贵州医科大学学报

2020, v.45;No.234(03) 315-320

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柯萨奇病毒B组2型SYBR Green Ⅰ RT-PCR检测方法的建立
Development of a SYBR Green Ⅰ RT-PCR Method to Detect Coxsackievirus B2

杨亚平;段素琴;杨凤梅;李艳艳;刘权;刘雨;赵远;马绍辉;和占龙;
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;

摘要(Abstract):

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 10~2~10~9 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 than1% in the repeated intergroup and within-group experiments. Conclusion: The SYBR Green I RTPCR 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(KeyWords): 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
hand,foot and mouth disease(HFMD);reverse transcription-polymerase chain reaction(RT-PCR);coxsackievirus infections;dye dilution technique

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 杨亚平;段素琴;杨凤梅;李艳艳;刘权;刘雨;赵远;马绍辉;和占龙;
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;

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DOI: 10.19367/j.cnki.1000-2707.2020.03.013

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