Simultaneous detection of multiple pathogens by multiplex PCR coupled with DNA biochip hybridization

Traditional serological enzyme-linked immunosorbent assay (ELISA) is routinely used to monitor pathogens during quarantine in most animal facilities to prevent possible infection. However, the ELISA platform is a single-target assay, and screening all targeted pathogens is time-consuming and laborious. In this study, to increase sensitivity and to reduce diagnosis time for high-throughput processes, multiplex PCR and DNA biochip techniques were combined to develop a multi-pathogen diagnostic method for use instead of routine ELISA. Eight primer sets were designed for multiplex PCR to detect genes from seven targeted bacterial and viral pathogens. DNA–DNA hybridization was conducted on a biochip following the multiple PCR analysis. Using this method, a total of 24 clinical samples were tested, and the result showed that not only single infection but also co-infection by multi-pathogens can be detected. In conclusion, multiplex PCR coupled with a DNA biochip is an efficient method for detecting multi-pathogens in a reaction. This platform is a useful tool for quarantine services and disease prevention in animal facilities.

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Multiplex PCR Primer Design for Simultaneous Detection of Multiple Pathogens

Methods in Molecular Biology - PCR Primer Design ◽

10.1007/978-1-4939-2365-6_6 ◽

2015 ◽

pp. 91-101 ◽

Cited By ~ 1

Author(s):

Wenchao Yan

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Primer Design ◽

Multiple Pathogens ◽

Pcr Primer

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Development of multiplex PCR for rapid detection of metallo-β-lactamase genes in clinical isolates of Acinetobacter baumannii

Iranian Journal of Microbiology ◽

10.18502/ijm.v12i2.2615 ◽

2020 ◽

Author(s):

Reza Ranjbar ◽

Shahin Zayeri ◽

Amir Mirzaie

Keyword(s):

Acinetobacter Baumannii ◽

Multiplex Pcr ◽

Rapid Detection ◽

Simultaneous Detection ◽

Cross Reactivity ◽

Clinical Isolates ◽

Clinical Samples ◽

Pcr Assay ◽

Multiplex Pcr Assay

Background and Objectives: Acinetobacter baumannii has been known as a major pathogen causing nosocomial infec- tions. The aim of this study was to develop multiplex PCR for rapid and simultaneous detection of metallo-β-lactamase (MBL) genes in clinical isolates of A. baumannii. Materials and Methods: In this study, we used three sets of primers to amplify the MBL genes including bla , bla and bla OXA-48 . The multiplex PCR assay was optimized for rapid and simultaneous detection of MBL genes in A. bau- OXA-23 NDM mannii strains recovered from clinical samples. Results: A. baumannii strains recovered from clinical samples were subjected to the study. The multiplex PCR produced 3 OXA-48 OXA-23 bands of 501 bp for bla , 744 bp for bla observed in multiplex PCR. OXA-48 and 623 bp for bla NDM genes. In addition to, no any cross-reactivity was Conclusion: Based on obtained data, the multiplex PCR had a good specificity without any cross reactivity and it appears that the multiplex PCR is reliable assay for simultaneous detection of MBL genes in A. baumannii strains.

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Detection of Aflatoxin-Producing Molds in Korean Fermented Foods and Grains by Multiplex PCR

Journal of Food Protection ◽

10.4315/0362-028x-67.11.2622 ◽

2004 ◽

Vol 67 (11) ◽

pp. 2622-2626 ◽

Cited By ~ 26

Author(s):

ZHENG-YOU YANG ◽

WON-BO SHIM ◽

JI-HUN KIM ◽

SEON-JA PARK ◽

SUNG-JO KANG ◽

...

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Penicillium Expansum ◽

Enzyme Linked Immunosorbent Assay ◽

Fermented Foods ◽

Elisa Assay ◽

Malt Extract ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Positive Results

An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.

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A Multiplex Snapback Primer System for the Enrichment and Detection ofJAK2V617F andMPLW515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

BioMed Research International ◽

10.1155/2014/458457 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Zhiyuan Wu ◽

Yunqing Zhang ◽

Xinju Zhang ◽

Xiao Xu ◽

Zhihua Kang ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Simultaneous Detection ◽

Melting Curve ◽

Clinical Samples ◽

Melting Curve Analysis ◽

Amplification Efficiency ◽

Primer Sets ◽

Low Ionic Strength ◽

Multiplex System

A multiplex snapback primer system was developed for the simultaneous detection ofJAK2V617F andMPLW515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets forJAK2andMPLmutation 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.

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

Acta Veterinaria Brno ◽

10.2754/avb201483s10s77 ◽

2014 ◽

Vol 83 (10) ◽

pp. S77-S83 ◽

Cited By ~ 4

Author(s):

Eva Renčová ◽

Zora Piskatá ◽

Darina Kostelníková ◽

Bohuslava Tremlová

Keyword(s):

Detection Limit ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Corylus Avellana ◽

Food Samples ◽

Pcr Analysis ◽

Food Protein ◽

Food Ingredients ◽

Pcr Method ◽

Food Matrices

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.

