negatively, depending on the duplex structure, specifically the degree correlations between layers is determinant. Our results give further quantitative insights in the promotion of cooperation under social pressure.

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.

An accurate, specific, sensitive, high-throughput method based on a microsphere immunoassay for multiplex detection of three viruses and bacterial fruit blotch bacterium in cucurbits.

Science.gov (United States)

Charlermroj, Ratthaphol; Makornwattana, Manlika; Himananto, Orawan; Seepiban, Channarong; Phuengwas, Sudtida; Warin, Nuchnard; Gajanandana, Oraprapai; Karoonuthaisiri, Nitsara

2017-09-01

To employ a microsphere immunoassay (MIA) to simultaneously detect multiple plant pathogens (potyviruses, Watermelon silver mottle virus, Melon yellow spot virus, and Acidovorax avenae subsp. citrulli) in actual plant samples, several factors need to be optimized and rigorously validated. Here, a simple extraction method using a single extraction buffer was successfully selected to detect the four pathogens in various cucurbit samples (cucumber, cantaloupe, melon, and watermelon). The extraction method and assay performance were validated with inoculated and field cucurbit samples. The MIA showed 98-99% relative accuracy, 97-100% relative specificity and 92-100% relative sensitivity when compared to commercial ELISA kits and reverse transcription PCR. In addition, the MIA was also able to accurately detect multiple-infected field samples. The results demonstrate that one common extraction method for all tested cucurbit samples could be applied to detect multiple pathogens; avoiding the need for multiple protocols to be employed. This multiplex method can therefore be instrumental for high-throughput screening of multiple plant pathogens with many advantages such as a shorter assay time (2.5h) with single assay format, a lower cost of detection ($5 vs $19.7 for 4 pathogens/sample) and less labor requirement. Its multiplex capacity can also be expanded to detect up to 50 different pathogens upon the availability of specific antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Multiplexed colorimetric detection of Kaposi's sarcoma associated herpesvirus and Bartonella DNA using gold and silver nanoparticles

Science.gov (United States)

Mancuso, Matthew; Jiang, Li; Cesarman, Ethel; Erickson, David

2013-01-01

Kaposi's sarcoma (KS) is an infectious cancer occurring most commonly in human immunodeficiency virus (HIV) positive patients and in endemic regions, such as Sub-Saharan Africa, where KS is among the top four most prevalent cancers. The cause of KS is the Kaposi's sarcoma-associated herpesvirus (KSHV, also called HHV-8), an oncogenic herpesvirus that while routinely diagnosed in developed nations, provides challenges to developing world medical providers and point-of-care detection. A major challenge in the diagnosis of KS is the existence of a number of other diseases with similar clinical presentation and histopathological features, requiring the detection of KSHV in a biopsy sample. In this work we develop an answer to this challenge by creating a multiplexed one-pot detection system for KSHV DNA and DNA from a frequently confounding disease, bacillary angiomatosis. Gold and silver nanoparticle aggregation reactions are tuned for each target and a multi-color change system is developed capable of detecting both targets down to levels between 1 nM and 2 nM. The system developed here could later be integrated with microfluidic sample processing to create a final device capable of solving the two major challenges in point-of-care KS detection.

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

Science.gov (United States)

Wu, Lucia R.; Chen, Sherry X.; Wu, Yalei; Patel, Abhijit A.; Zhang, David Yu

2018-01-01

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%. PMID:29805844

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.

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.

Multiplexing Short Primers for Viral Family PCR

Energy Technology Data Exchange (ETDEWEB)

Gardner, S N; Hiddessen, A L; Hara, C A; Williams, P L; Wagner, M; Colston, B W

2008-06-26

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets for large, diverse, and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ({approx}3700 18-mers or {approx}2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus, hemagglutinin and neuraminidase segments of influenza A virus, Norwalk virus, and HIV-1.

Gold nanoparticle immunochromatographic assay for quantitative detection of urinary RBP

Directory of Open Access Journals (Sweden)

XU Kuan

2013-04-01

Full Text Available A rapid quantitative detection of urinary RBP was established by using nano-gold immunochromatography (sandwich method and trisodium citrate reduction method and a rapid immunochromatographic test strip was developed. Theimmunochromatographic test strip can quantitatively detect RBP within 15 minutes. The detection limit was 150ng/mL and detection range was from 150 to 5000 ng/mL. There were no cross-reactions with others kidney disease markers,such as urinary albumin (ALB,transferrin protein (TRF,β2-microglobulin (β2-MG,urinary fiber connecting protein (FN,and lysozyme (LZM. The results indicate that it is a quick and simple method with strong specificity,high sensitivity,and wide detection range. The rapid detection method will have extensive clinical applications in the early diagnosis of proximal tubular damage,kidney disease,diabetic nephropathy,and process monitoring.

A Novel Method for Detection of Phosphorylation in Single Cells by Surface Enhanced Raman Scattering (SERS) using Composite Organic-Inorganic Nanoparticles (COINs)

OpenAIRE

Shachaf, Catherine M.; Elchuri, Sailaja V.; Koh, Ai Leen; Zhu, Jing; Nguyen, Lienchi N.; Mitchell, Dennis J.; Zhang, Jingwu; Swartz, Kenneth B.; Sun, Lei; Chan, Selena; Sinclair, Robert; Nolan, Garry P.

2009-01-01

Background Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. Methodology/Principal Findings To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using ?Composite Organic-Inorganic Nanoparticles? (COINs) Raman nanoparticles. COINs are Surface-Enhan...

Quantitative detection of DNA by autocatalytic enlargement of hybridized gold nanoprobes

DEFF Research Database (Denmark)

Zhan, Zongrui; Cao, Cuong; Sim, Sang Jun

2010-01-01

Quantitative detection of specific viral DNA has become a pressing issue for the earlier clinical diagnosis of viral infectious diseases. Therefore, in this paper, we report a simple, sensitive, and inexpensive quantitative approach for DNA detection based on the autocatalytic Au deposition of go...... to the concentration of the target DNA, could easily be confirmed by a UV–vis scanning spectrophotometer. Limit of detection could be obtained as low as 1.0 fM by this simple method....

Role of multiplex polymerase chain reaction in diagnosing tubercular meningitis

Directory of Open Access Journals (Sweden)

Anupam Berwal

2017-01-01

Full Text Available Tuberculous meningitis (TBM is one of the most serious manifestations of extrapulmonary tuberculosis. Timely and accurate diagnosis provides a favorable prognosis in patients with TBM. The study evaluated the use of multiplex polymerase chain reaction (PCR in the diagnosis of TBM. A study was conducted on 74 patients clinically suspected with TBM. The cerebrospinal fluid (CSF specimens were processed for smear microscopy, middle brook 7H9 culture, and multiplex PCR using primers directed against IS6110 gene and 38 kD protein for detection of Mycobacterium tuberculosis. The results were analyzed to assess the role of multiplex PCR in the diagnosis of TBM. A total of 26 (35.1% patients were diagnosed with TBM. Microscopy was negative in all while culture was positive in two cases only. Comparing with clinical diagnosis and CSF adenosine deaminase levels of ≥10 U/L, multiplex PCR showed sensitivity, specificity, positive predictive value, and negative predictive value of 71.4%, 89.6%, 83.3%, and 81.2%, respectively, in the diagnosis of TBM.

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.

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

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

Development of a chip-based multiplexed immunoassay using liposomal nanovesicles and its application in the detection of pathogens causing female lower genital tract infections

Directory of Open Access Journals (Sweden)

Wen-Hsiang Su

2013-03-01

Conclusion: This microarray chip was a rapid, easy, inexpensive and sensitive tool for detecting female lower genital tract Candida infection in a one-time vaginal sampling process, although the data on the four other pathogens were still unavailable. A larger population study is encouraged to test the validity of this multiplexed immunoassay chip.

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise

DEFF Research Database (Denmark)

Sanchez, Juan Jose; Børsting, C; Balogh, K

2008-01-01

base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA...

