应用实时荧光定量PCR检测多亚型EV71的研究

发布时间：2018-04-05 04:28

本文选题：肠道病毒　切入点：亚型　出处：《中国病原生物学杂志》2017年10期

【摘要】：目的建立SYBR Green实时荧光定量PCR方法,以期用于EV71亚型检测。方法根据中国大陆手足口病病原,设计多亚型EV71VP1基因特异性引物,PCR扩增VP1基因片段,并与pEASY-T1载体连接,构建pEASY-T1-VP1重组质粒,以此为标准品进行实时荧光定量PCR,绘制标准曲线,评价方法的敏感性、特异性和稳定性,并利用该方法检测门诊初诊手足口病患者的咽喉拭子标本。结果建立的实时荧光定量PCR标准曲线线性关系良好(R2=0.993),扩增效率为107.1%;方法的检测下限为(8.48×100)~(8.48×101)拷贝/μl之间,变异系数1%;非手足口病病原体无扩增信号,手足口病患者咽喉拭子阳性率为52.3%(45/86)。阳性样品测序后与NCBI比对,符合率为100%。结论建立的SYBR Green实时荧光定量PCR敏感、特异,可用于多亚型EV71感染的检测。
[Abstract]:Objective to establish a real-time quantitative SYBR Green PCR method for the detection of EV71 subtypes.Methods according to the pathogeny of hand, foot and mouth disease in mainland China, the VP1 gene fragment was amplified by designing multi-subtype EV71VP1 gene specific primers and ligated with pEASY-T1 vector to construct pEASY-T1-VP1 recombinant plasmid.The sensitivity, specificity and stability of the method were evaluated.Results the standard curve of real-time fluorescence quantitative PCR has a good linear relationship, the amplification efficiency is 107.1, the detection limit of the method is 8.48 脳 100 脳 10 ~ (-1) copy / 渭 l, the coefficient of variation is 1 / 渭 l, and the pathogen of non-hand, foot and mouth disease has no amplification signal.The positive rate of throat swabs was 52.3%.The positive samples were compared with NCBI after sequencing, and the coincidence rate was 100%.Conclusion the SYBR Green real-time quantitative PCR is sensitive and specific, and can be used for the detection of multiple subtypes of EV71 infection.
【作者单位】：吉林医药学院检验学院;
【基金】：吉林省卫生厅青年项目(No.2015Q042) 吉林省大学生创新创业训练计划项目(No.2016B326)
【分类号】：R440;R725.1

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