Establish of A Rapid and Visible Assay Method for Detection of Porcine Epidemic Diarrhea Virus Using Recombinase-aided Isothermal Nucleic Acid Amplification in Combination with Nucleic Acid Strips
The aim of this study was to establish a visualization detection method for porcine epidemic diarrhea virus(PEDV)based on recombinase-mediated isothermal amplification(RAA)technology combined with nucleic acid strip tests,which can be applied to on-site rapid detection in breeding farms.The conserved sequence of the PEDV N gene was selected as template.Specific primers and fluorescent probes were designed,and the primer sets were compared for screening.The reaction conditions,primer concentration,and probe concentration were optimized,and the specificity,sensitivity,and repeatability were evaluated.The established detection method was applied to clinical samples and compared with quantitative fluorescent PCR.Results showed that the established method could complete the detection in 10 minutes at an isothermal temperature of 38℃,plus 5 minutes of lateral flow strip testing.There was no cross-reactivity with porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV),porcine circovirus 2(PCV2),porcine deltacoronavirus(PDCoV),porcine rotavirus(PoRV),transmissible.gastroenteritis of swine virus(TGEV).The lowest detection limit was 101 copies/pL.The detection results were consistent with those of the quantitative fluorescent PCR in detection of 40 clinical samples,including 20 negative and 20 positive samples.The method established in this study has advantages of high specificity,sensitivity,short testing time,and simple instrumentation,making it suitable for on-site testing in grassroots areas.
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