H9N2亚型禽流感荧光定量RT-PCR检测方法的建立及应用  

Establishment and application of RT-PCR for detection of Avian influenza virus H9N2 subtype

作　　者：杨玲玲[1] 牛凯[1] 王涛[1] 常维山[1] 刘玉庆[2] 

机构地区：[1]山东农业大学动物科技学院预防兽医细胞免疫实验室,山东泰安271018 [2]山东省农业科学院畜牧兽医研究所,济南250100

出　　处：《黑龙江畜牧兽医》2017年第8期39-42,287,288,共6页Heilongjiang Animal Science And veterinary Medicine

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)

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。In order to study the organs distribution of H9N2 subtype Avian influenza virus in chicken,special primers based on H9N2 Avian in- fluenza virus HA gene were designed and a pair of primers of house - keeping gene 13- actin was chosen. The standard curve was made by the positive recombinant plasmid, and flUOreScent quantitative RT - PCR detection method was established. The trachea, thymus, lung, liver, spleen, kidney, digestive tract of SPF chickens were detected by the established motheds in three＇ replicates. The results showed a precise linear relationship with a correlation coefficient of R 〉0.99. The detection limits were 10 copies of DNA plasmid reaction, it was 1130 times more sen- sitive than RT- PCR, and excelent specificity. The variation coefficient between batch and batch repeat trials was less than 1% . The relative expressions of HA in the intestine and pancreas were highest, the proventriculus followed. The relative expressions of HA in spleen, liver, thy- mus, bursa were higher; trachea, lung and kidney were lower than that. Among them, the kidney content is lowest. It was concluded that the established RT - PCR assay was highly specific and ＇sensitiVe.

关 键 词：H9N2亚型禽流感病毒 HA基因 RT-PCR 检测 病毒分布 

分 类 号：Q78[生物学—分子生物学] S852.657[农业科学—基础兽医学]