柯萨奇病毒B组5型TaqMan一步法RT-qPCR检测方法的建立  

Establishment of TaqMan One-step Real-time Quantitative Polymerase Chain Reaction Assay to Detect Coxsackievirus B5

作　　者：刘煜菡 张名[1] 郭伟 许丹菡 冯昌增[1] 徐丽兰[1] 马绍辉[1] LIU Yuhan;ZHANG Ming;GUO Wei(Key Laboratory for Research and Development of Major Infectious Diseases Vaccine in Yunnan,Institute of Medical Biology,Chinese Academy of Medical Sciences,Yunnan 650118,China)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所、云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《医学研究杂志》2024年第7期84-88,共5页Journal of Medical Research

基　　金：云南省科技计划项目(202202AA100016)。

摘　　要：目的建立柯萨奇病毒B组5型(coxsackievirusB5,CV-B5)特异的TaqMan一步法实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测方法。方法根据CV-B5的VP1序列,设计特异性引物和TaqMan探针。将目的基因插入pMD18^(TM)载体,在DH5α感受态细胞中扩增,大肠杆菌体外转录后获得RNA标准品并建立标准曲线,最后对检测方法的重复性、敏感度和特异性进行评价。结果该方法在10^(3)~10^(11)拷贝/微升的模板范围内具有良好的线性关系(r^(2)>0.99),扩增效率E=100.9%,敏感度达1×10^(3)拷贝/微升,只对CV-B5有特异性扩增曲线,且重复性较好。结论本实验建立的TaqMan一步法RT-qPCR检测方法有较高的敏感度、特异性和重复性,有良好的抗干扰性,可用于CV-B5的临床样本检测和绝对定量分析。Objective To establish a TaqMan one-step real-time quantitative polymerase chain reaction(RT-qPCR)assay spe-cific for coxsackievirus B5(CV-B5).Methods Specific primers and TaqMan probes were designed based on the VPlsequence of CV-B5.The target gene was inserted into the pMD18TM vector,amplified in DH5αreceptor clls,and RNA standards were obtained from escherichia coli transcribed in vitro and a standard curve was established,and finally the reproducibility,sensitivity and specificity of the assay were evaluated.Results The method had good linearity(r^(2)>0.99)in the template range of 10^(3)~10^(11)copies/μl,amplifica-tion efficiency E=100.9%,sensitivity up to 1×10^(3)copies/μul,and specific amplification curve for CV-B5 only,and good reproduc-ibility.Conclusion The TaqMan one-step RT-qPCR assay established in this experiment has high sensitivity,specificity,reproducibili-ty,good anti-interference performance,which can be used for clinical sample detection and absolute quantitative analysis of CV-B5.

关 键 词：柯萨奇病毒B组5型 实时荧光定量聚合酶链反应 TaqMan探针法 

分 类 号：R373.9[医药卫生—病原生物学]