PMID- 35336998 OWN - NLM STAT- MEDLINE DCOM- 20220331 LR - 20220401 IS - 1999-4915 (Electronic) IS - 1999-4915 (Linking) VI - 14 IP - 3 DP - 2022 Mar 12 TI - Development and Application of a Reverse-Transcription Recombinase-Aided Amplification Assay for Porcine Epidemic Diarrhea Virus. LID - 10.3390/v14030591 [doi] LID - 591 AB - 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 degrees 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. FAU - Wu, Xiuhong AU - Wu X AD - Heyuan Branch, Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology, College of Animal Science, South China Agricultural University, Guangzhou 510642, China. AD - Guangdong Engineering Research Center for Vector Vaccine of Animal Virus, Guangzhou 510642, China. AD - South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642, China. FAU - Liu, Yuanjia AU - Liu Y AD - Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China. FAU - Gao, Liguo AU - Gao L AD - Heyuan Branch, Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology, College of Animal Science, South China Agricultural University, Guangzhou 510642, China. AD - Guangdong Engineering Research Center for Vector Vaccine of Animal Virus, Guangzhou 510642, China. AD - South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642, China. FAU - Yan, Zhuanqiang AU - Yan Z AD - Guangdong Enterprise Key Laboratory for Animal Health and Environmental Control, Wen's Foodstuff Group Co., Ltd., Yunfu 527439, China. FAU - Zhao, Qiqi AU - Zhao Q AD - Heyuan Branch, Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology, College of Animal Science, South China Agricultural University, Guangzhou 510642, China. AD - Guangdong Engineering Research Center for Vector Vaccine of Animal Virus, Guangzhou 510642, China. AD - South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642, China. FAU - Chen, Feng AU - Chen F AD - Heyuan Branch, Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology, College of Animal Science, South China Agricultural University, Guangzhou 510642, China. AD - Guangdong Engineering Research Center for Vector Vaccine of Animal Virus, Guangzhou 510642, China. AD - South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642, China. FAU - Xie, Qingmei AU - Xie Q AD - Heyuan Branch, Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology, College of Animal Science, South China Agricultural University, Guangzhou 510642, China. AD - Guangdong Engineering Research Center for Vector Vaccine of Animal Virus, Guangzhou 510642, China. AD - South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642, China. FAU - Zhang, Xinheng AU - Zhang X AUID- ORCID: 0000-0002-3232-4929 AD - Heyuan Branch, Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology, College of Animal Science, South China Agricultural University, Guangzhou 510642, China. AD - Guangdong Engineering Research Center for Vector Vaccine of Animal Virus, Guangzhou 510642, China. AD - South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642, China. LA - eng GR - 2020B020222001/Key Research and Development Program of Guangdong Province/ GR - 2019A1515012006/Guangdong Basic and Applied Basic Research Foundation/ GR - 31902252, 31972659/the National Natural Science Foundation of China/ GR - 2019KJ128/the Chief expert Project of Agricultural Industry Technology system in Guangdong Province/ PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20220312 PL - Switzerland TA - Viruses JT - Viruses JID - 101509722 RN - 0 (Recombinases) SB - IM MH - Animals MH - *Porcine epidemic diarrhea virus/genetics MH - Recombinases/genetics MH - Reverse Transcription MH - Sensitivity and Specificity MH - Swine MH - *Swine Diseases/diagnosis PMC - PMC8948910 OTO - NOTNLM OT - clinical diagnosis OT - constant temperature detection OT - porcine epidemic diarrhea virus OT - reverse-transcription recombinase-aided amplification assay COIS- The authors declare no conflict of interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. EDAT- 2022/03/27 06:00 MHDA- 2022/04/01 06:00 PMCR- 2022/03/12 CRDT- 2022/03/26 01:07 PHST- 2022/02/01 00:00 [received] PHST- 2022/02/25 00:00 [revised] PHST- 2022/03/08 00:00 [accepted] PHST- 2022/03/26 01:07 [entrez] PHST- 2022/03/27 06:00 [pubmed] PHST- 2022/04/01 06:00 [medline] PHST- 2022/03/12 00:00 [pmc-release] AID - v14030591 [pii] AID - viruses-14-00591 [pii] AID - 10.3390/v14030591 [doi] PST - epublish SO - Viruses. 2022 Mar 12;14(3):591. doi: 10.3390/v14030591.