Use of dual priming oligonucleotide system-based multiplex RT-PCR assay to detect five diarrhea viruses in pig herds in South China

Guangbin Si,Xia Zhou,Zhifei Chen,Jiawei Niu,Ruiai Chen,Yongsheng Xie,Gen Li,Dongsheng He

doi:10.1186/s13568-021-01255-z

Abstract

In this study, a specific and simple method based on the dual priming oligonucleotide (DPO) system was developed to simultaneously detect transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus A (PRV-A), porcine delta coronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), associated with the major enteric RNA viruses in pigs. The DPO system-based multiplex RT-PCR method simplified the primer design and did not require optimization of the annealing temperature. Specificity analysis revealed that the method could specifically detect TGEV, PEDV, PRV-A, PDCoV, and SADS-CoV without any cross-amplification of other circulating swine viruses. The limit of detection of the method was as low as 103–104 copies/μL plasmid of each virus. The method also had good repeatability, and obvious results were seen in three repeat experiments with an interval of 45 days. This optimized multiplex RT-PCR method was used to evaluate 181 clinical swine samples that were collected from four provinces of China between September 2016 and August 2018. The results showed that the positive detection rates of PEDV, PDCoV, SADS-CoV, PRV-A, and TGEV were 30.94% (56/181), 17.67% (32/181), 11.6% (21/181), 9.39% (17/181), and 0.55% (1/181), respectively. Mixed infection of two or more viruses was also common. The DPO system-based multiplex RT-PCR could be a useful tool for detecting enteric virus infections. This method has the advantages of easy operation, low cost, high detection efficiency, and short running time for early diagnosis in clinical cases.

Highlights

Diarrhea, especially viral diarrhea, seriously endangers the pig industry throughout the world; it is characterized by acute diarrhea, dehydration, and high mortality in the early stage of suckling piglets, and it results in severe economic losses in the global pig industry (Zhang et al 2016)
We developed a dual priming oligonucleotide (DPO) system-based multiplex RT-polymerase chain reaction (PCR) assay for the differential detection of Transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus A (PRV-A), porcine delta coronavirus (PDCoV), and SADS-CoV in the same reaction vial
Establishment of the single RT‐PCR of the DPO system‐based assay The specific amplifications of SADS-CoV, TGEV, PEDV, PDCoV, and PRV-A were observed at the expected sizes of 586, 472, 361, 263, and 123 bp (Fig. 1), respectively

Summary

Introduction

Especially viral diarrhea, seriously endangers the pig industry throughout the world; it is characterized by acute diarrhea, dehydration, and high mortality in the early stage of suckling piglets, and it results in severe economic losses in the global pig industry (Zhang et al 2016). Porcine deltacoronavirus (PDCoV) was first detected in pig feces in Hong Kong in 2012 (Woo et al 2012); subsequently, this virus spread, causing severe diarrhea and/ or vomiting and atrophy in pigs in the United States (Li et al 2014). In 2017, a novel swine enteric alphacoronavirus (SeACoV)—causing acute vomiting, watery diarrhea, and high mortality of newborn-piglets—was firstly reported in China (Gong et al 2017; Pan et al 2017). It was named the porcine enteric alphacoronavirus (PEAV) (Xu et al 2019) or the swine acute diarrhea syndrome coronavirus (SADS-CoV) (Zhou et al 2018). For the sake of consistency, the term SADS-CoV is used to refer to this newly-emerging virus in current research (Yang et al 2019)

Methods

Results

Conclusion