Journal Article

A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses

Chen, Jianing, Huang, Xin, Guo, Qingyong, Liu, Guangliang, Yao, Gang, Wang, Jinquan

2019

Overview

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 ( R 2 ) 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.

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: Animals / Applied Microbial and Cell Physiology / Assaying / Biomedical and Life Sciences / Biotechnology / Causes of / Coronaviridae / Coronavirus - classification / Coronavirus - isolation & purification / Coronavirus Infections - diagnosis / Coronavirus Infections - veterinary / Coronaviruses / Correlation coefficient / Correlation coefficients / COVID-19 / Deltacoronavirus / detection limit / Diagnosis / Diagnosis, Differential / Diarrhea / Diarrhea - virology / Differential diagnosis / Diseases / DNA Primers - genetics / Epidemics / Feces - virology / Gastroenteritis / Gastroenteritis, Transmissible, of Swine - diagnosis / Gastroenteritis, Transmissible, of Swine - virology / genes / Identification and classification / Life Sciences / Limit of Detection / Methods / Microbial Genetics and Genomics / Microbiology / mixed infection / Molecular diagnostic techniques / Multiplex Polymerase Chain Reaction - methods / Multiplexing / Pathogens / piglets / Polymerase chain reaction / Porcine epidemic diarrhea virus / Porcine epidemic diarrhea virus - genetics / Porcine epidemic diarrhea virus - isolation & purification / Primers / quantitative polymerase chain reaction / Real time / Real-Time Polymerase Chain Reaction - methods / Reproducibility of Results / reverse transcriptase polymerase chain reaction / Sensitivity and Specificity / Signs and symptoms / signs and symptoms (animals and humans) / Swine / Swine Diseases - diagnosis / Swine Diseases - virology / Transmissible gastroenteritis / Transmissible gastroenteritis virus / Transmissible gastroenteritis virus - genetics / Transmissible gastroenteritis virus - isolation & purification / Viral diarrhea / Viruses