利用基因芯片技术区分禽流感病毒主要亚型

Typing and subtyping Avian influenza virus using DNA microarrays

摘要:

【目的】研制可同时区分AIV的H5、H7、H9血凝素亚型及N1、N2神经氨酸酶亚型的基因诊断芯片。【方法】分别克隆了禽流感病毒的M基因，H5、H7、H9亚型HA基因，N1、N2亚型NA基因以及看家基因GAPDH的重组质粒。以重组质粒为模板，用PCR方法扩增制备探针，纯化后点于氨基修饰的片基上，制备基因芯片。在PCR过程中对待检样品进行标记，然后与芯片杂交，洗涤，扫描并进行结果分析。【结果】结果显示检测探针可特异性的与相应的标记样品进行杂交，呈现较强的杂交信号，且无交叉杂交。同时用RT-PCR、鸡胚接种和基因芯片方法对H1-H15亚型AIV参考毒株、30份人工感染样品、21份现地疑似样品进行检测，结果发现，对人工感染样品芯片检测方法与鸡胚接种和RT-PCR的符合率分别为100%和96%，现地样品符合率为100%。【结论】研究表明该方法可用于同步鉴别部分主要流行的禽流感亚型，是一种有效的新方法。

Abstract:

[Objective] Outbreaks of highly pathogenic avian influenza (HPAI) virus has caused great economic loss to the poultry industry and resulted in human deaths in Thailand and Vietnam since 2004. Rapid typing and subtyping of viruses, especially HPAI from clinical specimens, are desirable for taking prompt control measures to prevent spreading of the disease. We described a simultaneous approach using microarray to detect and subtype avian influenza virus (AIV). [Methods] We designed primers of probe genes and used reverse transcriptase PCR to prepare cDNAs of AIV M gene, H5, H7, H9 subtypes haemagglutinin genes and N1, N2 subtypes neuraminidase genes. They were cloned, sequenced, reamplified and spotted to form a glass-bound microarrays. We labeled samples using Cy3-dUTP by RT-PCR, hybridized and scanned the microarrays to typing and subtyping AIV. [Results] The hybridization pattern agreed perfectly with the known grid location of each probe, no cross hybridization could be detected. Examinating of HA subtypes 1 through 15, 30 infected samples and 21 field samples revealed the DNA microarray assay was more sensitive and specific than RT-PCR test and chicken embryo inoculation. [Conclusion] It can simultaneously detect and differentiate the main epidemic AIV. The results show that DNA microarray technology is a useful diagnostic method.