H9N2亚型流感病毒核酸参考品研制及荧光RT-PCR检测验证

Development and verification of reference nucleic acid materials of H9N2 influenza viruses by real-time RT-PCR

摘要:

从H9N2亚型流感病毒A/chicken/Hunan/04.14 (H9N2) 核酸中扩增了HA基因的编码序列，克隆测序后，采用体外转录方法制备RNA。用RNA保存液稀释至含量约109 copies/μL。分装后进行均匀性和稳定性检验，通过4家实验室协作标定，取平均值作为定值结果。此外，文中建立的实时荧光定量PCR (qPCR) 快速检测技术，对临床样品进行准确检测验证，检测限可达10个拷贝。结果表明，文中制备的核酸参考品可作为H9N2亚型流感病毒核酸快速检测方法的阳性定量参考品。

Abstract:

The HA gene of H9N2 influenza virus (A/chicken/Hunan/04.14 (H9N2)) was amplified and sequenced. The RNA was synthesized by in vitro transcription. The RNA transcription solutions were diluted to 109 copies/μL using the RNA storage solution. The aliquoted RNA solutions were used to evaluate the homogeneity and stability. The results were determined by the average value obtained from four independent laboratories. Furthermore, the fluorescence quantitative RT-PCR method was also developed to verify the detection accuracy of clinical samples. The detection limit of this method is approximately 10 copies. Taken together, the RNA transcription solution established in our study can used as positive standard reference for rapid detection of H9N2 influenza virus.