生物技术通报2024，Vol.40Issue(5)：103-111,9.DOI:10.13560/j.cnki.biotech.bull.1985.2023-1106
羊痘病毒与羊口疮病毒双重RPA-LFD检测方法的建立与应用
Establishment and Application of Dual RPA-LFD Detection Method for Capripox Virus and Orf Virus
摘要
Abstract
[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.关键词
羊痘病毒/羊口疮病毒/RPA-LFD/鉴别诊断/混合感染Key words
CaPV/ORFV/RPA-LFD/differential diagnosis/mixed infection引用本文复制引用
王玲玲,马爱红,宋前进,王小龙,曹晓真,刘治凤,陈亚飞,李继东..羊痘病毒与羊口疮病毒双重RPA-LFD检测方法的建立与应用[J].生物技术通报,2024,40(5):103-111,9.基金项目
宁夏回族自治区科技创新团队建设项目(2022BSB03107),宁夏银川市2023年推进产学研融合助力农业高质量发展项目 （2022BSB03107）
