实时荧光定量逆转录-聚合酶链反应(RT-PCR)法与RT-PCR法及细胞培养法检测甲_3型流行性感冒病毒的比较

Comparison of Real-time Fluorescence Reverse Transcription-PCR With Reverse Transcription-PCR and Cell Culture in Detection of Influenza A_3 Virus

目的比较实时荧光定量逆转录-聚合酶链反应(RT-PCR)法、RT-PCR法及细胞培养法检测甲3型流行性感冒(流感)病毒的灵敏度与特异性。方法采用建立的实时荧光定量RT-PCR、RT-PCR及经典的狗肾传代细胞病毒分离等3种方法,同时对流感监测点送检的60份疑似流感标本检测甲3型流感病毒。结果细胞病毒分离的阳性数为10份,RT-PCR与实时荧光定量RT-PCR的阳性数分别为12份和15份。实时荧光定量RT-PCR的灵敏度达0.01TCID50,且对甲1型流感病毒、乙型流感病毒、禽流感病毒H5、严重急性呼吸道综合征冠状病毒及其它呼吸道病毒均无交叉反应,从病毒核酸提取至完成检测仅需3h左右。结论实时荧光定量RT-PCR由于检测在密封环境中进行,避免了产物与环境间的交叉污染,且是3种方法中最为快速敏感的方法,适用于公共卫生应急疫情的实验室快速诊断。

Objective To compare the sensitivity and specitivity of real-time RT-PCR and RT-PCR and cell culture for influenza A3 virus detection. Method A one-tube real-time fluorescence reverse transcription （RT）-PCR with the MJ DNA Engine OpticonTM 2 instrument for influenza A3 virus detection was evaluated with 60 respiratory specimens. RT-PCR and MDCK（Madin-Darby canine kid- ney）cell culture were performed as well. All 60 influenza-like specimens were detected by cell culture, RT-PCR, and real-time RT-PCR, and influenza A3 virus-positive specimens were 10,12, and 15, respectively. Results The sensitivity of real-time fluorescence RT-PCR were 0.01 TCIDs0. Conclusion The high sensitivity and specificity and the rapid turnaround time made real-time RT-PCR valuable for the rapid diagnosis of influenza A3, especially in a public health laboratory. The closed real-time RT- PCR system avoided cross-contamination possible with RT-PCR and the excessive manipulations required for conventional RT-PCR analysis and saved time and labor.