H9N2亚型禽流感荧光定量RT-PCR检测方法的建立及应用

发布时间：2018-11-15 21:01

【摘要】：为了研究H9N2亚型禽流感病毒(AIV)在家禽体内各个器官的分布,试验针对H9N2亚型AIV的HA基因设计1对特异性引物,同时设计1对扩增内参基因β-actin的引物,以阳性重组质粒作为标准品做标准曲线,建立荧光定量RT-PCR检测方法,并对人工感染H9N2的SPF雏鸡气管、胸腺、肺脏、肝脏、脾脏、肾脏、肠等器官进行3次重复检测。结果表明:该法线性关系R值均在0.99以上,检测极限均为10拷贝质粒DNA,比RT-PCR灵敏100倍以上;特异性好,批内和批间重复性试验变异系数均小于1%。通过比较各器官HA相对表达量,得出肠和胰腺中HA相对表达量最高,腺胃次之,脾脏、肝脏、胸腺、法氏囊中含量也比较高,气管、肺脏、肾脏中含量较低,其中肾脏含量最低。说明研究建立的H9N2亚型禽流感病毒荧光定量RT-PCR方法灵敏度高,特异性强。
[Abstract]:In order to study the distribution of (AIV) of H9N2 subtype avian influenza virus in various organs of poultry, a pair of specific primers were designed for the HA gene of H9N2 subtype AIV, and a pair of primers were designed to amplify the 尾-actin gene of internal reference gene. Using the positive recombinant plasmid as the standard curve, a fluorescent quantitative RT-PCR method was established, and the trachea, thymus, lung, liver, spleen, kidney, intestine and other organs of SPF chicks artificially infected with H9N2 were detected for three times. The results showed that the linear relationship R values of the method were all above 0.99, the detection limit of 10 copies plasmid DNA, was more than 100 times more sensitive than RT-PCR, the specificity of the method was good, and the coefficient of variation of intra-and inter-assay reproducibility test was less than 1g. By comparing the relative expression of HA in various organs, it was found that the relative expression of HA was the highest in intestine and pancreas, followed by glandular stomach, spleen, liver, thymus, bursa of Fabricius, and lower in trachea, lung and kidney. The kidney content was the lowest. The results showed that the fluorescent quantitative RT-PCR method of H9N2 subtype avian influenza virus was highly sensitive and specific.
【作者单位】：山东农业大学动物科技学院预防兽医细胞免疫实验室;山东省农业科学院畜牧兽医研究所;
【基金】：山东省农业产业技术创新体系家禽团队岗位经费项目(SDAIT-11-09)
【分类号】：S855.3

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