H1N1亚型三个谱系猪流感病毒RT-PCR鉴别诊断方法的建立

Development of RT-PCR Assay for Differentiating Three Sublineage of H1N1 Subtype Swine Influenza Virus

根据Gen Bank中甲型、古典型和类禽型三个谱系H1N1猪流感病毒(swine influenza virus,SIV)的血凝素(Haemagglutinin,HA)基因保守序列,分别设计了3对特异性引物,建立了H1N1亚型三个谱系SIV的RT-PCR快速鉴别诊断方法。该方法能够对甲型、古典型和类禽型三个谱系的H1N1亚型SIV做出准确诊断,扩增片段大小分别为476 bp、293 bp和997 bp。敏感性试验结果显示,该方法对三个谱系病毒的最低检测限分别为1 896拷贝/μL、1 655拷贝/μL和1 459拷贝/μL;特异性试验显示,该方法可特异性检出古典型、类禽型和甲型H1N1三个谱系的SIV,而与H5、H7和H9亚型流感病毒、猪瘟病毒及猪繁殖与呼吸不障碍综合征病毒均无交叉反应;重复性试验表明,3次重复检测结果具有良好的一致性。以上结果表明,该RT-PCR分型诊断方法的敏感性、特异性和重复性良好,为有针对性地对猪流感采取综合防控措施提供重要技术支撑,具有良好地应用前景。

In this study, three RT-PCR methods were developed to distinguish the haemaggluti- nin（Hh） genes of three sublineages of H1N1 subtype swine influenza virus（Sir） by using three pairs of specific primers derived from the conserved regions of HA gene sequences from pandemic HIN1, classical H1N1 and avian-like H1N1 in GenBank. The results of the sensitivity test showed that the developed methods were able to specifically amplify 476 bp gene fragment in pandemic H1N1, 293 bp gene fragment in classical HIN1 and 997 bp gene fragment in avian-like H1N1 accurate- ly. And the detection limit was 1 896 copies/uL for pandemic H1N1, 1 655 copies/~ L for classi- cal HIN1 and 1 459 copies/~L for avian-like H1N1 sir. The specificity test suggested that these assays were able to specifically detect pandemic H1N1, classical HIN1 and avian-like H1N1 without cross-reaction with H5, H7 and H9 subtypes influenza virus, classical swine fever virus（CSFV） and porcine reproductive and respiratory syndrome virus（PRRSV）. The repeatability test indicated that the results from three repeated trials were consistent. All these results suggested that these RT-PCR methods were sensitive, specific and repeatable, which can be used to detect three major sublineages of H1N1 subtype SIV, fast and differentially.