实验动物与比较医学2017，Vol.37Issue(5)：372-377,6.DOI:10.3969/j.issn.1674-5817.2017.05.006
大鼠微小病毒和细小病毒双重荧光定量PCR检测方法建立
Establishment of TaqMan Real-time Fluorescent Quantitative PCR for Detecting Rat Minute Virus and Rat Parvovirus
摘要
Abstract
Objective To establish TaqMan real-time fluorescent quantitative PCR method which can detect rat minute virus (RMV) and rat parvovirus (RPV) quickly and accurately in clinic.Method According to the whole genome sequence of strain RMV NTU1 (Accession No.JX627317.1 in Genbank) the primers and TaqMan probe were designed from the 1705～1808 nt.And according to the whole genome sequence of strain RPV NTU1 (JX827169) the primers and TaqMan probe were designed from the 863-967nt.The stability,specificity,and sensitivity of the method were evaluated through realtime quantitative PCR method,which is based on the standardized plasmid constructed.50 clinical samples were detected using this fluorescence quantitative method,which validated with the traditional ELISA method.Result The real-time quantitative PCR for detecting RMV and RPV showed a perfect linear relationship of standard curve,and R2 value reached 0.99 with a high specificity.The sensitivity of the real-time PCR was 10 copies/μL at minimum.Due to dual specificity of primer and probe,TaqMan quantitative PCR is extremely accurate.One hundred liver samples and 50 serum samples were negative via ELISA and real time quantitative PCR respectively.Conclusion The TaqMan probe-based real-time PCR method is established with good specificity and sensitivity,which can make a powerful technical support for RMV and RPV investigation and detection.关键词
大鼠微小病毒(RMV)/大鼠细小病毒(RPV)/TaqMan探针/荧光定量PCRKey words
Rat minute virus (RMV)/Rat parvovirus (RPV)/TaqMan probe/Real-time PCR分类
生物科学引用本文复制引用
孙竹筠,蔡骁垚,熊炜,陈懿斐,张泉,李泽君,魏晓锋,陈鸿军..大鼠微小病毒和细小病毒双重荧光定量PCR检测方法建立[J].实验动物与比较医学,2017,37(5):372-377,6.基金项目
上海市科委实验动物专项资金资助(15140900600) （15140900600）