Videodensitometric quantitative angiography after coronary balloon angioplasty, compared to edge-detection quantitative angiography and intracoronary ultrasound imaging

NARCIS (Netherlands)

Peters, R. J.; Kok, W. E.; Pasterkamp, G.; von Birgelen, C.; Prins, M. [=Martin H.; Serruys, P. W.

2000-01-01

AIMS: To assess the value of videodensitometric quantification of the coronary lumen after angioplasty by comparison to two other techniques of coronary artery lumen quantification. METHODS AND RESULTS: Videodensitometric quantitative angiography, edge detection quantitative angiography and 30 MHz

Comparison and transfer testing of multiplex ligation detection methods for GM plants

Directory of Open Access Journals (Sweden)

Ujhelyi Gabriella

2012-01-01

Full Text Available Abstract Background With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i hybridisation and ligation of specific probes, ii amplification of the ligated probes and iii detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study. Results Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands. Conclusions From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the

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

Directory of Open Access Journals (Sweden)

Dehghan fatemeh

2014-05-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1994-09-01

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

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.

Multiplexed coding in the human basal ganglia

Science.gov (United States)

Andres, D. S.; Cerquetti, D.; Merello, M.

2016-04-01

A classic controversy in neuroscience is whether information carried by spike trains is encoded by a time averaged measure (e.g. a rate code), or by complex time patterns (i.e. a time code). Here we apply a tool to quantitatively analyze the neural code. We make use of an algorithm based on the calculation of the temporal structure function, which permits to distinguish what scales of a signal are dominated by a complex temporal organization or a randomly generated process. In terms of the neural code, this kind of analysis makes it possible to detect temporal scales at which a time patterns coding scheme or alternatively a rate code are present. Additionally, finding the temporal scale at which the correlation between interspike intervals fades, the length of the basic information unit of the code can be established, and hence the word length of the code can be found. We apply this algorithm to neuronal recordings obtained from the Globus Pallidus pars interna from a human patient with Parkinson’s disease, and show that a time pattern coding and a rate coding scheme co-exist at different temporal scales, offering a new example of multiplexed neuronal coding.

Distance-based microfluidic quantitative detection methods for point-of-care testing.

Science.gov (United States)

Tian, Tian; Li, Jiuxing; Song, Yanling; Zhou, Leiji; Zhu, Zhi; Yang, Chaoyong James

2016-04-07

Equipment-free devices with quantitative readout are of great significance to point-of-care testing (POCT), which provides real-time readout to users and is especially important in low-resource settings. Among various equipment-free approaches, distance-based visual quantitative detection methods rely on reading the visual signal length for corresponding target concentrations, thus eliminating the need for sophisticated instruments. The distance-based methods are low-cost, user-friendly and can be integrated into portable analytical devices. Moreover, such methods enable quantitative detection of various targets by the naked eye. In this review, we first introduce the concept and history of distance-based visual quantitative detection methods. Then, we summarize the main methods for translation of molecular signals to distance-based readout and discuss different microfluidic platforms (glass, PDMS, paper and thread) in terms of applications in biomedical diagnostics, food safety monitoring, and environmental analysis. Finally, the potential and future perspectives are discussed.

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.

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...

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

Simultaneous measurements of auto-immune and infectious disease specific antibodies using a high throughput multiplexing tool.

Directory of Open Access Journals (Sweden)

Atul Asati

Full Text Available Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders.

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

DEFF Research Database (Denmark)

Adriko, John; Aritua, V.; Mortensen, Carmen Nieves

2012-01-01

The present study developed a pathovar-specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265-bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment......-specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants...

Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

Science.gov (United States)

Noorani, Md Salik; Awasthi, Prachi; Sharma, Maheshwar Prasad; Ram, Raja; Zaidi, Aijaz Asgar; Hallan, Vipin

2013-10-01

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR. Copyright © 2013 Elsevier B.V. All rights reserved.

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 ...

A universal aptameric biosensor: Multiplexed detection of small analytes via aggregated perylene-based broad-spectrum quencher.

Science.gov (United States)

Hu, Rong; Zhang, Xi; Xu, Qiang; Lu, Dan-Qing; Yang, Yun-Hui; Xu, Quan-Qing; Ruan, Qiong; Mo, Liu-Ting; Zhang, Xiao-Bing

2017-06-15

A universal aptameric system based on the taking advantage of double-stranded DNA/perylene diimide (dsDNA/PDI) as the signal probe was developed for multiplexed detection of small molecules. Aptamers are single-stranded DNA or RNA oligonucleotides which are selected in vitro by a process known as systematic evolution of ligands by exponential enrichment. In this work, we synthesized a new kind of PDI and reported this aggregated PDI could quench the double-stranded DNA (dsDNA)-labeled fluorophores with a high quenching efficiency. The quenching efficiencies on the fluorescence of FAM, TAMRA and Cy5 could reach to 98.3%±0.9%, 97.2%±0.6% and 98.1%±1.1%, respectively. This broad-spectrum quencher was then adopted to construct a multicolor biosensor via a label-free approach. A structure-switching-triggered enzymatic recycling amplification was employed for signal amplification. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity towards small analytes. For other targets, changing the corresponding aptamer can achieve the goal. The quencher did not interfere with the catalytic activity of nuclease. The biosensor could be manipulated with similar sensitivity no matter in pre-addition or post-addition manner. Moreover, simultaneous and multiplexed analysis of several small molecules in homogeneous solution was achieved, demonstrating its potential application in the rapid screening of multiple biotargets. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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....

Comparative evaluation of uniplex, nested, semi-nested, multiplex and nested multiplex PCR methods in the identification of microbial etiology of clinically suspected infectious endophthalmitis.

Science.gov (United States)

Bharathi, Madasamy Jayahar; Murugan, Nandagopal; Rameshkumar, Gunasekaran; Ramakrishnan, Rengappa; Venugopal Reddy, Yerahaia Chinna; Shivkumar, Chandrasekar; Ramesh, Srinivasan

2013-05-01

This study is aimed to determine the utility of various polymerase chain reaction (PCR) methods in vitreous fluids (VFs) for detecting the infectious genomes in the diagnosis of infectious endophthalmitis in terms of sensitivity and specificity. This prospective and consecutive analysis included a total of 66 VFs that were submitted for the microbiological evaluation, which were obtained from 66 clinically diagnosed endophthalmitis patients presented between November 2010 and October 2011 at the tertiary eye care referral centre in South India. Part of the collected VFs were subjected to cultures and smears, and the remaining parts were utilized for five PCR methods: uniplex, nested, semi-nested, multiplex and nested multiplex after extracting DNA, using universal eubacterial and Propionibacterium acnes species-specific primer sets targeting 16S rRNA gene in all bacteria and P. acnes, and panfungal primers, targeting 28S rRNA gene in all fungi. Of the 66 VFs, five (7.5%) showed positive results in smears, 16 (24%) in cultures and 43 (65%) showed positive results in PCRs. Among the 43 positively amplified VFs, 10 (15%) were positive for P. acnes genome, one for panfungal genome and 42 (62%) for eubacterial genome (including 10 P. acnes positives). Among 42 eubacterial-positive VFs, 36 were positive by both uniplex (first round) and multiplex (first round) PCRs, while nested (second round) and nested multiplex (second round) PCRs produced positive results in 42 and 41 VFs, respectively. Of the 43 PCR-positive specimens, 16 (37%) had positive growth (15 bacterial and one fungal) in culture. Of 50 culture-negative specimens, 27 (54%) were showed positive amplification, of which 10 were amplified for both P. acnes and eubacterial genomes and the remaining 17 were for eubacterial genome alone. Nested PCRs are superior than uniplex and multiplex PCR. PCRs proved to be a powerful tool in the diagnosis of endophthalmitis, especially for detecting uncultured microbes.

Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

Science.gov (United States)

Raghav, Sunil Kumar; Deplancke, Bart

2012-01-01

Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction

Directory of Open Access Journals (Sweden)

Natália Malaguti

2015-01-01

Full Text Available Bacterial vaginosis (BV is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs related to symptomatic BV: Gardnerella vaginalis, Mobiluncus curtisii, Mobiluncus mulieris, Bacteroides fragilis, Mycoplasma hominis, Atopobium vaginae, Ureaplasma urealyticum, Megasphaera type I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs 1, 2, and 3, Sneathia sanguinegens, and Mycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%, and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future.

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

[Combined G-banded karyotyping and multiplex ligation-dependent probe amplification for the detection of chromosomal abnormalities in fetuses with congenital heart defects].

Science.gov (United States)

Liu, Yang; Xie, Jiansheng; Geng, Qian; Xu, Zhiyong; Wu, Weiqin; Luo, Fuwei; Li, Suli; Wang, Qin; Chen, Wubin; Tan, Hongxi; Zhang, Hu

2017-02-10

To assess the value of G-banded karyotyping in combination with multiplex ligation-dependent probe amplification (MLPA) as a tool for the detection of chromosomal abnormalities in fetuses with congenital heart defects. The combined method was used to analyze 104 fetuses with heart malformations identified by ultrasonography. Abnormal findings were confirmed with chromosomal microarray analysis (CMA). Nineteen (18%) fetuses were found to harbor chromosomal aberrations by G-banded karyotyping and MLPA. For 93 cases, CMA has detected abnormalities in 14 cases including 10 pathogenic copy number variations (CNVs) and 4 CNVs of uncertain significance (VOUS). MLPA was able to detect all of the pathogenic CNVs and 1 VOUS CNV. Combined use of G-banded karyotyping and MLPA is a rapid, low-cost and effective method to detect chromosomal abnormalities in fetuses with various heart malformations.

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

A novel multiplex bead-based platform highlights the diversity of extracellular vesicles.

Science.gov (United States)

Koliha, Nina; Wiencek, Yvonne; Heider, Ute; Jüngst, Christian; Kladt, Nikolay; Krauthäuser, Susanne; Johnston, Ian C D; Bosio, Andreas; Schauss, Astrid; Wild, Stefan

2016-01-01

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell-derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.

Comparative study of colloidal gold and quantum dots as labels for multiplex screening tests for multi-mycotoxin detection

Energy Technology Data Exchange (ETDEWEB)

Foubert, Astrid, E-mail: astrid.foubert@hotmail.com; Beloglazova, Natalia V.; De Saeger, Sarah

2017-02-22

Quantum dots (QDs) and colloidal gold nanoparticles (CG) were evaluated as labels for multiplex lateral flow immunoassay (LFIA) for determination of mycotoxins deoxynivalenol (DON), zearalenone (ZEN) and T2/HT2-toxin (T2/HT2) in cereal matrices. Both developed assays were based on the same immunoreagents (except for the labels), therefore their analytical characteristics could be objectively compared. For both LFIAs antigens (DON-ovalbumin (OVA), ZEN-OVA and T2-OVA) and rabbit anti-mouse immunoglobulin were immobilized on a nitrocellulose membrane as three test lines and one control line, respectively. Depending on the LFIA, monoclonal antibodies (mAb) against DON, ZEN and T2 were conjugated with CdSeS/ZnS QDs or CG. T2 and HT2 were detected by one test line (T2-OVA) with an anti-T2 mAb which showed 110% cross-reactivity with HT2. Both tests were developed in accordance with the legal limits and were developed in such a way that they had the same cut-off limits of 1000 μg kg{sup −1}, 80 μg kg{sup −1} and 80 μg kg{sup −1} for DON, ZEN and T2/HT2, respectively in order to allow a correct comparison. Applicability of these assays was demonstrated by analysis of naturally contaminated wheat samples. The results demonstrate that both the LFIAs can be used as rapid, cost-effective and convenient qualitative tool for on-site screening for simultaneous detection of DON, ZEN and HT2/T2 in wheat without special instrumentation. However, the QD-based LFIA consumed less immunoreagents and was more sensitive and economically beneficial. In addition, the results were easier to interpret, resulting in a lower false negative rate (<5%) which was in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes. - Highlights: • Development of colloidal gold- and quantum dot-based multiplex lateral flow immunoassay. • Lateral flow immunoassays allow simultaneous detection of four mycotoxins. �

Nanoparticle discrimination based on wavelength and lifetime-multiplexed cathodoluminescence microscopy

NARCIS (Netherlands)

Garming, Mathijs W H; Weppelman, I Gerward C; de Boer, Pascal; Martínez, Felipe Perona; Schirhagl, Romana; Hoogenboom, Jacob P; Moerland, Robert J

2017-01-01

Nanomaterials can be identified in high-resolution electron microscopy images using spectrally-selective cathodoluminescence. Capabilities for multiplex detection can however be limited, e.g., due to spectral overlap or availability of filters. Also, the available photon flux may be limited due to

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.

Towards Multiplex Molecular Diagnosis—A Review of Microfluidic Genomics Technologies

Directory of Open Access Journals (Sweden)

Ismail Hussain Kamal Basha

2017-08-01

Full Text Available Highly sensitive and specific pathogen diagnosis is essential for correct and timely treatment of infectious diseases, especially virulent strains, in people. Point-of-care pathogen diagnosis can be a tremendous help in managing disease outbreaks as well as in routine healthcare settings. Infectious pathogens can be identified with high specificity using molecular methods. A plethora of microfluidic innovations in recent years have now made it increasingly feasible to develop portable, robust, accurate, and sensitive genomic diagnostic devices for deployment at the point of care. However, improving processing time, multiplexed detection, sensitivity and limit of detection, specificity, and ease of deployment in resource-limited settings are ongoing challenges. This review outlines recent techniques in microfluidic genomic diagnosis and devices with a focus on integrating them into a lab on a chip that will lead towards the development of multiplexed point-of-care devices of high sensitivity and specificity.

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

Development of a multiplex polymerase chain reaction protocol for the simultaneous detection of Salmonella enterica serovar Typhi and Class 1 integron

Directory of Open Access Journals (Sweden)

Juthika Mandal

2014-09-01

Full Text Available Objective: To develop a multiplex polymerase chain reaction (PCR protocol for the simultaneous detection of Salmonella enterica serovar Typhi (S. Typhi and Class 1 integron, so as to aid rapid diagnosis of S. Typhi cases and help in the selection of treatment options based on the presence of the Class 1 integron that can carry resistance cassettes to a range of antibiotics. Methods: PCR for amplification of specific regions was done using fliC-d and intl primers and agarose gel electrophoresis was used for resolution of PCR products. Results: The fliC-d primer (S. Typhi specific amplified a 587 bp region and the intl primer (Class 1 integron specific amplified two bands approximately 500 and 550 bps. The developed method was specific for S. Typhi and did not amplify any products with Salmonella enterica serovar Typhimurium ATCC 14028, Salmonella enterica serovar Paratyphi and Escherichia coli O157:H7. Conclusions: The developed multiplex PCR protocol can be used for rapid diagnosis and aid in proper treatment strategies for patients infected with S. Typhi.

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.

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.

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

Development and use of tuf gene-based primers for the multiplex PCR detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum in commercial dairy products.

Science.gov (United States)

Sheu, Sen-Je; Hwang, Wen-zhe; Chen, Hsin-Chih; Chiang, Yu-Cheng; Tsen, Hau-Yang

2009-01-01

PCR primers specific for the detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum were designed based on the elongation factor Tu gene (tuf). The specificity of these four primer sets were confirmed by PCR with 88 bacterial strains of Lactobacillus, Enterococcus, Bifidobacterium, and other bacterial species. Results indicated that these primer sets generated predicted PCR products of 397, 230, 202, and 161 bp for L. acidophilus, L. delbrueckii, L. casei group, and B. longum, respectively. Bacterial species other than the target organisms tested did not generate false-positive results. When these four primer sets were combined for the simultaneous detection of the lactic acid bacteria (LAB) in fermented milk products including yogurt, the LAB species listed on the labels of these products could be identified without the preenrichment step. The identification limit for each LAB strain with this multiplex PCR method was N X 10(3) CFU/ml in milk samples. The results of our multiplex PCR method were confirmed by PCR assay using primers based on the 16S rDNA or the 16S-23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. When this multiplex PCR method was used with the determination of counts of total viable LAB and bifidobacteria, the quality of commercial fermented milk products could be assured.

Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

Science.gov (United States)

Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S; Overall, Christopher M

2010-05-01

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses

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)

A lab-on-a-chip-based multiplex platform to detect potential fraud of introducing pig, dog, cat, rat and monkey meat into the food chain.

Science.gov (United States)

Razzak, Md Abdur; Hamid, Sharifah Bee Abd; Ali, Md Eaqub

2015-01-01

Food forgery has posed considerable risk to public health, religious rituals, personal budget and wildlife. Pig, dog, cat, rat and monkey meat are restricted in most religions, but their sporadic adulteration are rampant. Market controllers need a low-cost but reliable technique to track and trace suspected species in the food chain. Considering the need, here we documented a lab-on-a-chip-based multiplex polymerase chain reaction (PCR) assay for the authentication of five non-halal meat species in foods. Using species-specific primers, 172, 163, 141, 129 and 108-bp sites of mitochondrial ND5, ATPase 6 and cytochrome b genes were amplified to detect cat, dog, pig, monkey and rat species under complex matrices. Species-specificity was authenticated against 20 different species with the potential to be used in food. The targets were stable under extreme sterilisation (121°C at 45 psi for 2.5 h) which severely degrades DNA. The assay was optimised under the backgrounds of various commercial meat products and validated for the analysis of meatballs, burgers and frankfurters, which are popular fast food items across the globe. The assay was tested to detect 0.1% suspected meats under commercial backgrounds of marketed foods. Instead of simplex PCR which detects only one species at a time, such a multiplex platform can reduce cost by at least fivefolds by detecting five different species in a single assay platform.

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

Science.gov (United States)

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

2017-01-01

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

[Detection of recombinant-DNA in foods from stacked genetically modified plants].

Science.gov (United States)

Sorokina, E Iu; Chernyshova, O N

2012-01-01

A quantitative real-time multiplex polymerase chain reaction method was applied to the detection and quantification of MON863 and MON810 in stacked genetically modified maize MON 810xMON 863. The limit of detection was approximately 0,1%. The accuracy of the quantification, measured as bias from the accepted value and the relative repeatability standard deviation, which measures the intra-laboratory variability, were within 25% at each GM-level. A method verification has demonstrated that the MON 863 and the MON810 methods can be equally applied in quantification of the respective events in stacked MON810xMON 863.

ICP-MS analysis of lanthanide-doped nanoparticles: A quantitative and multiplexing approach to investigate biodistribution, blood clearance, and targeting

Science.gov (United States)

Crayton, Samuel

The rapidly progressing field of nanotechnology promises to revolutionize healthcare in the 21st century, with applications in the prevention, diagnosis, and treatment of a wide range of diseases. However, before nanoparticulate agents can be brought into clinical use, they must first be developed, optimized, and evaluated in animal models. In the typical pre-clinical paradigm, almost all of the optimization is done at the in vitro level, with only a few select agents reaching the level of animal studies. Since only one experimental nanoparticle formulation can be investigated in a single animal, and in vivo experiments have relatively higher complexity, cost, and time requirements, it is not feasible to evaluate a very large number of agents at the in vivo stage. A major drawback of this approach, however, is that in vitro assays do not always accurately predict how a nanoparticle will perform in animal studies. Therefore, a method that allows many agents to be evaluated in a single animal subject would allow for much more efficient and predictive optimization of nanoparticles. We have found that by incorporating lanthanide tracer metals into nanoparticle formulations, we are successfully able to use inductively coupled plasma mass spectrometry (ICP-MS) to quantitatively determine a nanoparticle's blood clearance kinetics, biodistribution, and tumor delivery. This approach was applied to evaluate both passive and active tumor targeting, as well as metabolically directed targeting of nanoparticles to low pH tumor microenvironments. Importantly, we found that these in vivo measurements could be made for many nanoparticle formulations simultaneously, in single animals, due to the high-order multiplexing capability of mass spectrometry. This approach allowed for efficient and reproducible comparison of performance between different nanoparticle formulations, by eliminating the effects of subject-to-subject variability. In the future, we envision that this "higher

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

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

Development of a multiplex real-time PCR for the simultaneous detection of herpes simplex and varicella zoster viruses in cerebrospinal fluid and lesion swab specimens.

Science.gov (United States)

Wong, Anita A; Pabbaraju, Kanti; Wong, Sallene; Tellier, Raymond

2016-03-01

Herpes simplex viruses (HSV) and varicella zoster virus (VZV) can have very similar and wide-ranging clinical presentations. Rapid identification is necessary for timely antiviral therapy, especially with infections involving the central nervous system, neonates, and immunocompromised individuals. Detection of HSV-1, HSV-2 and VZV was combined into one real-time PCR reaction utilizing hydrolysis probes. The assay was validated on the LightCycler(®) (Roche) and Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific Inc.) to detect alphaherpesviruses in cerebral spinal fluid (CSF) and lesion swab specimens, respectively. Validation data on blood and tissue samples are also presented. The multiplex assay showed excellent sensitivity, specificity and reproducibility when compared to two singleplex real-time PCR assays for CSF samples and direct fluorescent antigen/culture for lesion swab samples. Implementation of the multiplex assay has facilitated improved sensitivity and accuracy as well as reduced turn-around-times and costs. The results from a large data set of 16,622 prospective samples tested between August 16, 2012 to February 1, 2014 at the Provincial Laboratory for Public Health (Alberta, Canada) are presented here. Copyright © 2015 Elsevier B.V. All rights reserved.

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)....

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.

Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR

Directory of Open Access Journals (Sweden)

Rodolakis Annie

2009-07-01

Full Text Available Abstract Background Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus and Coxiella burnetii (C. burnetii. Chlamydophila pecorum (Cp. pecorum is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals. Results Specific PCR primers were designed and a sensitive and specific m-PCR was developed to detect simultaneously, in one tube reaction, three specific fragments of 821, 526 and 687-bp long for Cp. abortus, Cp. pecorum and C. burnetii respectively. This m-PCR assay was performed on 253 clinical samples taken from infected ruminant's flocks that have showed problems of abortion diseases. Thus, 67 samples were infected by either one of the three pathogens: 16 (13 vaginal swabs and 3 placentas were positive for Cp. abortus, 2 were positive for Cp. pecorum (1 vaginal swab and 1 placenta and 49 samples (33 vaginal swabs, 11 raw milks, 4 faeces and 1 placenta were positive for C. burnetii. Two vaginal swabs were m-PCR positive of both Cp. abortus and C. burnetii and none of the tested samples was shown to be infected simultaneously with the three pathogens. Conclusion We have successfully developed a rapid multiplex PCR that can detect and differentiate Cp. abortus, Cp. pecorum and C. burnetii; with a good sensitivity and specificity. The diagnosis of chlamydiosis and Q fever may be greatly simplified and performed at low cost. In addition, the improvement in diagnostic techniques will enhance our knowledge regarding the prevalence and

Synthetic internal control sequences to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

Science.gov (United States)

Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi, the soybean rust pathogen. Because of the extreme sensitivity of Q-PCR, the DNA of a single u...

A Dual Filtration-Based Multiplex PCR Method for Simultaneous Detection of Viable Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on Fresh-Cut Cantaloupe.

