柯萨奇B组3型病毒SYBR GreenⅠRT-PCR检测方法的建立  

Establishment of SYBR GreenⅠRT-PCR Detection Assay for Coxsackievirus B3

作　　者：张伟 李咏洁 杨凤梅[1] 李艳艳[1] 靳玮华 徐鸿界 李明学 刘权 赵远[1] 和占龙[1] ZHANG Wei;LI Yongjie;YANG Fengmei(In-stitute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College/Yunnan Key Laboratory of Vaccine Re-search and Development on Severe Infectious Diseases,Yunnan 650118,China)

机构地区：[1]中国医学科学院医学生物学研究所,昆明650118

出　　处：《医学研究杂志》2022年第1期55-59,共5页Journal of Medical Research

基　　金：中国医学科学院医学与健康科技创新工程项目(2016-I2M-2-001);云南省重大科技专项(2017ZF020);昆明市科技创新和服务能力提升计划重点项目(2016-2R-07674)。

摘　　要：目的建立一种灵敏、特异的柯萨奇B组3型(CV-B3)病原SYBR GreenⅠRT-PCR实时荧光定量检测方法,用于快速、准确地检测CV-B3。方法设计柯萨奇B组病毒通用引物以及VP1基因的T7启动子特异性引物,以体外转录获得的RNA为标准品,绘制标准曲线,建立CV-B3的荧光定量PCR检测方法;对检测方法的敏感度、特异性、重复性进行评价。结果该检测方法在10~10^(9)拷贝/微升范围内具有良好的扩增效率和线性关系,最低检测限为10拷贝/微升,标准曲线r^(2)=0.999,扩增效率为95.7%;且对柯萨奇病毒A组6型(CV-A6)、柯萨奇病毒A组10型(CV-A10)、肠道病毒71型(EV-71)均无交叉反应,重复性实验变异系数(CV%)<2%。结论本实验建立的检测方法敏感度较高,同时具备良好的特异性及重复性,可用于CV-B3病原的临床及实验室样本的定性定量检测。Objective To Establish a sensitive and specific SYBR GreenⅠreal-time quantitative PCR(RT-qPCR)for rapid and accurate detection of coxsackievirus B3(CV-B3).Methods Universal primers for Coxsackie B virus and specific primers for the VP1 gene with T7 promoter of this Serotype strain was designed.RNA obtained from in vitro transcription was used as the standard.Standard curve was drawn,and a RT-qPCR method was established to detect CV-B3.The sensitivity,specificity and repeatability of this detection method was evaluated.Results The method showed a good amplification efficiency and linear relationship in the range of 10-10^(9) copies/μl.The lowest detection limit was 10copies/μl,the r^(2) coefficient of the standard curve was 0.999,and the amplification efficiency was 95.7%.There was no cross reaction was found with coxsackievirus A6(CV-A6),coxsackievirus A10(CV-A10)and enterovirus 71(EV-71)in the method and the coefficient of variation(CV%)value of repeatability experiment was less than 2%.Conclusion The detection method established in this experiment has strong sensitivity,specificity,and reproducibility,and can be used for the qualitative and quantitative detection of the clinical and laboratory samples.

关 键 词：柯萨奇病毒B组3型 SYBR GreenⅠ RT-PCR 

分 类 号：R373[医药卫生—病原生物学]