札如病毒实时荧光RT-PCR的建立与应用

目的 建立一种可快速、全面、准确检测人感染札如病毒的TaqMan探针荧光实时RT-PCR方法。方法 利用Bioedit2.0、MAGE6.0软件对可感染人的GⅠ、GⅡ、GⅣ、GⅤ基因群札如病毒进行保守序列比对,应用Primer6.0、Primer Express 3.0进行引物和TaqMan探针的设计与评价,分别设计了两套引物及TaqMan探针,克隆相应靶基因并构建阳性标准品,建立及优化一步法实时荧光RT-PCR反应体系,并进行灵敏度、特异度、临床标本验证试验。结果 根据基因序列比对分析,札如病毒ORF1与ORF2连接区具有相对高保守序列,针对GⅠ、GⅡ、GⅣ及GⅤ设计了一条共用的下游引物、以及两套上游引物和TaqMan探针,能覆盖当前GenBank可见的感染人的札如病毒基因型,在同一反应管中建立了同时检测札如病毒四个基因群的实时荧光RT-PCR,并可将GⅤ与GⅠ、GⅡ、GⅣ进行初步分型,对人柯萨奇病毒、诺如病毒、轮状病毒、腺病毒等非靶标病原体无扩增,仅对札如病毒有特异扩增,最低检测下限为10拷贝/反应。结论 本研究建立了札如病毒一步法实时荧光RT-PCR反应体系,可将GⅤ与GⅠ、GⅡ、GⅣ区分开来,为腹泻疫情防控和风险评估提供快速、准确的病原学依据。

Establishment and application of real-time RT-PCR for Sapovirus

Objective To establish a rapid, comprehensive and accurate TaqMan probe-based real-time fluorescence RT-PCR assay for human infected Sapovirus. Methods Conservative sequence alignment for GⅠ, GⅡ, GⅣ and GⅤ genomes of Sapovirus was performed by Bioedit2.0, MEGA6.0 softwares. Two sets of primers and TaqMan probes were designed and evaluated by Primer 6.0, Primer Express 3.0 softwares, and the two target genes were amplified for plasmid cloning. The one-step real-time fluorescence RT-PCR reaction was established and optimized, and the tests for sensitivity, specificity and clinical sample validation were performed. Results The conservative sequences are in the ORF1-ORF2 junction of Sapovirus based on gene sequence alignment. A common reverse primer for GⅠ, GⅡ, GⅣ and GⅤ, two sets of forward primers and TaqMan probes, were designed, which can cover the current GenBank visible human infected genotype of Sapovirus, the one-step real-time RT-PCR assay was established, which could detect the four genomes of human infected Sapovirus in one tube at the same time, and can preliminary classify GⅤ from GⅠ, GⅡ, GⅣ. The assay was highly specific for Sapovirus and has no cross-response for other non-targeted virus, such as Human coxsackie virus, Norovirus, Rotavirus and Adenovirus. The minimum test limit for this assay could be 10 copies per reaction. Conclusion Our study established a one-step real-time fluorescence RT-PCR reaction system for sapoviruses, which can distinguish GⅤ from GⅠ, GⅡ, GⅣ genomes, providing rapid and accurate pathogen basis for diarrhoea epidemic prevention and risk assessment.