Directory of Open Access Journals (Sweden)

Ke Feng

Full Text Available Fresh-cut cantaloupe is particularly susceptible to contamination with pathogenic bacteria, such as Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus. Therefore, development of rapid, yet accurate detection techniques is necessary to ensure food safety. In this study, a multiplex PCR system and propidium monoazide (PMA concentration were optimized to detect all viable pathogens in a single tube. A dual filtration system utilized a filtration membrane with different pore sizes to enrich pathogens found on fresh-cut cantaloupe. The results revealed that an optimized multiplex PCR system has the ability to effectively detect three pathogens in the same tube. The viable pathogens were simultaneously detected for PMA concentrations above 10 μg/ml. The combination of a nylon membrane (15 μm and a micro pore filtration membrane (0.22 μm formed the dual filtration system used to enrich pathogens. The achieved sensitivity of PMA-mPCR based on this dual filtration system was 2.6 × 103 cfu/g for L. monocytogenes, 4.3 × 10 cfu/g for E. coli O157:H7, and 3.1 × 102 cfu/g for S. aureus. Fresh-cut cantaloupe was inoculated with the three target pathogens using concentrations of 103, 102, 10, and 1 cfu/g. After 6-h of enrichment culture, assay sensitivity increased to 1 cfu/g for each of these pathogens. Thus, this technique represents an efficient and rapid detection tool for implementation on fresh-cut cantaloupe.

Development of touch down-multiplex PCR for the diagnosis of toxoplasmosis

Directory of Open Access Journals (Sweden)

V Hallur

2015-01-01

Full Text Available Purpose: The diagnosis of toxoplasmosis is challenging since conventional methods like culture and immunofluorescence are not universally available. Serology, which is used regularly might be negative during early phase of infection and in immunosuppressed patients or may remain positive for a long time. Several molecular tests have been used for the diagnosis of toxoplasmosis, but none of them have an internal control which would inform us regarding the presence of polymerase chain reaction (PCR inhibitors thus, undermining the confidence of a laboratory physician. Materials and Methods: We designed a multiplex PCR containing primers targeting human beta globin gene which would act as internal control and two primers against the B1 gene and 5s gene which aid in sensitive detection of T. gondii. Results: Multiplex PCR had a sensitivity of 83.3% and specificity of 100%. Conclusion: Multiplex PCR may provide a sensitive and specific tool for diagnosis of human toxoplasmosis.

Reliability and short-term intra-individual variability of telomere length measurement using monochrome multiplexing quantitative PCR.

Directory of Open Access Journals (Sweden)

Sangmi Kim

Full Text Available Studies examining the association between telomere length and cancer risk have often relied on measurement of telomere length from a single blood draw using a real-time PCR technique. We examined the reliability of telomere length measurement using sequential samples collected over a 9-month period.Relative telomere length in peripheral blood was estimated using a single tube monochrome multiplex quantitative PCR assay in blood DNA samples from 27 non-pregnant adult women (aged 35 to 74 years collected in 7 visits over a 9-month period. A linear mixed model was used to estimate the components of variance for telomere length measurements attributed to variation among women and variation between time points within women. Mean telomere length measurement at any single visit was not significantly different from the average of 7 visits. Plates had a significant systematic influence on telomere length measurements, although measurements between different plates were highly correlated. After controlling for plate effects, 64% of the remaining variance was estimated to be accounted for by variance due to subject. Variance explained by time of visit within a subject was minor, contributing 5% of the remaining variance.Our data demonstrate good short-term reliability of telomere length measurement using blood from a single draw. However, the existence of technical variability, particularly plate effects, reinforces the need for technical replicates and balancing of case and control samples across plates.

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...

Performance of Multiplexed XY Resistive Micromegas detectors in a high intensity beam

Science.gov (United States)

Banerjee, D.; Burtsev, V.; Chumakov, A.; Cooke, D.; Depero, E.; Dermenev, A. V.; Donskov, S. V.; Dubinin, F.; Dusaev, R. R.; Emmenegger, S.; Fabich, A.; Frolov, V. N.; Gardikiotis, A.; Gninenko, S. N.; Hösgen, M.; Karneyeu, A. E.; Ketzer, B.; Kirsanov, M. M.; Konorov, I. V.; Kramarenko, V. A.; Kuleshov, S. V.; Levchenko, E.; Lyubovitskij, V. E.; Lysan, V.; Mamon, S.; Matveev, V. A.; Mikhailov, Yu. V.; Myalkovskiy, V. V.; Peshekhonov, V. D.; Peshekhonov, D. V.; Polyakov, V. A.; Radics, B.; Rubbia, A.; Samoylenko, V. D.; Tikhomirov, V. O.; Tlisov, D. A.; Toropin, A. N.; Vasilishin, B.; Arenas, G. Vasquez; Ulloa, P.; Crivelli, P.

2018-02-01

We present the performance of multiplexed XY resistive Micromegas detectors tested in the CERN SPS 100 GeV/c electron beam at intensities up to 3 . 3 × 105e- /(s ṡcm2) . So far, all studies with multiplexed Micromegas have only been reported for tests with radioactive sources and cosmic rays. The use of multiplexed modules in high intensity environments was not explored due to the effect of ambiguities in the reconstruction of the hit point caused by the multiplexing feature. For the specific mapping and beam intensities analyzed in this work with a multiplexing factor of five, more than 50% level of ambiguity is introduced due to particle pile-up as well as fake clusters due to the mapping feature. Our results prove that by using the additional information of cluster size and integrated charge from the signal clusters induced on the XY strips, the ambiguities can be reduced to a level below 2%. The tested detectors are used in the CERN NA64 experiment for tracking the incoming particles bending in a magnetic field in order to reconstruct their momentum. The average hit detection efficiency of each module was found to be ∼96% at the highest beam intensities. By using four modules a tracking resolution of 1.1% was obtained with ∼85% combined tracking efficiency.

Explaining HIV Risk Multiplexity: A Social Network Analysis.

Science.gov (United States)

Felsher, Marisa; Koku, Emmanuel

2018-04-21

Risk multiplexity (i.e., overlap in drug-use, needle exchange and sexual relations) is a known risk factor for HIV. However, little is known about predictors of multiplexity. This study uses egocentric data from the Colorado Springs study to examine how individual, behavioral and social network factors influence engagement in multiplex risk behavior. Analyses revealed that compared to Whites, Hispanics were significantly more likely to engage in risk multiplexity and Blacks less so. Respondents who were similar to each other (e.g., in terms of race) had significantly higher odds of being in risk multiplex relationships, and respondents' risk perceptions and network size were significantly associated with engaging in multiplex risk behaviors. Findings from interaction analysis showed the effect of knowing someone with HIV on the odds of multiplexity depends partly on whether respondents' know their HIV status. Findings suggest that demographics, HIV behaviors and network factors impact engagement in multiplex risk behaviors, highlighting the need for multi-level interventions aimed at reducing HIV risk behavior.

Authentication of Meat Species in Sucuk by Multiplex PCR

Directory of Open Access Journals (Sweden)

Osman İrfan İLHAK

2015-01-01

Full Text Available The identification of meat species used in meat products is important by reason of economic considerations, religious factors, verification of label, and prevention of unfair-market competition. In this paper, multiplex PCR method was experienced for routine detection of equine (horse and donkey, poultry (chicken and turkey, pig and cattle meat in sucuk (sausage. The primers used for these animals generated specific fragments, and they did not show cross reactions with the DNA from the other genus of animal. After multiplex PCR was successfully optimized, a field study was carried out to investigate the presence of horse, donkey, chicken, turkey and pig meat in 50 sucuks (30 beef and 20 beef + poultry collected from markets. The result of the field study indicated that 23.3% of 30 beef sucuk samples were containing poultry meat. None of the 50 sucuk samples was containing pig meat, but one (2% of the samples generated equine fragment. The present study showed that the multiplex PCR method can be used for routine analysis of meat species identification, verification and control of label information of meat products.

A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay.

Directory of Open Access Journals (Sweden)

Mark Jesus M Magbanua

Full Text Available Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19 and lung cancer patients (n = 21, and healthy controls (n = 30 using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs, circulating mesenchymal cells (CMCs, putative circulating stem cells (CSCs, and circulating endothelial cells (CECs. Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85% and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001. Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03. Fifty-three percent (53% of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001. In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch® vs. our microfluidic filter method revealed moderate correlation (R2 = 0.46, kappa = 0.47. Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.

A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay.

