Abstract
Porcine epidemic diarrhea virus (PEDV) is a coronavirus currently widespread worldwide in the swine industry. Since PEDV was discovered in China in 1984, it has caused huge economic losses in the swine industry. PEDV can infect pigs of all ages, but piglets have the highest infection with a death rate as high as 100%, and the clinical symptoms are watery diarrhea, vomiting, and dehydration. At present, there is not any report on PEDV detection by RT-RAA. In this study, we developed an isothermal amplification technology by using reverse-transcription recombinase-aided amplification assay (RT-RAA) combined with portable instruments to achieve a molecular diagnosis of PEDV in clinical samples from China. By designing a pair of RT-RAA primers and probes based on the PEDV N gene, this method breaks the limitations of existing detection methods. The assay time was within 30 min at 41 °C and can detect as few as 10 copies of PEDV DNA molecules per reaction. Sixty-two clinical tissue samples were detected by RT-qPCR and RT-RAA. The positive and negative rates for the two methods were 24.19% and 75.81%, respectively. Specificity assay showed that the RT-RAA had specifically detected PEDV and was not reactive for porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), swine flu virus (SIV), or porcine Japanese encephalitis virus (JEV). The results suggested that RT-RAA had a strong specificity and high detection sensitivity when combined with a portable instrument to complete the detection under a constant temperature of 30 min, which are more suitable for preventing and controlling PEDV onsite in China.
Highlights
Porcine epidemic diarrhea virus (PEDV) is the pathogen of porcine epidemic diarrhea (PED), which can cause epidemic diseases in the global swine industry
The porcine pseudorabies virus (PRV, batch no. 20210388), classical swine fever virus (CSFV, batch no. 20210210) and swine flu virus (SIV, batch no. 20210101) were commercially available vaccines purchased from Wuhan Keqian Biology Co., Ltd., (Wuhan, China)
Sixty-two clinical samples in this study were provided by Wen’s Foodstuff Group Co., Ltd., (Yunfu, China). They were the intestinal tissues of pigs that died of diarrhea and were suspected of being infected with PEDV
Summary
Porcine epidemic diarrhea virus (PEDV) is the pathogen of porcine epidemic diarrhea (PED), which can cause epidemic diseases in the global swine industry. PEDV is an enveloped virus, and a member of the Coronaviridae family and genus Alphacoronavirus. Its single- and positive-stranded RNA is similar to other coronaviruses, with a full-length genome of 28,033–28,038 nt [1]. The mainly clinical features of PED are acute watery diarrhea, vomiting, dehydration, and weight loss. Pigs of all ages are susceptible to PEDV, and the morbidity and mortality of suckling piglets reach 80–100%, causing huge economic losses in the swine industry [2,3]. Since PED was discovered in the UK in 1971, the disease has spread to European countries and Asian regions, especially China and South
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