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Development of multiplex PCR for simultaneous detection and differentiation of six DNA and RNA viruses from clinical samples of sheep and goats

Journal of Virological Methods ◽

10.1016/j.jviromet.2017.01.012 ◽

2017 ◽

Vol 243 ◽

pp. 44-49 ◽

Cited By ~ 3

Author(s):

Ya-Peng He ◽

Qi Zhang ◽

Ming-Zhe Fu ◽

Xin-Gang Xu

Keyword(s):

Multiplex Pcr ◽

Rna Viruses ◽

Simultaneous Detection ◽

Clinical Samples ◽

Sheep And Goats ◽

Dna And Rna

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PCR Detection of Group A Bovine Rotaviruses in Feces

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879300500403 ◽

1993 ◽

Vol 5 (4) ◽

pp. 516-521 ◽

Cited By ~ 11

Author(s):

Jarasvech Chinsangaram ◽

Geoffrey Y. Akita ◽

Anthony E. Castro ◽

Bennie I. Osburn

Keyword(s):

Ethidium Bromide ◽

Clinical Samples ◽

Pcr Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Pcr Assay ◽

Fecal Samples ◽

Bovine Rotavirus ◽

Viral Particles ◽

Pcr Inhibitor ◽

Group A

A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3′ ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 × 104 viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 × 102 viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.

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Distribution and diversity of root-lession nematode (Pratylenchus spp.) associated with Miscanthus × giganteus and Panicum virgatum used for biofuels, and species identification in a multiplex polymerase chain reaction

Nematology ◽

10.1163/138855410x538153 ◽

2011 ◽

Vol 13 (6) ◽

pp. 673-686 ◽

Cited By ~ 16

Author(s):

Michael Gray ◽

Horacio Lopez-Nicora ◽

Tesfamariam Mekete ◽

Terry Niblack ◽

Kimberly Reynolds

Keyword(s):

Multiplex Pcr ◽

Panicum Virgatum ◽

Sequence Data ◽

Simultaneous Detection ◽

Morphological Identification ◽

28S Rrna ◽

Detection Rates ◽

The Usa ◽

Primer Sets ◽

Species Specific

AbstractThe distribution of Pratylenchus spp. from bioenergy field plots in six states (Illinois, Iowa, South Dakota, Kentucky, Tennessee, Georgia) of the USA were surveyed. The species were identified based on morphology and morphometrics and further characterised based on fragment sequences of the 28S rRNA of the D2-D3 region. The region revealed variations in sequencing information that supports the morphological identification. In this work, six Pratylenchus spp. were detected: Pratylenchus brachyurus, P. crenatus, P. hexincisus, P. neglectus, P. penetrans and P. scribneri. Pratylenchus scribneri, P. crenatus, and P. penetrans were distributed most widely, with detection of 34, 29 and 15%, respectively. Pratylenchus hexincisus, P. brachyurus and P. neglectus were distributed sporadically, with detection rates of 10.0, 2.6 and 2.0%, respectively. A one-step multiplex PCR was developed for the simultaneous detection of P. scribneri, P. crenatus and P. penetrans. Sequence data from this research and NCBI were used to generate different primer sets that are species-specific. We have therefore designed three sets of primers that discriminate P. scribneri, P. crenatus and P. penetrans in multiplex PCR. All the tested primers have shown specificity and have no cross-reaction with the non-target species. When used in a uniplex, duplex and triplex PCR, the selected three primers gave a unique electrophoretic DNA banding pattern characterised by a single DNA fragment for P. scribneri (ca 750), P. crenatus (ca 690), and P. penetrans (ca 520). The method could be used for routine diagnostic programmes.

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Simultaneous Detection of Seven Human Coronaviruses by Multiplex PCR and MALDI-TOF MS

COVID ◽

10.3390/covid2010002 ◽

2021 ◽

Vol 2 (1) ◽

pp. 5-17

Author(s):

Tingting Liu ◽

Lin Kang ◽

Yanwei Li ◽

Jing Huang ◽

Zishuo Guo ◽

...

Keyword(s):

Multiplex Pcr ◽

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Detection Sensitivity ◽

Clinical Samples ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Human Coronaviruses ◽

Tof Ms

Human coronaviruses (HCoVs) are associated with a range of respiratory symptoms. The discovery of severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome, and SARS-CoV-2 pose a significant threat to human health. In this study, we developed a method (HCoV-MS) that combines multiplex PCR with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), to detect and differentiate seven HCoVs simultaneously. The HCoV-MS method had high specificity and sensitivity, with a 1–5 copies/reaction detection limit. To validate the HCoV-MS method, we tested 163 clinical samples, and the results showed good concordance with real-time PCR. Additionally, the detection sensitivity of HCoV-MS and real-time PCR was comparable. The HCoV-MS method is a sensitive assay, requiring only 1 μL of a sample. Moreover, it is a high-throughput method, allowing 384 samples to be processed simultaneously in 30 min. We propose that this method be used to complement real-time PCR for large-scale screening studies.

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