肠道病毒TaqMan荧光定量RT-PCR法快速检测

Rapid detection of enterovirus with TaqMan real-time RT-PCR

目的建立一种特异、灵敏、快速的荧光定量RT-PCR方法检测肠道病毒核酸,应用于暴发疫情的实验室应急检测。方法根据GenBank登录的肠道病毒基因序列,应用生物学软件进行序列比对,在肠道病毒基因的保守区设计特异性引物和TaqMan探针;对荧光RT-PCR反应条件进行优化,验证方法的特异性和灵敏度。同时对疑似肠道病毒感染者的临床样本进行检测。结果该方法对肠道病毒的检测有高度的特异性,可检出脊髓灰质炎、柯萨奇和埃可等肠道病毒,与腮腺炎、麻疹、风疹、乙型脑炎等病毒均无交叉反应。检测的灵敏度达0.1病毒效价滴定(TCID50);可直接从疑似肠道病毒感染者的脑脊液、疱疹液和粪便等标本中检测肠道病毒核酸,从病毒核酸提取至完成检测仅需3 h左右。结论所建立的TaqMan荧光定量RT-PCR是一种快速检测肠道病毒特异、敏感的方法,适用于由肠道病毒感染引起的应急疫情的实验室早期诊断。

Objective To establish a specific, sensitive and rapid method for detecting enterovirus nucleic acid with quantitative real-time RT-PCR and laboratory diagnosis for rapid response of enterovirus outbreaks. Methods Based on the alignment result of enterovirus sequences downloaded from GenBank, the conserved region was used for the design of primers and TaqMan probe. The reaction conditions were optimized using different concentrations of primers and probe. Specificity and sensitivity evaluations of the TaqMan real-time RT-PCR were also included. Laboratory diagnoses of some clinical specimens from suspected cases of enterovirus infection were conducted using the real-time RT-PCR method. Results The real-time RT-PCR was proved to be a high specificity method which could detect enteroviruses such as poliovirus, coxsackie virus, EHCO virus and so on. Cross-reactivity with mumps, measles, rubella or Japanese encephalitis virus was not observed. The sensitivity for detection was 0.1 TCID50. The detection of enteroviruses could be performed using nucleic acid directly extracted from cerebrospinal fluid, herpes fluid or stool samples of suspected cases. The time duration was about 3h from the viral RNA extraction to the completion of detection. Condusion The established TaqMan quantitative real-time RT-PCR was a specific and sensitive method for rapid detection of enteroviruses. It could be used for early laboratory diagnosis in response to enterovirus epidemics.