A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses

SIMPLE SUMMARY: Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause similar manifestations of diarrhea, vomiting, and dehydration. In this study, a quadruplex real-time quantitative PCR (qRT-PCR) assay was developed for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV. A total of 3236 clinical fecal samples from Guangxi province, China, were tested to evaluate the application of the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26%, 0.46%, 13.16%, and 0.15%, respectively. The developed assay showed extreme specificity, high sensitivity, and excellent reproducibility for the simultaneous detection and differentiation of PEDV, TGEV, PDCoV, and SADS-CoV. ABSTRACT: Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/μL (final reaction concentration of 12.1 copies/μL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.