小鼠巨细胞病毒荧光定量PCR检测方法的建立及应用

Establishment and Application of Fluorescent Quantitative PCR Assay for Mice Cytomegalovirus

目的建立小鼠巨细胞病毒(Mouse Cytomegalovirus,MCMV)荧光定量PCR方法并对其进行初步应用。方法选取NCBI发表的MCMV Smith株DNA polymerase基因保守序列设计引物探针,建立MCMV的荧光定量PCR方法,对方法的特异性、敏感性、重复性及稳定性进行验证,并应用该方法检测掺入MCMV的小鼠血液样品及2018年度送检的409份小鼠血液样本。结果建立的MCMV荧光定量PCR方法标准曲线Slope为-3.418,R2值为0.999,扩增效率为96.137%,可定量检测到的MCMV最低含量为47 copies/μL。以大鼠巨细胞病毒,猴巨细胞病毒,人单纯疱疹病毒,伪狂犬病毒及猫疱疹病毒I型为模板均无扩增曲线,特异性良好。方法组内和组间变异系数分别为0.39%~0.68%和0.48%~1.01%,重复性和稳定性好。可检测到掺入小鼠血液样品中MCMV病毒的最大稀释度为1∶1000(100.75 TCID50/0.1 m L),409份小鼠血液样品经检测均为阴性。结论建立的小鼠巨细胞病毒荧光定量PCR方法有很好的敏感性、特异性及稳定性,可有效地检测小鼠中MCMV,为实验小鼠MCMV的监测及相关标准的补充完善提供了技术参考。

Objective To establishment and apply fluorescent quantitative PCR method for mouse cytomegalovirus.Method Primer probes were designed by using the conserved sequence of MCMV Smith strain DNA polymerase gene published by NCBI to establish a quantitative PCR method for MCMV.The specificity,sensitivity,repeatability and stability of the method were verified.The sample was applied to detect mice samples mixed with MCMV and 409 mice blood samples sent in 2018.Result The established FQ-PCR method for MCMV has a standard curve Slope of-3.418,R2 value of 0.999,and an amplification efficiency of 96.137%.The minimum detectable amount of MCMV was 47 copies/μL.There was no amplification curve when rat cytomegalovirus,monkey cytomegalovirus,human herpes simplex virus,pseudorabies virus and cat herpesvirus type I were used as templates,which showing the good specificity.The intra-and inter-assay coefficients of variation were 0.39%-0.68%and 0.48%-1.01%,respectively,with good repeatability and stability.The maximum dilution of MCMV virus in the mouse blood samples was detected to be 1∶1000(100.75 TCID50/0.1 m L),and 409 mice blood samples were tested negative.Conclusion The established FQ-PCR method for mouse cytomegalovirus has good sensitivity,specificity and stability,and can effectively detect MCMV in mice,which provides technical reference for the monitoring of MCMV in lab mice and improvement of related standards.