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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。
OBJECTIVE: To establish a new method for the rapid detection of the mutation of NA gene in influenza A (H1N1) virus by dual base extension and Fluorescence polarization (FP). Methods The 30 neuraminidase gene sequences of H1N1 were randomly downloaded from GenBank. Three specific primers were designed by MEGA and Primer Premier software. One of the primers was used to amplify the neuraminidase gene containing H275Y Of the NA gene, the third primer is the use of double base extension termination method and fluorescent polarization detection of NA gene mutations in the drug resistance site Darfur H275Y mutation, and then randomly selected 50 strains of H1N1 influenza virus detection, Compared with the sequencing results to verify the accuracy of test results. Results The amino acid at amino acid 275 of NA protein of all the 50 influenza A (H1N1) viruses was histidine, and no substitution of H Y occurred. The sensitivity and specificity of the amino acid sequence of the NA protein in the NA gene of the 50 strains were both 100% identical to the traditional sequencing results. Conclusion The method is simple, sensitive and specific, the interpretation of the results intuitive and clear, low detection costs, enabling rapid and accurate detection of drug-resistant site mutations can be applied to SNP-related diseases, rapid typing and mutation Point of real-time monitoring and clinical testing.