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

作　　者：宋洁[1,2] 李翠 李晶[1,2] 张爽[1] 范文辉 刘丽蓉[1] 贾泓毅[4] 俞蔼毕 郝客 牛春艳 王晶 赵启祖[3] 刘文军[1,2] Jie Song1,2, Cui Li3, Jing Li1,2, Shuang Zhang1, Wenhui Fan1, Lirong Liu1, Hongyi Jia4, Aibi Yu4, Ke Hao4, Chunyan Niu5, Jing Wang5, Qizu Zhao3, and Wenjun Liu1,2(1 Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China ;2 University of Chinese Academy of Sciences, Beijing 100049, China ;3 China Institute of Veterinary Drug Control, Beijing 100081, China; 4 Beijing Haidian Foreign Language Experiment School, Beijing 100195, China; 5 National Institute ofMetrology, Beijing 100013, China)

机构地区：[1]中国科学院微生物研究所中国科学院病原微生物与免疫学重点实验室,北京100101 [2]中国科学院大学,北京100049 [3]中国兽医药品监察所,北京100081 [4]北京海淀外国语实验学校,北京100195 [5]中国计量科学研究院,北京100013

出　　处：《生物工程学报》2018年第10期1579-1586,共8页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(No.2018YFC1200502)资助~~

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10~9 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。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 10～9 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.

关 键 词：H9N2亚型流感病毒 HA基因 参考品 荧光RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]