Science.gov (United States)

Magbanua, Mark Jesus M; Pugia, Michael; Lee, Jin Sun; Jabon, Marc; Wang, Victoria; Gubens, Matthew; Marfurt, Karen; Pence, Julia; Sidhu, Harwinder; Uzgiris, Arejas; Rugo, Hope S; Park, John W

2015-01-01

Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19) and lung cancer patients (n = 21), and healthy controls (n = 30) using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs), circulating mesenchymal cells (CMCs), putative circulating stem cells (CSCs), and circulating endothelial cells (CECs). Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85%) and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001). Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03). Fifty-three percent (53%) of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001). In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch® vs. our microfluidic filter method revealed moderate correlation (R2 = 0.46, kappa = 0.47). Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.

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.

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.

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples

Science.gov (United States)

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal and polyclonal anti...

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.

Pilot-multiplexed continuous-variable quantum key distribution with a real local oscillator

Science.gov (United States)

Wang, Tao; Huang, Peng; Zhou, Yingming; Liu, Weiqi; Zeng, Guihua

2018-01-01

We propose a pilot-multiplexed continuous-variable quantum key distribution (CVQKD) scheme based on a local local oscillator (LLO). Our scheme utilizes time-multiplexing and polarization-multiplexing techniques to dramatically isolate the quantum signal from the pilot, employs two heterodyne detectors to separately detect the signal and the pilot, and adopts a phase compensation method to almost eliminate the multifrequency phase jitter. In order to analyze the performance of our scheme, a general LLO noise model is constructed. Besides the phase noise and the modulation noise, the photon-leakage noise from the reference path and the quantization noise due to the analog-to-digital converter (ADC) are also considered, which are first analyzed in the LLO regime. Under such general noise model, our scheme has a higher key rate and longer secure distance compared with the preexisting LLO schemes. Moreover, we also conduct an experiment to verify our pilot-multiplexed scheme. Results show that it maintains a low level of the phase noise and is expected to obtain a 554-Kbps secure key rate within a 15-km distance under the finite-size effect.

Edge detection versus densitometry for assessing coronary stenting quantitatively

NARCIS (Netherlands)

B.H. Strauss (Bradley); Y. Juilliere; B.J.W.M. Rensing (Benno); J.H.C. Reiber (Johan); P.W.J.C. Serruys (Patrick)

1991-01-01

textabstractThe optimal method used to analyze quantitatively the immediate angiographic results of coronary stenting in the coronary arteries has not been studied. Accordingly, minimal luminal cross-sectional area was determined by 2 methods, edge detection and densitometry, in 19 patients who

Gold Nanoparticle Labeling Based ICP-MS Detection/Measurement of Bacteria, and Their Quantitative Photothermal Destruction

Science.gov (United States)

Lin, Yunfeng

2015-01-01

Bacteria such as Salmonella and E. coli present a great challenge in public health care in today’s society. Protection of public safety against bacterial contamination and rapid diagnosis of infection require simple and fast assays for the detection and elimination of bacterial pathogens. After utilizing Salmonella DT104 as an example bacterial strain for our investigation, we report a rapid and sensitive assay for the qualitative and quantitative detection of bacteria by using antibody affinity binding, popcorn shaped gold nanoparticle (GNPOPs) labeling, surfance enchanced Raman spectroscopy (SERS), and inductively coupled plasma mass spectrometry (ICP-MS) detection. For qualitative analysis, our assay can detect Salmonella within 10 min by Raman spectroscopy; for quantitative analysis, our assay has the ability to measure as few as 100 Salmonella DT104 in a 1 mL sample (100 CFU/mL) within 40 min. Based on the quantitative detection, we investigated the quantitative destruction of Salmonella DT104, and the assay’s photothermal efficiency in order to reduce the amount of GNPOPs in the assay to ultimately to eliminate any potential side effects/toxicity to the surrounding cells in vivo. Results suggest that our assay may serve as a promising candidate for qualitative and quantitative detection and elimination of a variety of bacterial pathogens. PMID:26417447

Remote (250 km Fiber Bragg Grating Multiplexing System

Directory of Open Access Journals (Sweden)

Manuel Lopez-Amo

2011-09-01

Full Text Available We propose and demonstrate two ultra-long range fiber Bragg grating (FBG sensor interrogation systems. In the first approach four FBGs are located 200 km from the monitoring station and a signal to noise ratio of 20 dB is obtained. The second improved version is able to detect the four multiplexed FBGs placed 250 km away, offering a signal to noise ratio of 6–8 dB. Consequently, this last system represents the longest range FBG sensor system reported so far that includes fiber sensor multiplexing capability. Both simple systems are based on a wavelength swept laser to scan the reflection spectra of the FBGs, and they are composed by two identical-lengths optical paths: the first one intended to launch the amplified laser signal by means of Raman amplification and the other one is employed to guide the reflection signal to the reception system.

One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacterium and Virus Occurring on Brassicaceae Crop Seeds

Directory of Open Access Journals (Sweden)

Kyusik Jeong

2011-04-01

Full Text Available The aim of this research was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant pathogenic bacteria and seed borne virus in commercial Brassicaceae crop seeds, Xanthomonns campestris pv. campestris (Xcc and Lettuce Mosaic Virus (LMV. Bacterial and virus diseases of Brassicaceae leaves are responsible for heavy losses. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcc and LMV in commercial Brassicaceae crop seeds (lettuce, kohlrabi, radish, chinese cabbage and cabbage, two pairs of specific primer (LMV-F/R, Xcc-F/R were synthesized by using primer-blast program (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/. The multiplex PCR for the two pathogens in Brassicaceae crop seeds could detect specifically without interference among primers and/or cDNA of other plant pathogens. The pathogen detection limit was determined at 1 ng of RNA extracted from pathogens. In the total PCR results for pathogen detection using commercial kohlrabi (10 varieties, lettuce (50 varieties, radish (20 varieties, chinese cabbage (20 varieties and cabbage (20 varieties, LMV and Xcc were detected from 39 and 2 varieties, respectively. In the PCR result of lettuce, LMV and Xcc were simultaneously detected in 8 varieties.

One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacteria and Viruses in Pepper and Tomato Seeds

Directory of Open Access Journals (Sweden)

Kyusik Jeong

2011-04-01

Full Text Available The aim of this study was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant seed infection pathogenic bacteria and virus, Xanthomonns campestris pv. vesicatoria (Xcv, Clavibacter michiganensis subsp. michiganensis (Cmm, Erwinia carotovora subsp. carotovora (Ecc, Pepper mild mottle virus (PMMoV and Tobacco mild green mosaic virus (TMGMV in pepper and tomato seeds. Most of pepper and tomato bacterial and virus diseases are responsible for germination and growth obstruction. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcv, Cmm, Ecc, PMMoV and TMGMV in pepper and tomato seeds, five pairs (Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMV-F/R of specific primer were synthesized by primer-blast program. The multiplex PCR for the five pathogens in pepper and tomato seeds could detect specially without interference among primers and/or cDNA of plant seeds and other plant pathogens. The PCR result for pathogen detection using 20 commercial pepper and 10 tomato seed samples, Ecc was detected from 4 pepper and 2 tomato seed samples, PMMoV was detected from 1 pepper seed sample, and PMMoV and TMGMV were simultaneously detected from 1 pepper seed sample.

Detection of medically important Candida species by absolute quantitation real-time polymerase chain reaction.

Science.gov (United States)

Than, Leslie Thian Lung; Chong, Pei Pei; Ng, Kee Peng; Seow, Heng Fong

2015-01-01

The number of invasive candidiasis cases has risen especially with an increase in the number of immunosuppressed and immunocom promised patients. The early detection of Candida species which is specific and sensitive is important in determining the correct administration of antifungal drugs to patients. This study aims to develop a method for the detection, identification and quantitation of medically important Candida species through quantitative polymerase chain reaction (qPCR). The isocitrate lyase (ICL) gene which is not found in mammals was chosen as the target gene of real-time PCR. Absolute quantitation of the gene copy number was achieved by constructing the plasmid containing the ICL gene which is used to generate standard curve. Twenty fungal species, two bacterial species and human DNA were tested to check the specificity of the detection method. All eight Candida species were successfully detected, identified and quantitated based on the ICL gene. A seven-log range of the gene copy number and a minimum detection limit of 10(3) copies were achieved. A one-tube absolute quantification real-time PCR that differentiates medically important Candida species via individual unique melting temperature was achieved. Analytical sensitivity and specificity were not compromised.

