呼吸道合胞病毒TaqMan~实时定量RT-PCR检测方法的建立

Development of TaqMan~ real-time quantitative RT-PCR for detection of respiratory syncytial virus

目的建立呼吸道合胞病毒(respiratory syncytial virus,RSV)的TaqMan~实时定量RT-PCR检测方法。方法根据GenBank中RSV相关序列,以A、B亚型毒株基因组保守区域设计并合成RSV特异性引物和探针,采用体外转录RNA标准品,建立TaqMan~实时定量RT-PCR方法,同时验证方法特异性、检测范围、灵敏度和精密度。采用建立的方法检测50份下呼吸道感染患儿的痰样品。结果 RNA标准品建立的标准曲线R2> 0. 99,扩增效率在90%～110%之间。建立的方法可特异性扩增RSV Long株和RSV A2株核酸样本,IFA和HPIV-3核酸样本均无扩增;最高可检测到1. 3×10~9 copies/μL的RNA标准品,最低可检测到单拷贝的RNA标准品;4名实验员检测同一样品Ct值的试验内CV小于1. 72%,试验间的CV在2. 44%～3. 39%之间。50份下呼吸道感染患儿痰样品中,24份为RSV阳性,阳性率为48%。结论成功建立了RSV的TaqMan~实时定量RT-PCR方法,且该方法快速、特异、灵敏,可用于临床样本中RSV的有效检测。

Objective To develop a TaqMan^（R）real-time quantitative RT-PCR for detection of respiratory syncytial virus（RSV）. Methods Primers and probes specific for RSV subtypes A and B were designed and synthesized according to the conservative regions in GenBank,based on which a TaqMan^（R）real-time quantitative RT-PCR was developed by using in vitro transcription RNA standard and validated for specificity,detection range,sensitivity and precision. Fifty sputum samples from children with lower respiratory tract infection were detected by the developed method. Results The R2 value of standard curve plotted by the RNA standard was more than 0. 99,while the amplification efficacy was 90% ～110%. The RNAs in RSV Long and A2 strains were amplified specifically by the developed method,while no RNAs were amplified from IFA and HPIV-3 strains. The detection range of RNA standard was from a single copy to 1. 3 × 10^9 copies/μL.The CVs of Ct values in intra-assay by four laboratory personnel were less than 1. 72%,while those in inter-assay were2. 44% ～ 3. 39%. Of the 50 sputum samples from children with lower respiratory tract infection,24 were positive for RSV,indicating a positive rate of 48%. Conclusion A TaqMan^（R）real-time quantitative RT-PCR method for RSV was developed successfully,which was rapid,specific,sensitive,and might be used for detection of RSV in clinical samples.