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

作　　者：王玲玲[1] 马爱红 宋前进 王小龙 曹晓真 刘治凤 陈亚飞 李继东[1] WANG Ling-ling;MA Ai-hong;SONG Qian-jin;WANG Xiao-long;CAO Xiao-zhen;LIU Zhi-feng;CHEN Ya-fei;LI Ji-dong(College of Animal Science and Technology,Ningxia University,Yinchuan 750021;Yinchuan Animal Husbandry Technology Extension Service Center,Yinchuan 750006)

机构地区：[1]宁夏大学动物科技学院,银川750021 [2]银川市畜牧技术推广服务中心,银川750006

出　　处：《生物技术通报》2024年第5期103-111,共9页Biotechnology Bulletin

基　　金：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。

摘　　要：【目的】旨在建立一种基于重组聚合酶(recombinase polymerase amplification,RPA)检测技术结合测流层析试纸条(lateral flow dipstick,LFD)快速鉴别检测羊痘病毒(capripox virus,CaPV)与羊口疮病毒(orf virus,ORFV)的双重RPA-LFD检测方法。【方法】选取CaPV P32基因及ORFV 011基因的保守片段,进行目的片段扩增并构建重组质粒;设计CaPV及ORFV RPA引物各3对,CaPV-HP、ORFV-HP探针各1条;进行单重基础RPA引物筛选试验;对双重RPA-LFD的反应时间及反应温度进行优化;探索双重RPA-LFD最佳引物配比体系及最佳探针配比体系;进行双重RPA-LFD灵敏度、特异性及重复性试验;用所建立的方法对采集的55份临床样品进行检测。【结果】引物筛选试验结果显示,引物对CaPV-RPA-F3/R3、ORFV-RPA-F1/R1的特异性最强、扩增效率最高;该方法在反应温度为39℃、反应时间为11 min的反应条件下扩增效率最佳;CaPV-RPA-F3/R3、ORFV-RPA-F1/R1的最佳引物配比为0.8 μL∶1.6 μL,CaPV-HP、ORFV-HP的最佳探针配比为0.1 μL∶0.9 μL;双重RPA-LFD灵敏性试验最低检出限为CaPV/ORFV 4.65/3.94×10~0 copies/μL;特异性试验结果显示与PPRV、IBRV、Sau、SPN均无交叉反应;临床样品检测结果显示RPA-LFD检测阳性率与PCR检测阳性率相符合。【结论】成功建立了CaPV与ORFV双重RPA-LFD快速检测方法,可用于临床中CaPV与ORFV混合感染的快速鉴别诊断。【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 0.8μL∶1.6μL,and the optimal probe ratio of CaPV-HP and ORFV-HP was 0.1μL∶0.9μL.The minimum detection limit of 0 dual RPA-LFD sensitivity test was CaPV/ORFV 4.65/3.94×10 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 

分 类 号：S85[农业科学—兽医学]