This study aims to develop a recombinase aided amplification(RAA)technology to de-tect porcine epidemic diarrhea virus(PEDV).A set of RAA primers and probes with high amplifi-cation efficiency and specificity was designed,specifically targeting the M gene.The amplification process was monitored in real-time using a fluorescent constant temperature detector to facilitate the rapid,sensitive,and specific detection of PEDV nucleic acids.The results showed that the es-tablished method exhibited excellent sensitivity,with a minimum detection limit of 8.86 × 101 copies/μL.Furthermore,the detection method has good specificity and reproducibility,with no cross-reactions observed with other porcine viruses such as transmissible gastroenteritis virus(TGE),porcine coronavirus,and porcine circovirus.The method also showed clear amplification curves at constant temperatures ranging from 37.0 to 41 ℃,highlighting its good temperature a-daptability.The establishment of fluorescent RAA technology for PEDV detection method provides a method for on-site rapid detection of PEDV.

Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.

This study aims to develop a recombinase aided amplification(RAA)technology to de-tect porcine epidemic diarrhea virus(PEDV).A set of RAA primers and probes with high amplifi-cation efficiency and specificity was designed,specifically targeting the M gene.The amplification process was monitored in real-time using a fluorescent constant temperature detector to facilitate the rapid,sensitive,and specific detection of PEDV nucleic acids.The results showed that the es-tablished method exhibited excellent sensitivity,with a minimum detection limit of 8.86 × 101 copies/μL.Furthermore,the detection method has good specificity and reproducibility,with no cross-reactions observed with other porcine viruses such as transmissible gastroenteritis virus(TGE),porcine coronavirus,and porcine circovirus.The method also showed clear amplification curves at constant temperatures ranging from 37.0 to 41 ℃,highlighting its good temperature a-daptability.The establishment of fluorescent RAA technology for PEDV detection method provides a method for on-site rapid detection of PEDV.

Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.