柯萨奇病毒B组2型SYBR GreenⅠRT-PCR检测方法的建立  

Development of a SYBR Green Ⅰ RT-PCR Method to Detect Coxsackievirus B2

作　　者：杨亚平 段素琴[1] 杨凤梅[1] 李艳艳[1] 刘权 刘雨 赵远[1] 马绍辉[1] 和占龙[1] YANG Yaping;DUAN Suqin;YANG Fengmei;LI Yanyan;LIU Quan;LIU Yu;ZHAO Yuan;MA Shaohui;HE Zhanlong(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,Yunnan,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,云南昆明650118

出　　处：《贵州医科大学学报》2020年第3期315-320,共6页Journal of Guizhou Medical University

基　　金：中国医学科学院医学与健康科技创新工程项目(2016-I2M-2-001,2018-I2M-3-002);中央级公益性科研院所基本科研业务费(2016ZX310179-4);云南省重大科技专项(2017ZF020);云南省科技创新人才计划(2015HC027);昆明市科技创新和服务能力提升计划重点项目(2016-2-R-07674)。

摘　　要：目的:建立一种简单快速、灵敏、特异检测柯萨奇病毒B组2型(CVB2)的SYBR Green I逆转录聚合酶链反应(RT-PCR)方法。方法:根据CVB2病毒衣壳蛋白1(VP1)的基因保守序列设计带有T7启动子的特异性引物,构建CVB2-T载体的重组质粒,以体外转录获得的RNA为标准品,绘制标准曲线;建立检测CVB2的SYBR Green I RT-PCR方法,对该方法特异性、灵敏性及重复性进行评价。结果:该方法在102~109拷贝/μL范围内具有良好的线性关系,最低检测限度为102拷贝/μL,标准曲线中R^2为0.996,扩增效率为102.2%;且该方法对肠道病毒71型(EVA71)、柯萨奇病毒A组16型(CVA16)、柯萨奇病毒A组10型(CVA10)和柯萨奇病毒A组6型(CVA6)均无交叉反应;组间和组内重复性实验中变异系数(CV)均小于1%。结论:本研究建立的检测CVB2的SYBR Green I RT-PCR方法具有良好的灵敏性、特异性和重复性,可用于CVB2病毒的快速检测和定量分析。Objective:To establish a simple,fast,sensitive and specific one-step SYBR Green I reverse transcription polymerase chain reaction(RT-PCR)for rapid and accurate detection of Coxsackievirus B2(CVB2).Methods:Specific primers with T7 promoter were designed in accordance with the conserved sequence of CVB2 capsid protein 1(VP1)gene,and the recombinant plasmid of CVB2-T vector was constructed.The ribonucleic acid(RNA)obtained from in vitro transcription was used as the standard to draw a standard curve,and SYBR Green I RT-PCR method was established to detect CVB2.The sensitivity,specificity and repeatability of this method were evaluated.Results:The established method showed a good amplification curve in the range of 102~109 copies/μL template.The lowest detection limit was about 102 copies/μL,the R 2 coefficient in the standard curve was 0.996,and the amplification efficiency was 102.2%.No cross-reaction was found with enterovirus A71(EVA71),coxsackievirus A16(CVA16),coxsackievirus A10(CVA10)and coxsackievirus A6(CVA6)in the method.The variation coefficients value of the method was less than 1%in the repeated intergroup and within-group experiments.Conclusion:The SYBR Green I RT-PCR method established to detect CVB2 in the experiment possesses good sensitivity,specificity and repeatability,and can be used for rapid detection and quantitative analysis of CVB2.

关 键 词：手足口病 逆转录聚合酶链反应 柯萨奇病毒感染 染料稀释技术 

分 类 号：R373.23[医药卫生—病原生物学]