A组轮状病毒G1和G9亚型的二重qPCR检测

The duplex qPCR assay for G1 and G9 subtypes of group A Rotavirius

目的建立快速检测A组轮状病毒G1和G9亚型的二重实时荧光定量PCR(qPCR)检测法。方法针对A组轮状病毒G1和G9亚型VP7基因的保守区域与高变区域,设计特异的引物、探针。构建含A组轮状病毒G1和G9亚型VP7基因的质粒,将其作为该检测系统的阳性标准品,优化A组轮状病毒G1和G9亚型的qPCR检测系统。结果该方法检测A组轮状病毒G1和G9亚型阳性样本的灵敏度为103copies/μL,对其他亚型的标准质粒检测呈阴性。结论本实验建立的A组轮状病毒G1和G9亚型的二重qPCR检测方法具有良好的特异度、敏感度和重复性,该方法的应用有助于A组轮状病毒G1与G9亚型感染的早期快速诊断。

Objective To establish a real-time quantitative PCR（qPCR） assay for G1 and G9 subtypes of group A Rotavirus.Methods Primers and probes were designed according to the conservative and hypervariable region of VP7 gene carried by group A Rotavirus.2 pairs of specific primers and 2 types of TaqMan probes were designed.Standard plasmids carring VP7 gene of the 2 subtypes of group A Rotavirus were constructed respectively in vitro,which were used as standard template to optimize the duplex qPCR assay.Results The sensitivity of the assay was 103 copies/μL,and negative results were observed when the duplex qPCR assay was applied to positive specimens of other subtypes of group A Rotavirus.Conclusion The duplex qPCR assay established for the detection of G1 and G9 subtypes of group A Rotavirius has good specificity,sensitivity and repeatability.The application of this assay is helpful in rapid diagnosis for early infection of G1 and G9 subtypes of group A rotavirus.