红细胞富集与多重RT-PCR联合检测禽流感病毒

Combination of red blood cell enrichment and multiplex RT-PCR to detect avian Influenza viruses

目的对禽流感病毒H5、H7亚型进行更快速、更灵敏检测。方法根据禽流感病毒(AIV)核蛋白基因和血凝素基因设计3对特异性引物,建立在结合红细胞富集AIV病毒的基础上进行多重RT-PCR检测方法。结果该方法在1个反应体系中不仅能够鉴定A型流感病毒,而且可以同时确定是否为H5和H7亚型AIV;该方法的检测下限可达2.5pg的病毒RNA;对于参考毒株都可以扩增出预期大小的基因片段,而对新城疫病毒和传染性支气管炎病毒的平行对照结果均呈阴性。结合AIV能够吸附鸡红细胞并在一定条件下解离的特点,利用鸡红细胞对48份已确诊样品进行病毒富集,然后用建立的多重RT-PCR方法检测富集产物结果显示48个样品中,30个为H5亚型AIV阳性,与病毒分离结果完全一致。结论由以上结果初步表明本研究建立的检测方法快速,特异,在禽流感临床筛检中显示出良好的应用前景。

The multiplex reverse transcription-PCR（RT-PCR） used for the rapid detection of H5 and H7 subtype of avian influenza viruses （AIV） was developed with three pairs of specific primers based on the sequences of nucleoprotein and hemagglutinin gene of AIV. The results showed that it could identify H5 and H7 AIV subtype in one reaction of multiplex RTPCR, and the sensitivity of this method is 2.5pg RNA of AIV. The expected segments were amplified using this procedure to detect the reference strains AIV. It showed no cross-reactions with infectious bronchitis virus and Newcastle disease virus. Based on the trait of adsorption and desorption of chicken red blood cell（CRBC） with AIV under some conditions, virus enrich- ment was performed in 48 samples diagnosed by CRBC, and the enrichment products were detected by the multiplex RT-PCR In whih, 30 of 48 were showed to be H5 subtype AIV, completely accorded with the result of same samples tested by embryonated eggs propagation and hemagglutinin inhibition assay. All the results of the experiment demonstrate this multiplex RTPCR is rapid and specific, and very useful for clinical screening of avian influenza disease.