风疹病毒RNA Taq Man实时荧光定量RT-PCR方法及参考标准的建立

Establishment of a TaqMan real-time quantitative reverse transcription-polymerase chain reaction assay for rubella virus RNA

目的建立风疹病毒RNA TaqMan实时荧光定量RT-PCR方法,制备风疹病毒RT-PCR扩增子作为该方法的参考标准品。方法设计引物与探针,优化PCR条件,建立风疹病毒RNA实时荧光定量RT-PCR方法,并制备风疹病毒RT-PCR扩增子作为该方法的参考标准品。结果制备的风疹病毒特异性扩增子参考标准品为503 bp的片断,原液浓度为2.75×109copies/μl,纯度为92%;采用该特异性扩增子参考标准品建立的标准曲线相关系数r为-0.9993(r2=0.9986),P<0.001。结论本研究建立的风疹病毒特异性扩增子参考标准品稳定性好,纯度高;特异性扩增子参考标准品浓度的对数值与Ct值之间具有良好的线性关系,证实了本研究建立的风疹病毒RNATaqMan实时荧光定量RT-PCR方法定量的准确性。因而,建立的风疹病毒RNA实时荧光定量RT-PCR方法简便快捷、定量准确,可用于临床标本中风疹病毒RNA的定量检测。

Objective：To establish a real-time quantitative reverse transcription-polymerase chain reaction（RT-PCR） for rubella virus（RV）,a rubella virus specific PCR amplicon as external standard to analytically estimate this real time quantitative assay.Methods： Firstly,the primer and TaqMan probe concentrations,as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA.Next,and a RV specific PCR amplicon was made as external standard.Results： The RV specific PCR amplicon prepared for evaluation of the assay was 503bp,and its original concentration was 2.75×109 copies/μl.The real time quantitative assay was shown to have good linearity（r2=0.9986,P0.001）.Conclusion： The established quantitative RT-PCR is a simple,rapid,quantitative and accurate assay for RV RNA.