Abstract：Abstract Porcine delta coronavirus (PDCoV) is a newly emerging swine intestinal coronavirus. In this study, a reverse transcription polymerase chain reaction (RT-PCR) method based on M gene for detecting PDCoV was developed and clinically used for epidemiology survey of PDCoV in Sichuan province and detecting the tissue distribution of PDCoV in challenged piglets. Based on the results of PDCoV M gene sequence multiple alignment and comparison with M gene sequences of Porcine epidemic diarrhea virus (PEDV) and Porcine transmissible gastroenteritis virus (TGEV), a pair of specific primers was designed and the RT-PCR (reverse transcription PCR) method for detecting PDCoV was established by determining the optimum reaction conditions and the specificity, sensitivity and reproducibility was evaluated, respectively, epidemiological investigation of PDCoV in Sichuan was conducted by detecting 226 clinical diarrhea samples, and the tissue distribution of PDCoV in infected piglets after challenging 7-day-old piglets was assessed. The results showed the target M gene fragment was 654 bp by RT-PCR, the optimum annealing temperature is 60 ℃, the upstream and downstream primer volume was 1.75 μL.The method had high sensitivity and specificity with a limited detection of 8.82×109 IU/mL for PDCoV, respectively, and no cross-reaction with other reference virus such as PEDV, TGEV, Japanese encephalitis virus (JEV), Porcine rota virus (RV), Porcine respiratory and reproductive syndrome virus (PRRSV) or Classical swine fever virus (CSFV). The positive rate of PDCoV in 226 clinical samples from Sichuan was 7.1% (16/226). The M gene of PDCoV CHN-SC2015 strain was sequenced and compared with other 28 reference M genes of PDCoV strains collected from GenBank. The results showed that the nucleotide and amino similarity was 98%~100% and 99%~100%, respectively. From the challenged piglets, PDCoV could be detected from heart, liver, spleen, lung, kidney, colon, ileum and jejunum, indicating that PDCoV had a broad tissue tropism. The established RT-PCR in this study is a simple, rapid and specific assay suitable for clinically detecting PDCoV.
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