A novel IPTV program multiplex access system to EPON

Science.gov (United States)

Xu, Xian; Liu, Deming; He, Wei; Lu, Xi

2007-11-01

With the rapid development of high speed networks, such as Ethernet Passive Optical Network (EPON), traffic patterns in access networks have evolved from traditional text-oriented service to the mixed text-, voice- and video- based services, leading to so called "Triple Play". For supporting IPTV service in EPON access network infrastructure, in this article we propose a novel IPTV program multiplex access system to EPON, which enables multiple IPTV program source servers to seamlessly access to IPTV service access port of optical line terminal (OLT) in EPON. There are two multiplex schemes, namely static multiplex scheme and dynamic multiplex scheme, in implementing the program multiplexing. Static multiplex scheme is to multiplex all the IPTV programs and forward them to the OLT, regardless of the need of end-users. While dynamic multiplex scheme can dynamically multiplex and forward IPTV programs according to what the end-users actually demand and those watched by no end-user would not be multiplexed. By comparing these two schemes, a reduced traffic of EPON can be achieved by using dynamic multiplex scheme, especially when most end-users are watching the same few IPTV programs. Both schemes are implemented in our system, with their hardware and software designs described.

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

Quantifying the Role of Homophily in Human Cooperation Using Multiplex Evolutionary Game Theory.

Directory of Open Access Journals (Sweden)

Alessandro Di Stefano

Full Text Available Nature shows as human beings live and grow inside social structures. This assumption allows us to explain and explore how it may shape most of our behaviours and choices, and why we are not just blindly driven by instincts: our decisions are based on more complex cognitive reasons, based on our connectedness on different spaces. Thus, human cooperation emerges from this complex nature of social network. Our paper, focusing on the evolutionary dynamics, is intended to explore how and why it happens, and what kind of impact is caused by homophily among people. We investigate the evolution of human cooperation using evolutionary game theory on multiplex. Multiplexity, as an extra dimension of analysis, allows us to unveil the hidden dynamics and observe non-trivial patterns within a population across network layers. More importantly, we find a striking role of homophily, as the higher the homophily between individuals, the quicker is the convergence towards cooperation in the social dilemma. The simulation results, conducted both macroscopically and microscopically across the network layers in the multiplex, show quantitatively the role of homophily in human cooperation.

High sensitive photonic crystal multiplexed biosensor array using H0 sandwiched cavities

Directory of Open Access Journals (Sweden)

Arafa Safia

2017-01-01

Full Text Available We theoretically investigate a high sensitive photonic crystal integrated biosensor array structure which is potentially used for label-free multiplexed sensing. The proposed device consists of an array of three sandwiched H0 cavities patterned above silicon on insulator (SOI substrate; each cavity has been designed for different cavity spacing and different resonant wavelength. Results obtained by performing finite-difference time-domain (FDTD simulations, indicate that the response of each detection unit shifts independently in terms of refractive index variations. The optimized design makes possible the combination of sensing as a function of location, as well as a function of time in the same platform. A refractive index sensitivity of 520nm/RIU and a quality factor over 104 are both achieved with an accompanied crosstalk of less than -26 dB. In addition, the device presents an improved detection limit (DL of 1.24.10-6 RIU and a wide measurement range. These features make the designed device a promising element for performing label-free multiplexed detection in monolithic substrate for medical diagnostics and environmental monitoring.

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

Science.gov (United States)

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

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

Preliminary study of visual effect of multiplex hologram

Science.gov (United States)

Fu, Huaiping; Xiong, Bingheng; Yang, Hong; Zhang, Xueguo

2004-06-01

The process of any movement of real object can be recorded and displayed by a multiplex holographic stereogram. An embossing multiplex holographic stereogram and a multiplex rainbow holographic stereogram have been made by us, the multiplex rainbow holographic stereogram reconstructs the dynamic 2D line drawing of speech organs, the embossing multiplex holographic stereogram reconstructs the process of an old man drinking water. In this paper, we studied the visual result of an embossing multiplex holographic stereogram made with 80 films of 2-D pictures. Forty-eight persons of aged from 13 to 67 were asked to see the hologram and then to answer some questions about the feeling of viewing. The results indicate that this kind of holograms could be accepted by human visual sense organ without any problem. This paper also discusses visual effect of the multiplex holography stereograms base on visual perceptual psychology. It is open out that the planar multiplex holograms can be recorded and present the movement of real animal and object. Not only have the human visual perceptual constancy for shape, just as that size, color, etc... but also have visual perceptual constancy for binocular parallax.

Detection of three porcine vesicular viruses using multiplex real-time primer-probe energy transfer

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Uttenthal, Åse; Aguero, M.

2006-01-01

Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous...

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.

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.

Demonstration of hybrid orbital angular momentum multiplexing and time-division multiplexing passive optical network.

Science.gov (United States)

Wang, Andong; Zhu, Long; Liu, Jun; Du, Cheng; Mo, Qi; Wang, Jian

2015-11-16

Mode-division multiplexing passive optical network (MDM-PON) is a promising scheme for next-generation access networks to further increase fiber transmission capacity. In this paper, we demonstrate the proof-of-concept experiment of hybrid mode-division multiplexing (MDM) and time-division multiplexing (TDM) PON architecture by exploiting orbital angular momentum (OAM) modes. Bidirectional transmissions with 2.5-Gbaud 4-level pulse amplitude modulation (PAM-4) downstream and 2-Gbaud on-off keying (OOK) upstream are demonstrated in the experiment. The observed optical signal-to-noise ratio (OSNR) penalties for downstream and upstream transmissions at a bit-error rate (BER) of 2 × 10(-3) are less than 2.0 dB and 3.0 dB, respectively.

Online Continuous Trace Process Analytics Using Multiplexing Gas Chromatography.

Science.gov (United States)

Wunsch, Marco R; Lehnig, Rudolf; Trapp, Oliver

2017-04-04

The analysis of impurities at a trace level in chemical products, nutrition additives, and drugs is highly important to guarantee safe products suitable for consumption. However, trace analysis in the presence of a dominating component can be a challenging task because of noncompatible linear detection ranges or strong signal overlap that suppresses the signal of interest. Here, we developed a technique for quantitative analysis using multiplexing gas chromatography (mpGC) for continuous and completely automated process trace analytics exemplified for the analysis of a CO 2 stream in a production plant for detection of benzene, toluene, ethylbenzene, and the three structural isomers of xylene (BTEX) in the concentration range of 0-10 ppb. Additional minor components are methane and methanol with concentrations up to 100 ppm. The sample is injected up to 512 times according to a pseudorandom binary sequence (PRBS) with a mean frequency of 0.1 Hz into a gas chromatograph equipped with a flame ionization detector (FID). A superimposed chromatogram is recorded which is deconvoluted into an averaged chromatogram with Hadamard transformation. Novel algorithms to maintain the data acquisition rate of the detector by application of Hadamard transformation and to suppress correlation noise induced by components with much higher concentrations than the target substances are shown. Compared to conventional GC-FID, the signal-to-noise ratio has been increased by a factor of 10 with mpGC-FID. Correspondingly, the detection limits for BTEX in CO 2 have been lowered from 10 to 1 ppb each. This has been achieved despite the presence of detectable components (methane and methanol) with a concentration about 1000 times higher than the target substances. The robustness and reliability of mpGC has been proven in a two-month field test in a chemical production plant.

