Multiplex PCR for simultaneous detection and differentiation of sheeppox, goatpox and orf viruses from clinical samples of sheep and goats

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

Laboratory Animals ◽

10.1177/0023677217718864 ◽

2017 ◽

Vol 52 (2) ◽

pp. 186-195 ◽

Cited By ~ 2

Author(s):

Hsiang-Yun Tung ◽

Wei-Chen Chen ◽

Bor-Rung Ou ◽

Jan-Ying Yeh ◽

Yeong-Hsiang Cheng ◽

...

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Clinical Samples ◽

Single Infection ◽

Pcr Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Viral Pathogens ◽

Single Target ◽

Primer Sets ◽

Multiple Pathogens

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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Development of a multiplex TaqMan qPCR assay for simultaneous detection and differentiation of four DNA and RNA viruses from clinical samples of sheep and goats

Journal of Virological Methods ◽

10.1016/j.jviromet.2019.01.015 ◽

2019 ◽

Vol 266 ◽

pp. 58-64 ◽

Cited By ~ 3

Author(s):

Xingang Xu ◽

Feng Yang ◽

Qi Zhang ◽

Ying Xu ◽

Jiali Huang ◽

...

Keyword(s):

Rna Viruses ◽

Simultaneous Detection ◽

Qpcr Assay ◽

Clinical Samples ◽

Sheep And Goats ◽

Dna And Rna ◽

Taqman Qpcr Assay ◽

Taqman Qpcr

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Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples

Journal of Medical Microbiology ◽

10.1099/jmm.0.46997-0 ◽

2007 ◽

Vol 56 (4) ◽

pp. 480-486 ◽

Cited By ~ 75

Author(s):

Luis G. C. Pacheco ◽

Roberta R. Pena ◽

Thiago L. P. Castro ◽

Fernanda A. Dorella ◽

Robson C. Bahia ◽

...

Keyword(s):

Multiplex Pcr ◽

Corynebacterium Pseudotuberculosis ◽

Clinical Samples ◽

Rrna Gene ◽

Sheep And Goats ◽

Multiplex Pcr Assay ◽

Pure Cultures ◽

Biochemical Identification ◽

Biochemical Testing ◽

Mpcr Assay

Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a debilitating disease of sheep and goats. Accurate diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene, rpoB and pld. This method allowed efficient identification of 40 isolates of this bacterium that had been identified previously by biochemical testing. Analysis of taxonomically related species did not generate the C. pseudotuberculosis mPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg of C. pseudotuberculosis genomic DNA was detected by this mPCR assay, demonstrating the sensitivity of the method. The detection limit in clinical samples was estimated to be 103 c.f.u. C. pseudotuberculosis could be detected directly in pus samples from infected sheep and goats (n=56) with a high diagnostic sensitivity (94.6 %). The developed assay significantly improves rapid C. pseudotuberculosis detection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.

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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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Automation of a fluorescence-based multiplex PCR for the laboratory confirmation of common bacterial pathogens

Journal of Medical Microbiology ◽

10.1099/jmm.0.05416-0 ◽

2004 ◽

Vol 53 (2) ◽

pp. 115-117 ◽

Cited By ~ 16

Author(s):

Karen Smith ◽

Mathew A. Diggle ◽

Stuart C. Clarke

Keyword(s):

Public Health ◽

Streptococcus Pneumoniae ◽

Multiplex Pcr ◽

High Throughput ◽

Health Management ◽

Simultaneous Detection ◽

Clinical Samples ◽

Liquid Handling ◽

Laboratory Confirmation ◽

Extraction Liquid

A fluorescence-based multiplex PCR was automated for the simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae in clinical samples from patients with suspected meningitis. Sensitivity of one to two genome copies per 100 μl sample and specificity of 100 % for each organism were shown. Automation of DNA extraction, liquid handling, PCR and analysis are achieved on a single platform, which enables a high throughput and rapid turnaround of clinical samples that, in turn, leads to faster diagnosis. This is ultimately beneficial to the treatment of the patient and for public health management.

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Simultaneous Detection of Bacteroides forsythus and Prevotella intermedia by 16S rRNA Gene-Directed Multiplex PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1621-1624.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1621-1624 ◽

Cited By ~ 27

Author(s):

Georg Conrads ◽

Thomas F. Flemmig ◽

Ilse Seyfarth ◽

Friedrich Lampert ◽

Rudolf Lütticken

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Clinical Samples ◽

Rrna Gene ◽

Subgingival Plaque ◽

Prevotella Intermedia ◽

Forward Primer

In a 16S rRNA gene-directed multiplex PCR, Prevotella intermedia- and Bacteroides forsythus-specific reverse primers were combined with a single conserved forward primer. A 660-bp fragment and an 840-bp fragment that were specific for both species could be amplified simultaneously. A total of 152 clinical samples, subgingival plaque and swabs of three different oral mucosae, from 38 periodontitis patients were used for the evaluation.

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