一步法实时荧光RT-PCR在快速检测甲型流感病毒中的应用

The Application of One-Step Real-Time Reverse Transcription PCR on Rapid Detection of Influenza Virus A

目的建立一种能快速检测甲型流感病毒的一步法实时荧光PCR方法,对样本进行准确检测。方法以甲型流感病毒特异M基因的保守区为靶区域设计特异引物和TaqMan荧光探针,建立一步法实时荧光RT-PCR方法,并对乙型流感病毒、呼吸道合胞病毒Long株、冠状病毒OC43及229E、副流感病毒1、2、3型以及季节性流感病毒H1、H3进行检测,验证该方法的特异性和灵敏度,并对临床样本进行检测应用。结果所建立的一步法实时荧光RT-PCR可对甲型流感病毒进行特异扩增,操作方便快速,对H1和H3亚型的最低检出限可达0.01TCID50/ml;对384例临床咽拭子样本的检测准确度达99.5%。结论本研究建立的一步法实时荧光RT-PCR方法可实现对甲型流感病毒快速准确的检测,有助于对临床流感病例的快速诊断。

Objective To set up a method based one-step real-time PCR to detect influenza virus A in specimens of patients with respiratory infections.Methods Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A. The applicability of this one-step RT-PCR was evaluated with influenza virus A,7 respiratory pathogens reference strains,isolates and clinical samples. 7 respiratory pathogens reference strains including influenza virus B,respiratory syncytial virus long strain,adenovirus coronaviruses OC43 and 229E,and human parainfluenza virus 1,2,3.Results The sensitivity of one-step RT-PCR is 0.01 50% tissue culture infective doses detecting for influenza virus A subtypes H1 and H3. The diagnostic efficacy of one-step RT-PCR was 99.5%,which was determined by testing 384 clinical samples. Conclusion This study describes a rapid,sensitive detection method that can be applied to routine diagnosis.