小鼠肝炎病毒逆转录-重组酶聚合酶扩增检测方法的建立、验证及应用  

Development,verification and application of reverse transcription-isothermal recombinase polymerase amplification method for detection of mouse hepatitis virus

作　　者：闫雪 李明 张旭[2] 张振[2] 王霄 马跃宇 马鸣潇 费东亮 YAN Xue;LI Ming;ZHANG Xu;ZHANG Zhen;WANG Xiao;MA Yue-yu;MA Ming-xiao;FEI Dong-liang(College of Animal Husbandry and Veterinary Medicine,Jinzhou Medical University,Jinzhou 121000,Liaoning Province,China;不详)

机构地区：[1]锦州医科大学畜牧兽医学院,辽宁锦州121000 [2]锦州市疾病预防控制中心,辽宁锦州121000

出　　处：《中国生物制品学杂志》2022年第6期729-733,共5页Chinese Journal of Biologicals

基　　金：辽宁省重点研发计划项目(2020JH2/10300117)。

摘　　要：目的建立小鼠肝炎病毒(mouse hepatitis virus,MHV)的逆转录-重组酶聚合酶扩增(reverse-trancription recombinase ploymerase amplification,RT-RPA)检测方法。方法以GenBank中登录的MHV-FJ株(FJ6647223)M基因保守区段(82~317 bp)为靶基因,设计合成6对RPA扩增引物(M-MHV-1~6),筛选1对最适引物,同时优化反应温度(31、33、35、37、39、41℃)和时间(20、25、30、35 min),并验证该方法的特异性和敏感性。采用优化方法检测32份野生田鼠肝脏组织样本,并与RT-PCR法检测结果进行对比。结果筛选最适引物为M-MHV-2,最适反应温度和时间分别为37℃和30 min。该方法的检测下限为4.75×10;copies/μL,对鼠肺炎病毒(Pneumonia virus of mice,PVM)、仙台病毒(Sendai virus,SV)、汉坦病毒(Hantaan virus,HV)、鼠细小病毒(minute virus of mice,MVM)、呼肠弧病毒Ⅲ型(reovirus typeⅢ,ReoⅢ)无交叉反应。32份野生田鼠肝脏组织样本中,检测出阳性样本1份,检出率为3.13%(1/32),与RT-PCR检测结果一致。结论建立了MHV的RT-RPA检测方法,该方法的特异性及灵敏性良好,且具有反应迅速、操作简单等优点。本研究为MHV快速检测方法的进一步研发奠定了基础。Objective To develop a reverse transcription-recombinase polymerase amplification(RT-RPA)method for detection of mouse hepatitis virus(MHV).Methods Based on the conservative segment(82~317 bp)of M gene of MHV-FJ strain(FJ6647223)in GenBank,six pairs of RPA primers(M-MHV-1~6)were designed and synthesized,from which a pair of optimal primers were screened.Meanwhile,the temperature(31,33,35,37,39 and 41℃)and time(20,25,30 and 35 min)for reaction were optimized.The developed method was verified for specificity and sensitivity,by which 32 liver specimens of wild mice were determined,and the results were compared with those by RT-PCR.Results The screened optimal primer was M-MHV-2,while the optimal temperature and time for reaction were 37℃and30 min respectively.The minimum detection limit of the method was 4.75×10~2copies/μL.No cross reactions with pneumonia virus of mice(PVM),Sendai virus(SV),Hantaan virus(HV),minute virus of mice(MVM)and Reovirus typeⅢ(ReoⅢ)were observed.One positive specimen was found in the 32 liver specimens of wild mice,indicating a detection rate of 3.13%,which was consistent with that by RT-PCR.Conclusion The RT-RPA rapid detection method for mouse hepatitis virus has been successfully established.This method has the advantages of good specificity,rapid sensitivity,rapid response,simple operation,etc.,which provides help for the further development of MHV rapid detection equipment.

关 键 词：小鼠肝炎病毒 重组酶聚合酶 优化 验证 

分 类 号：S855.3[农业科学—临床兽医学]