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

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.