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A multiplex RT-PCR assay for rapid and simultaneous detection of four RNA viruses in swine.

ABSTRACT: A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. The conserved target sequences directed to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) were selected based on alignments of genomic sequences and then specific primers were designed. The mRT-PCR assay was developed and evaluated for its specificity and sensitivity. The expected product from the single viral template was amplified by mRT-PCR and no spurious PCR amplification occurred from the genomic RNA or DNA of other pathogens. For single virus or different combinations of two viruses the detection limit of mRT-PCR was consistent with a single RT-PCR wtith 1?×?103 copies. For different combinations of the three viruses or four viruses, sensitivity of PEDV detection partially decreased. All of positive clinical specimens by the mRT-PCR were identically confirmed using Taqman RT-qPCR. Therefore, the mRT-PCR is a useful tool for epidemiological studies and laboratory diagnosis of single virus and/or mixed infections in swine.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC7113762 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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A multiplex RT-PCR assay for rapid and simultaneous detection of four RNA viruses in swine.

Zhao Yan Y Liu Feifei F Li Qingmei Q Wu Mengfan M Lei Lei L Pan Zishu Z

Journal of virological methods 20190402

A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. The conserved target sequences directed to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) were selected based on alignments of genomic sequences and then specific primers were designed. The mRT-PCR assay was developed ...[more]

PMID: 30951787

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Project description:Influenza and other acute respiratory infections are of great concern for public health, causing excessive morbidity and mortality throughout the world. Influenza virus A(H2N2), which caused a pandemic of so called "Asian flu" in 1957 was expelled from the human population by the new pandemic virus subtype H3N2 in 1968, however, influenza A(H2) viruses continue to circulate in wild birds and poultry. The lack of immunity in human population and the continued circulation of influenza A(H2) among animals makes emergence of a new pandemic virus possible. One of the basic techniques of molecular diagnostics of infectious diseases is the realtime polymerase chain reaction (PCR). The aim of this work was to design oligonucleotide primers and probes for the rapid detection of influenza A virus subtype H2 by realtime reverse transcription - polymerase chain reaction (rRT-PCR). Analysis of 539 sequences of influenza A(H2N2) virus hemagglutinin gene from GISAID EpiFlu database revealed conservative regions suitable for use as binding sites for primers and probes. 191 probes were designed and 2 sets of primers and probes (H2-1 and H2-2) were selected for further experimental evaluation. Detection limit of RT-PCR system was 50 copies of DNA per 25 ?l reaction when 10-fold dilutions of pCI-neo-H2 plasmid used as template. Analytical specificity of selected sets of primers and probes were tested on wide range of influenza strains and non-influenza respiratory viruses. H2-2 set found to have insufficient specificity detecting seasonal influenza A(H1N1) viruses and was excluded from further analysis. Analytical sensitivity was further tested on vaccine strain A/17/California/66/395 (H2N2) and A/Japan/305/1957 (H2N2), limit of detection for primers-probe set H2-1 was 3.2 (CI95%: 3.07-3.48) lg EID50/ml. Designed primers and probes for the realtime RT-PCR universal detection of influenza A(H2) viruses could be used in clinical trials of vaccines against influenza A(H2) and screening for H2 in cases of unsubtypeable influenza A in humans.

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A multiplex RT-PCR assay for rapid and simultaneous detection of four RNA viruses in swine.

Publications

A multiplex RT-PCR assay for rapid and simultaneous detection of four RNA viruses in swine.

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