柯萨奇A组6型病毒TaqMan探针实时荧光RT-PCR的建立

Development and application of TaqMan probe-based real-time RT-PCR for Coxsackie virus A6

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 TaqMan probe-based real-time fluorescence RT-PCR for Coxsackie virus A6,and be used in the rapid diagnosis and monitoring of HFMD. Methods Based on the conservativesequences of VP1 gene of Coxsackie virus A6 published on GenBank,specific primers and TaqMan probes were designed and optimized for the real-time RT-PCR assay.The specificity,sensitivity,and stability of real time RT-PCR assay were evaluatedand applied in surveillance of hand ,foot and mouth disease ,and compared with commercial kits. Some positive samples were sequenced for further verification. Results The TaqMan probe-based quantitative real-time RT-PCR assay should cost 60 minutes for a running,and had no cross-reaction with other serum types of enterovirus and other common respiratory pathogens. The sensitivity for the assay could reach the dilution of 10^0 copies/μL. The coefficients of variation for the Ct value of two concentration of nucleic acid were 0.29% and 0.38%. Of the 280 clinical samples ,142 were identified as positive by this real-time RT-PCR,being consistent with the result of commercial reagents kit.15 positive samples were selected randomly and identified with the sequence result of VP1 gene of Coxsackie virus A6. Conclusion The rapid,specific and sensitive TaqMan probe-based real-time RT-PCR for Coxsackie virus A6 could be used in the classification in non-EV71 and non-CA16 enteroviruses.