荧光RAA检测小鼠微小病毒的研究和应用  

Application of recombinase-aid nucleic acid amplification for detection of minute virus of mice

作　　者：赵晓瑜 佟巍[1] 郭智 向志光[1] 魏强[1] ZHAO Xiaoyu;TONG Wei;GUO Zhi;XIANG Zhiguang;WEI Qiang(Institute of Laboratory Animal Sciences,CAMS/PUMC,Beijing 100021,China)

机构地区：[1]中国医学科学院医学实验动物研究所,北京100021

出　　处：《中国比较医学杂志》2023年第4期63-68,82,共7页Chinese Journal of Comparative Medicine

基　　金：国家重点研发计划青年科学家项目(2021YFF0702900)。

摘　　要：目的 病毒培养、组织学、免疫学及普通PCR方法等在小鼠微小病毒(minute virus of mice, MVM)的检测方面都存在一定的不足,不能满足目前实验动物饲养机构现场检测MVM病毒的需要,因此探索开发一种新的、简单易用,便于现场检测MVM的检测方法具有重要的意义。方法 根据Genbank公布的MVM序列,对MVM高度保守序列区域Ns1基因设计荧光重组酶介导链替换核酸扩增的引物和探针,建立了一种MVM荧光法重组酶介导链替换核酸扩增技术(Real-time RAA)检测方法,对其特异性、室温储存稳定性、灵敏度及临床样本验证方面与qPCR法进行对比分析。结果 荧光RAA法在39℃20 min内即可检测到MVM病毒特异性扩增曲线;其灵敏度为10 copies/μL;与Reo-3、MHV-1、MHV-3、MHV-A59、MHV-JHM病毒均无交叉反应;检测试剂室温储存20 d无明显的扩增性能下降;分别对15份阳性模拟样品进行荧光RAA法与qPCR法的检测比对,结果显示两种方法的符合率为15/15;对两种方法的相关性进行统计学分析,结果显示相关系数R^(2)=0.85,两种方法具有显著的线性相关性。结论 本研究建立的MVM荧光RAA法,操作简单,特异性强、灵敏度高,能够快速有效地检出临床样本。Objective Virus culture,histology,immunology,and PCR have certain shortcomings in the detection of minute virus of mice(MVM),which cannot meet the current needs.Exploring a new method for Rapid and reliable detection of MVM is a major problem that needs to be solved.Methods On the basis of the MVM sequence published by GenBank,we designed primers and probes for Real-time recombinase-aid nucleic acid amplification(RAA)of the NS1 gene,a highly conserved sequence region of MVM,and established a real-time RAA detection method of MVM.We analyzed its specificity,sensitivity,storage stability,and clinical sample validation in comparison with qPCR.Results The sensitivity was 10 copies/μL,and there was no cross-reactivity with Reo-3,MHV-1,MHV-3,MHV-A59,or MHVJHM viruses.There was no significant deterioration in amplification performance of the RAA stored at room temperature for 20 days.Additionally,the Real-time RAA method was compared with qPCR in 15 positive simulative samples,and the compliance rate was 15/15.Statistical analysis of the correlation between the two method revealed a correlation coefficient of R^(2)=0.85,which indicates that the two method were significantly correlated.Conclusions The MVM real-time RAA method in this study quickly and effectively detects MVM virus,which also has the advantages of simple operation,strong specificity,and high sensitivity.

关 键 词：小鼠微小病毒 荧光RAA法 快速检测 

分 类 号：R-33[医药卫生]