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
刘煜菡 1张名 1郭伟 1许丹菡 1冯昌增 1徐丽兰 1马绍辉1
作者信息
- 1. 650118 昆明,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
- 折叠
摘要
目的 建立柯萨奇病毒B组5型(coxsackievirus B5,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pMD18TM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103～1011拷贝/微升的模板范围内具有良好的线性关系(r2＞0.99),扩增效率E=100.9％,敏感度达1 × 103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.
Abstract
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 VP1 sequence of CV-B5.The target gene was inserted into the pMD18TM vector,amplified in DH5α receptor cells,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(r2＞0.99)in the template range of 103-1011 copies/µl,amplifica-tion efficiency E=100.9％,sensitivity up to 1 × 103 copies/µl,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Key words
Coxsackievirus B5/Real-time quantitative polymerase chain reaction/TaqMan probe method引用本文复制引用
基金项目
云南省科技计划项目(202202AA100016)
出版年
2024
NETL
NSTL
万方数据