A polarization-division multiplexing SSB-OFDM system with beat interference cancellation receivers

Science.gov (United States)

Yang, Peiling; Ma, Jianxin; Zhang, Junyi

2018-06-01

In this paper, we have proposed a polarization-division multiplexing (PDM) single-sideband optical orthogonal frequency division multiplexing (SSB-OOFDM) scheme with signal-signal beat interference cancellation receivers with balanced detection (ICRBD). This system can double channel capacity and improve spectrum efficiency (SE) with the reduced guard band (GB) due to the PDM. Multiple input multiple output (MIMO) technique is used to solve polarization mode dispersion (PMD) associated with channel estimation and equalization. By simulation, we demonstrate the efficacy of the proposed technique for a 2 ×40 Gbit/s 16-QAM SSB-PDM-OOFDM system according to the error vector magnitude (EVM) and the constellation diagrams.

Real-time multiplexed digital cavity-enhanced spectroscopy

International Nuclear Information System (INIS)

Boyson, Toby K.; Dagdigian, Paul J.; Pavey, Karl D.; Fitzgerald, Nicholas J.; Spence, Thomas G.; Moore, David S.; Harb, Charles C.

2015-01-01

Cavity-enhanced spectroscopy is a sensitive optical absorption technique but one where the practical applications have been limited to studying small wavelength ranges. In addition, this Letter shows that wideband operation can be achieved by combining techniques usually reserved for the communications community with that of cavity-enhanced spectroscopy, producing a multiplexed real-time cavity-enhanced spectrometer. We use multiple collinear laser sources operating asynchronously and simultaneously while being detected on a single photodetector. This is synonymous with radio frequency (RF) cellular systems in which signals are detected on a single antenna but decoded uniquely. Here, we demonstrate results with spectra of methyl salicylate and show parts-per-billion per root hertz sensitivity measured in real-time

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)

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.

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.

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

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

Science.gov (United States)

Velasco, Valeria; Sherwood, Julie S.; Rojas-García, Pedro P.; Logue, Catherine M.

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using

Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL genes from selective enrichments from animals and retail meat.

Directory of Open Access Journals (Sweden)

Valeria Velasco

Full Text Available The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat. The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus, mecA (associated with methicillin resistance and PVL (virulence factor, and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68-0.88 (from substantial to almost perfect agreement and 0.29-0.77 (from fair to substantial agreement for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0-0.49 (from no agreement beyond that expected by chance to moderate agreement for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA

Supramolecular assembly affording a ratiometric two-photon fluorescent nanoprobe for quantitative detection and bioimaging.

Science.gov (United States)

Wang, Peng; Zhang, Cheng; Liu, Hong-Wen; Xiong, Mengyi; Yin, Sheng-Yan; Yang, Yue; Hu, Xiao-Xiao; Yin, Xia; Zhang, Xiao-Bing; Tan, Weihong

2017-12-01

Fluorescence quantitative analyses for vital biomolecules are in great demand in biomedical science owing to their unique detection advantages with rapid, sensitive, non-damaging and specific identification. However, available fluorescence strategies for quantitative detection are usually hard to design and achieve. Inspired by supramolecular chemistry, a two-photon-excited fluorescent supramolecular nanoplatform ( TPSNP ) was designed for quantitative analysis with three parts: host molecules (β-CD polymers), a guest fluorophore of sensing probes (Np-Ad) and a guest internal reference (NpRh-Ad). In this strategy, the TPSNP possesses the merits of (i) improved water-solubility and biocompatibility; (ii) increased tissue penetration depth for bioimaging by two-photon excitation; (iii) quantitative and tunable assembly of functional guest molecules to obtain optimized detection conditions; (iv) a common approach to avoid the limitation of complicated design by adjustment of sensing probes; and (v) accurate quantitative analysis by virtue of reference molecules. As a proof-of-concept, we utilized the two-photon fluorescent probe NHS-Ad-based TPSNP-1 to realize accurate quantitative analysis of hydrogen sulfide (H 2 S), with high sensitivity and good selectivity in live cells, deep tissues and ex vivo -dissected organs, suggesting that the TPSNP is an ideal quantitative indicator for clinical samples. What's more, TPSNP will pave the way for designing and preparing advanced supramolecular sensors for biosensing and biomedicine.

Limits of qualitative detection and quantitative determination

International Nuclear Information System (INIS)

Curie, L.A.

1976-01-01

The fact that one can find a series of disagreeing and limiting definitions of the detection limit leads to the reinvestigation of the problems of signal detection and signal processing in analytical and nuclear chemistry. Three cut-off levels were fixed: Lsub(C) - the net signal level (sensitivity of the equipment), above which an observed signal can be reliably recognized as 'detected'; Lsub(D) - the 'true' net signal level, from which one can a priori expect a detection; Lsub(Q) - the level at which the measuring accuracy is sufficient for quantitative determination. Exact definition equations as well as a series of working formulae are given for the general analytical case and for the investigation of radioactivity. As it is assumed that the radioactivity of the Poisson distribution is determined, it is dealt with in such a manner that precise limits can be derived for short-lived and long-lived radionuclides with or without disturbance. The fundamentals are made clear by simple examples for spectrophotometry and radioactivity and by a complicated example for activation analysis in which one must choose between alternative nuclear reactions. (orig./LH) [de

Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes.

Science.gov (United States)

Novo, Pedro; Prazeres, Duarte Miguel França; Chu, Virginia; Conde, João Pedro

2011-12-07

Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.

Accounting for imperfect detection in ecology: a quantitative review.

Science.gov (United States)

Kellner, Kenneth F; Swihart, Robert K

2014-01-01

Detection in studies of species abundance and distribution is often imperfect. Assuming perfect detection introduces bias into estimation that can weaken inference upon which understanding and policy are based. Despite availability of numerous methods designed to address this assumption, many refereed papers in ecology fail to account for non-detection error. We conducted a quantitative literature review of 537 ecological articles to measure the degree to which studies of different taxa, at various scales, and over time have accounted for imperfect detection. Overall, just 23% of articles accounted for imperfect detection. The probability that an article incorporated imperfect detection increased with time and varied among taxa studied; studies of vertebrates were more likely to incorporate imperfect detection. Among articles that reported detection probability, 70% contained per-survey estimates of detection that were less than 0.5. For articles in which constancy of detection was tested, 86% reported significant variation. We hope that our findings prompt more ecologists to consider carefully the detection process when designing studies and analyzing results, especially for sub-disciplines where incorporation of imperfect detection in study design and analysis so far has been lacking.

Quantitative Digital Tomosynthesis Mammography for Improved Breast Cancer Detection and Diagnosis

National Research Council Canada - National Science Library

Zhang, Yiheng

2008-01-01

.... When fully developed, the DTM can provide radiologists improved quantitative, three-dimensional volumetric information of the breast tissue, and assist in breast cancer detection and diagnosis...

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.

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...

Comparison between specific and multiplex reverse transcription-polymerase chain reaction for detection of hepatitis A virus, poliovirus and rotavirus in experimentally seeded oysters

Directory of Open Access Journals (Sweden)

C Coelho

2003-06-01

Full Text Available Outbreaks of gastroenteritis have occurred among consumers of raw or undercooked shellfish harvested from faecally polluted waters. A multiplex reverse transcription-polymerase chain reaction (RT-PCR was applied for the simultaneous detection of hepatitis A virus (HAV, poliovirus (PV and simian rotavirus (RV-SA11 and compared with specific primers for each genome sequence. Three amplified DNA products representing HAV (192 bp, PV (394 bp and RV (278 bp were identified when positive controls were used. However, when tested on experimentally contaminated raw oysters, this method was not able to detect the three viruses simultaneously. This is probably due to the low concentration of viral RNAs present in oyster extract which were partially lost during the extracts preparation.

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

Multiplex PCR detection of Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica directly from dried stool samples from Guinea-Bissauan children with diarrhoea.

Science.gov (United States)

Mero, Sointu; Kirveskari, Juha; Antikainen, Jenni; Ursing, Johan; Rombo, Lars; Kofoed, Poul-Erik; Kantele, Anu

2017-09-01

In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea. The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.

Multiplex real-time PCR for identification of canine parvovirus antigenic types.

Science.gov (United States)

Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Narang, Deepti

2016-07-01

Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types. Copyright © 2016 Elsevier B.V. All rights reserved.

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...

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.