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目的建立柯萨奇A组6型病毒TaqMan探针实时荧光RT-PCR,用于手足口病例快速诊断及监测。方法根据GenBank中柯萨奇A组6型病毒VP1高保守序列设计引物和探针,建立和优化实时荧光RT-PCR反应体系,评估其特异性、灵敏度、稳定性,在手足口病监测中应用,并与商品化试剂比较,抽取部分阳性标本测序验证。结果本研究建立的柯萨奇A组6型病毒实时荧光RT-PCR可在60 min内完成检测,与其它型别的肠道病毒和病原体无交叉反应,灵敏度可达10~0 copies/μL梯度,对两个不同核酸浓度样本Ct值的变异系数分别为0.29%、0.38%。280份非EV71、CA16的标本中检出142份CA6阳性标本,与商品化试剂检测结果一致。随机抽取15份阳性标本产物测序,与CA6靶基因序列同源性99.8%以上。结论建立的柯萨奇A组6型病毒实时荧光RT-PCR方法特异、灵敏、稳定,可用于非EV71、非CA16的其它肠道病毒标本分型。
Objective To establish a real-time fluorescent RT-PCR for detection of Coxsackievirus A type 6 TaqMan probe for the rapid diagnosis and monitoring of hand-foot-mouth disease. Methods Primers and probes were designed according to the high conservation sequence of VP1 of Coxsackievirus A genotype 6 in GenBank. The real-time fluorescence RT-PCR reaction system was established and optimized to evaluate its specificity, sensitivity and stability. The method was applied to the monitoring of HFMD , And compared with commercial reagents, part of the positive samples were extracted and verified. Results Real-time RT-PCR of Coxsackievirus A genotype 6 virus was completed within 60 min, and no cross-reactivity with other types of enterovirus and pathogen was detected with a sensitivity of 10 ~ 0 copies / μL The coefficients of variation of Ct values for two different nucleic acid concentration samples were 0.29% and 0.38%, respectively. A total of 142 CA6 positive samples were detected in 280 non-EV71 and CA16 samples, which was consistent with the commercial reagents. Fifteen positive samples were randomly selected and sequenced. The homology was 99.8% with the CA6 target gene sequence. Conclusion The established Coxsackievirus A group 6 real-time fluorescent RT-PCR method is sensitive, stable and can be used for other enterovirus typing of non-EV71 and non-CA16.