A型流感病毒分型基因芯片在流感病毒各亚型监测中的应用

Application of Influenza A Virus Genotyping Microarray in Surveillance of Influenza Virus Subtypes

为验证自行研制的A型流感病毒分型基因芯片方法在实际中的应用效果,了解A型流感病毒各亚型在我国不同地区和不同宿主中的分布情况,对我国20个地区、不同宿主源(鸡、鸭、鹌鹑、鸽子、猪、人等)拭子和组织样品共16 852份进行了检测。利用自行研制的A型流感病毒分型基因芯片方法,对样品进行RNA提取、多重不对称RT-PCR扩增、芯片杂交、洗涤和扫描分析;同时将基因芯片检测阳性的样品,用普通RT-PCR扩增后进行序列测定。结果显示:利用该方法检出阳性样品2 582份,总检出率为15.32%(2 582/16 852),共检测到H1N1、H2N9、H3N2、H4N6、H5N1、H7N1、H7N9、H9N2、H10N5、H16N3等10个亚型,且均与阳性样品测序结果一致。结果表明,该方法可准确检测不同地区、不同宿主中的A型流感病毒不同亚型。同时,该方法能在同一张芯片上准确鉴定A型流感病毒的多种亚型,解决了其他常规方法无法检测已发现和可能发生变异的所有亚型的棘手问题,从而为A型流感诊断和分子流行病学监测提供了一种新技术。

In order to verify the effect of influenza A virus genotyping microarray in practice,and to make clear the distribution of all subtypes of influenza A virus in various hosts in different regions in China,16 852 swabs and tissue samples collected from different hosts(chickens,ducks,quails,dove,pigs and human)in 20 regions were tested,and then RNA extraction,multiple asymmetric RT-PCR amplification,chip hybridization,cleaning and scanning analysis were carried out by the self-developed genotyping microarray. Meanwhile,the positive samples were amplified by conventional RT-PCR followed by sequencing. The results showed that 2 582 positive samples were detected out,with a total detection rate of 15.23%(2 582/16 852),involving 10 subtypes including H1 N1,H2 N9,H3 N2,H4 N6,H5 N1,H7 N1,H7 N9,H9 N2,H10 N5 and H16 N3,which was consistent with the result of conventional RT-PCR. It was concluded that different subtypes of influenza A virus could be detected in various hosts in different regions in China by the genotyping microarray that could also accurately identify various subtypes in the same microarray and solve the difficulty in detecting all identified or variant subtypes compared to the conventional methods,so a new technology was developed for confirmation of influenza A virus and molecular epidemiological surveillance.