羊痘病毒与羊口疮病毒双重RPA-LFD检测方法的建立与应用OA北大核心CSTPCD

[Objective]Based on recombinase polymerase amplification(RPA)detection technology combined with(lateral flow dipstick,LFD),it is aimed to establish dual RPA-LFD method for the rapid identification of capripox virus(CaPV)and orf virus(ORFV).[Method]The conserved fragments of CaPV P32 gene and ORFV 011 gene were selected for the amplification of target fragments and the construction of recombinant plasmids.Three pairs of primers for CaPV and ORFV RPA,and one probe for CAPV-HP and ORFV-HP were designed.Single base RPA primer screening test was performed.The reaction time and temperature of double RPA-LFD were optimized.The optimal primers and probe matching systems of dual RPA-LFD were explored.Dual RPA-LFD sensitivity,specificity and repeatability tests were performed.The method was used to detect 55 clinical samples.[Result]The results of primer screening test showed that the primers had the strongest specificity and the highest amplification efficiency for CaPV-RPA-F3/R3 and ORFV-RPA-F1/R1.The method had the best amplification efficiency at reaction temperature of 39℃and reaction time of 11 min.The optimal primer ratio of CaPV-RPA-F3/R3 and ORFV-RPA-F1/R1 was 0.8 μL∶1.6 μL,and the optimal probe ratio of CaPV-HP and ORFV-HP was 0.1 μL∶0.9 μL.The minimum detection limit of dual RPA-LFD sensitivity test was CaPV/ORFV 4.65/3.94×100 copies/μL.The specific test results showed no cross-reaction with PPRV,IBRV,Sau and SPN.The positive rate of RPA-LFD was consistent with that of PCR.[Conclusion]This study successfully established a dual RPA-LFD detection method for CaPV and ORFV,which can be used for the rapid differential diagnosis of CaPV and ORFV mixed infection in clinic.