双碱基延伸法结合荧光偏振法快速检测新甲型H1N1耐药位点方法的建立
房师松1 ,柴燕文2 ,王昕1 ,吕星1 ,吴春利1 ,程小雯1 ,张仁利1 ,薛红2 ,程锦泉1
1.深圳市疾病预防控制中心,深圳 518055;
Detection of oseltamivir-resistant pandemic influenza A/H1N1 2009 viruses using two-base extension termination and fluorescence polarization methods
FANG Shi-song1 ,CHAI Yan-wen2 ,WANG Xin1 ,LU Xing1 ,WU Chun-li1 ,CHENG Xiao-wen1 ,ZHANG Ren-li1 ,XUE Hong2 ,CHENG Jin-quan1
1. Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China;
摘要 目的 建立一种利用双碱基延伸法与荧光偏振技术(Fluorescence polarization,FP)快速检测甲型H1N1流感病毒NA基因耐药突变位点的新方法。方法 从GenBank随机下载30条甲型H1N1的NA(neuraminidase)基因序列,通过MEGA分析,利用Primer Premier软件设计3条特异引物,其中一对引物通过RT-PCR用于扩增含有耐药位点H275Y的NA基因,第3条引物是应用双碱基延伸终止法与荧光偏振技术检测NA基因对达菲类药物耐药位点H275Y的突变情况,再随机选取50株甲型H1N1流感病毒进行检测,与测序结果进行比对,验证检测结果的准确性。结果 50株甲型H1N1流感病毒的NA蛋白第275位氨基酸均为组氨酸,未发生H Y的替换,在灵敏度与特异性方面,与传统的基因测序结果一致性均达100%。结论 本方法操作简便、敏感性及特异性高、判读结果直观明确、检测费用低,能实现对耐药位点突变的快速、准确检测,可应用于与SNP相关疾病的快速分型和突变位点的实时监测和临床检测。
关键词 ：
新甲型H1N1病毒 ,
NA基因 ,
SNP ,
双碱基延伸 ,
荧光偏振 ,
耐药
Abstract ：A novel method based on two-base extension termination and fluorescence polarization was established for the detection of oseltamivir-resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase (NA) gene of influenza A/H1N1 2009. Firstly, 30 neuraminidase gene sequences coding for influenza A/H1N1 were randomly downloaded from GenBank and analyzed by software MEGA. Three specific primers for NA gene of influenza A/H1N1 were designed by Primer Premier--one pair of primer was used to amplify the NA gene containing the His275Tyr mutation region by RT-PCR, and the third primer was detected the H275Y mutation of oseltamivir-resistance by two-base extension termination and fluorescence polarization. Additionally, 50 sample of influenza A/H1N1 were tested using Sanger sequencing, which is the reference method for the novel one to verify the accuracy. Results indicated that the 275th amino acids of NA protein in 50 influenza A/H1N1 were histidine, while no replace found between H and Y. In comparison to Sanger sequencing, the sensitivity and specificity of the novel method were 100% (50/50) and 100% (50/50) respectively. The novel method is simpler, high in sensitivity and specificity, and low-cost. It can be used to achieve the rapid and accurate detection of drug-resistant mutation and applied into the other SNP related disease, which also need the fast classification, real-time monitoring and clinical testing of mutation.
Key words ：
influenza A/H1N1 2009
NA gene
SNP
two-base extension
fluorescence polarization
drug-resistance
收稿日期: 2013-05-19
基金资助: 深圳市科技计划项目(No.201102108)资助
引用本文:
房师松,柴燕文,王昕,吕星,吴春利,程小雯,张仁利,薛红,程锦泉. 双碱基延伸法结合荧光偏振法快速检测新甲型H1N1耐药位点方法的建立[J]. 中国人兽共患病学报, 2014, 30(2): 175-179.
FANG Shi-song,CHAI Yan-wen,WANG Xin,LU Xing,WU Chun-li,CHENG Xiao-wen,ZHANG Ren-li,XUE Hong,CHENG Jin-quan. Detection of oseltamivir-resistant pandemic influenza A/H1N1 2009 viruses using two-base extension termination and fluorescence polarization methods. Chinese Journal of Zoonoses, 2014, 30(2): 175-179.
链接本文:
http://www.rsghb.cn/CN/10.3969/cjz.j.issn.1002-2694.2014.02.014 或 http://www.rsghb.cn/CN/Y2014/V30/I2/175
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