甲型流感病毒流行毒株检测和分型基因芯片的研制

Microarray for detecting and subtyping of circulating influenza A virus

【目的】研制一种可同时对甲型流感病毒H1N1、H1N2、H3N2、H5N1和H9N2等5种流行亚型进行检测和分型的基因芯片。【方法】根据National Center for Biotechnology Information中Influenza Virus Resource数据库,针对H1N1、H1N2、H3N2、H5N1和H9N2等5种亚型甲型流感病毒的HA和NA基因设计46条特异性寡核苷酸探针和1条质控探针,点制成基因芯片。利用通用引物扩增流感病毒HA和NA基因,使用Klenow酶对扩增产物进行荧光标记和片段化,将标记后产物和芯片杂交,清洗、扫描后根据荧光信号判定检测结果。用18株不同种属来源的甲型流感病毒分离毒株和186份咽拭子对芯片特异性、敏感性和临床应用进行初步评价。【结果】所有18株分离毒株均能被芯片准确检测并分型,芯片检测灵敏度能达约1×104个病毒基因拷贝。同时8份咽拭子检测结果为H1N1阳性,4份咽拭子为H3N2阳性。【结论】研究表明该芯片具有较高的特异性和灵敏度,可为甲型流感病毒的监测提供一种有效的方法。

[Objective]We designed and characterized an oligonucleotide microarray for detecting and subtyping of circulating influenza A virus including subtypes H1N1,H1N2,H3N2,H5N1 and H9N2. [Methods] Based on the sequences of influenza A viruses obtained from the Influenza Virus Resource database of National Center for Biotechnology Information,46 oligonucleotides probes and one quality control probe were designed to fabricate the microarray. The full-length cDNAs of hemagglutinin and neuraminidase genes were amplified by RT-PCR using universal primers,and the resulting PCR products were labeled and fragmented using Klenow fragment before hybridized with the microarray. A total of 18 different influenza A virus strains representing 5 subtypes and 186 clinical samples were used to validate the specificity and sensitivity of the microarray. [Results] All 18 strains were accurately detected and subtyped by the microarray and no cross hybridization could be detected. The limit of detection for the microarray was approximately 1×10^4 gene copies of in vitro transcribed RNA. Of the 186 clinical samples,8 were successfully subtyped as H1N1 and 4 were subtyped as H3N2. [Conclusion] The results show that the microarray is a useful diagnostic method with high specificity and sensitivity,and could be used for influenza surveillance.