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.
作者
张伟
李咏洁
杨凤梅
李艳艳
靳玮华
徐鸿界
李明学
刘权
赵远
和占龙
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)
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)。
