A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses
- 26 April 2019
- journal article
- research article
- Published by Springer Science and Business Media LLC in Applied Microbiology and Biotechnology
- Vol. 103 (12), 4943-4952
- https://doi.org/10.1007/s00253-019-09835-7
Abstract
Swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. To develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (TGEV) N, porcine epidemic diarrhea virus (PEDV) M, porcine deltacoronavirus (PDCoV) M, and porcine enteric alphacoronavirus (PEAV) N genes respectively. A TaqMan-probe-based multiplex real-time RT-qPCR assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. The results showed that the limit of detection can reach as low as 10 copies in singular real-time RT-qPCR assays and 100 copies in multiplex real-time RT-qPCR assay, with all correlation coefficients (R2) at above 0.99, and the amplification efficiency at between 90 and 120%. This multiplex real-time RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. The multiplex real-time RT-qPCR assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. The results manifested that TGEV and PDCoV were the main pathogens in these samples, accompanied by co-infections. This well-established multiplex real-time RT-qPCR assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses.Keywords
Funding Information
- National Key R&D Program of China (2016YFD0500103)
- National Natural Science Foundation of China (31572498, 31702209)
- Elite Youth Program of CAAS (N/A)
This publication has 25 references indexed in Scilit:
- Generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsidApplied Microbiology and Biotechnology, 2019
- Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat originNature, 2018
- Development of rapid immunochromatographic strip test for the detection of porcine epidemic diarrhoea virusVeterinary Record, 2017
- Discovery of a novel swine enteric alphacoronavirus (SeACoV) in southern ChinaVeterinary Microbiology, 2017
- Development and application of a recombinant M protein-based indirect ELISA for the detection of porcine deltacoronavirus IgG antibodiesJournal of Virological Methods, 2017
- New Chimeric Porcine Coronavirus in Swine Feces, Germany, 2012Emerging Infectious Diseases, 2016
- A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirusThe Journal of Veterinary Medical Science, 2016
- Newly Emerged PorcineDeltacoronavirusAssociated With Diarrhoea in Swine in China: Identification, Prevalence and Full-Length Genome Sequence AnalysisTransboundary and Emerging Diseases, 2015
- Origin, Evolution, and Virulence of Porcine Deltacoronaviruses in the United StatesmBio, 2015
- Multiplex Reverse Transcription-PCR for Rapid Differential Detection of Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Group a RotavirusJournal of Veterinary Diagnostic Investigation, 2006