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为了建立特异、灵敏、快速的荧光定量RT-PCR方法用于检测埃可病毒9型病毒核酸,并初步应用于埃可病毒9型的临床标本检测。根据GenBank登录的埃可病毒9型毒株VP1基因序列,应用生物学软件进行序列比对,在保守区设计特异性引物和TaqMan探针。对反应条件进行优化,验证方法的特异性、灵敏度和重复性,同时对疑似埃可病毒9型病例标本进行检测。该方法对埃可病毒9型的检出有高度特异性,与EV71、CA16、CA24v、CA6、CA10、埃可病毒30型等均无交叉反应,检测灵敏度均达0.1TCID50/mL,可从疑似埃可病例粪便标本中直接检测病毒核酸,检测仅需3~4h。本研究建立的埃可病毒9型TaqMan荧光定量RT-PCR检测方法特异、灵敏,适用于临床早期诊断。
In order to establish a specific, sensitive and rapid real-time fluorescent quantitative RT-PCR method for the detection of the echovirus type 9 virus nucleic acid and preliminary application in the detection of clinical samples of the 9 type of echovirus. According to the sequence of VP1 gene of echovirus type 9 strain registered in GenBank, sequence alignment was performed with biological software and specific primers and TaqMan probes were designed in conserved regions. The reaction conditions were optimized to verify the specificity, sensitivity and reproducibility of the method, while the detection of suspected cases of echovirus type 9 was performed. The method was highly specific for the detection of echovirus type 9 and had no cross-reaction with EV71, CA16, CA24v, CA6, CA10, echovirus type 30 and the like. The detection sensitivity was 0.1 TCID50 / mL, AEC cases of stool specimens directly detect the virus nucleic acid, testing only 3 ~ 4h. The echovirus 9 TaqMan quantitative real-time RT-PCR assay established in this study is specific and sensitive and is suitable for early clinical diagnosis.