A型H1N1及H3N2亚型流感病毒实时荧光RT-PCR检测方法的建立

Development of real-time RT-PCR for simultaneous detection of influenza A virus and subtypes of H1N1 and H3N2

根据A型H1N1亚型流感病毒2009年北美变异株和H3N2亚型流行株的基因序列,设计了3套特异性引物、TaqMan探针,分别用不同的荧光基团标记,以构建的A型流感病毒及H1N1和H3N2亚型流感病毒基因重组质粒作为阳性对照,经反应条件优化,特异性、敏感性和重复性试验,筛选出3套不同的反应体系及同一个反应参数,建立了可同时检测A型流感病毒、H1N1和H3N2亚型流感病毒的实时荧光RT-PCR方法。结果表明,该方法可特异地检测H1N1(人源、猪源)、H3N2(猪源),禽流感病毒H5、H9等A型流感病毒,且与新城疫病毒、减蛋综合征病毒等其他病毒不发生交叉反应,具有特异、敏感、重复性好、快速、费用低廉等优点,试验耗时仅3h。表明,建立的方法,一次试验即可完成A型流感病毒的初筛和H1N1、H3N2亚型流感病毒的初步确认定型。

To develop a real-time RT-PCR for preliminary detection and differentiation of influenza A virus and subtypes H1N1 and H3N2 at one test,based on gene nucleotide sequences of Influenza A Virus/H1N1/2009 strain and Influenza A Virus/H3N2 strain in GenBank,three sets of specific primers and TaqMan probes were designed and labelled with different fluorescence reporters respectively,their recombinant plasmids were constructed to be used as positive control,and three different reaction mixtures and same RT-PCR condition were screened out.The real-time RT-PCR was developed by optimizing reaction conditions and carrying out a specificity,sensitivity and reproducibility test.By the real-time RT-PCR,influenza A virus,H1N1 and H3N2 were able to be specifically detected in one test only for 3h.In result,the established method could specifically detect influenza A virus H1N1（human/pig）,H3N2（pig）,AIV H5 and AIV H9,and not cross react with Newcastle disease virus and egg drop syndrome virus with advantages of specificity,sensitivity,reproducibility,rapidity and cheapness.The results showed that the real-time RT-PCRcould preli minarily detect and differentiate influenza Avirus,H1N1 and H3N2 in one test.