多重实时定量PCR检测G1—G4型轮状病毒方法探索

The method exploration of multiplex real-time quantitative PCR for detection of type G1-G4 rotaviruses

目的:对多重实时定量PCR(real-time quantitative PCR,qPCR)进行反应体系筛选和方法探索,使其用于G1—G4型轮状病毒VP7基因的快速分型和定量分析。方法:设计G1—G4型轮状病毒VP7基因特异性引物和探针,以特异性体外转录RNA为模板,筛选多重qPCR反应体系,建立多重qPCR方法。采用单因素方差分析和配对样本t检验对多重与单重qPCR检测结果进行比较。结果:经筛选,得到6组三重qPCR反应体系和1组四重qPCR反应体系。所建立的三重和四重qPCR中,除四重qPCR的G1型参数〔标准曲线决定系数(R2)为0.982、扩增效率为89.221%〕略低于要求外,其余标准曲线R2均>0.99,扩增效率均在90%至110%之间;检测灵敏度达102拷贝/μl。多重与单重qPCR检测同一样本的相关性较好(R2>0.95)。三重与单重qPCR(t值为1.420~25.786)、四重与单重qPCR(t值为2.505~4.851)检测结果间的差异均无统计学意义(P值均>0.05)。结论:建立的多重qPCR可同时对3种以上目的基因进行分型和定量检测,为未来多价轮状病毒疫苗及混合病毒样本中毒株的快速分型和定量检测方法的建立提供了借鉴。

Objective To screen reaction system and explore method of multiplex real-time quantitative PCR(qPCR)for rapid genotyping and quantitative testing VP7 genes of G1-G4 rotaviruses.Methods Primers,probes and in vitro transcripted RNAs specific for VP7 genes of G1-G4 rotaviruses were used to screen multiplex qPCR reaction systems and establish multiplex qPCR methods.The detection results between multiplex qPCRs and simplex qPCR were compared by one-way ANOVA and paired-sample t-test.Results After screening,6 reaction systems of triplex qPCR and 1 reaction system of quadruplex qPCR were obtained.The triplex qPCR and quadruplex qPCR were established with determination coefficients(R2)of standard curves>0.99,amplification efficiencies ranging from 90%to 110%,except for type G1 in quadruplex qPCR(R2=0.982,amplification efficiency=89.221%)slightly below the requirement.The detection sensitivity was 102 copies/μl.The correlation was good(R2>0.95)when the multiplex qPCRs and simplex qPCR were used to detect same sample.There was no significant difference in detection results between triplex qPCR and simplex qPCR(t=1.420-25.786,all P>0.05)and between quadruplex qPCR and simplex qPCR(t=2.505-4.851,all P>0.05).Conclusion The multiplex qPCR established is capable of typing and quantitative detection of more than 3 target genes in the same reaction tube,providing a reference for the establishment of rapid typing and quantitative detection of multivalent rotavirus vaccine and mixed virus samples in the future.