J亚群禽白血病病毒嵌套式PCR检测方法的建立及其应用  

Development and application of nested-PCR for detection of ALV-J

作　　者：王丽萍[1,2] 龚振华[2] 张康[1] 臧京帅[1] 王洪伟 侯广宇[2] 李蕾[2] 李金平[2] 于建敏[2] 单虎[1] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]诸城市畜牧兽医管理局,山东诸城262200

出　　处：《中国兽医科学》2013年第12期1285-1290,共6页Chinese Veterinary Science

基　　金：青岛市科技计划基础研究项目(11-2-4-5-<11>-jch)

摘　　要：根据GenBank中ALV-J亚群HPRS103株(登录号:Z46390.1)gp85蛋白基因序列,设计合成了2对引物,建立了针对ALV-J亚群的嵌套式PCR检测方法。外侧引物PCR扩增片段的大小为960bp,内侧引物PCR扩增片段的大小为766bp。筛选嵌套式PCR的最佳反应条件,并进行了特异性试验、敏感性试验、重复性试验和临床检测比较。结果显示,建立的嵌套式PCR方法能扩增ALV-J亚群特异性片段,而对禽网状内皮组织增殖病病毒、马立克氏病病毒、H9亚型禽流感病毒、新城疫病毒、传染性腔上囊病病毒和减蛋综合征病毒的扩增结果均为阴性;该方法第一轮和第二轮扩增的敏感性分别为1fg/μL和0.1fg/μL,第二轮扩增的敏感性比第一轮的高10倍;通过对32份临床样品的检测,嵌套式PCR的检出率与病毒分离方法的检出率相同,且高于参考PCR的检出率18.7%。结果表明,本研究建立的嵌套式PCR方法具有敏感性高、重复性好、特异性强等优点,可用于ALV-J亚群的临床诊断。Based on the gp85 gene sequence of HPRS103 strain（No. ：Z46390.1）,a subgroup J avian leukosis virus（ALV-J）,two pairs of special primers of nested PCR for detection of ALV-J were designed, and were expected to have the amplified targets at 960 bp by first-round PCR and 766 bp by second-round PCR. The nested-PCR was established for detection of ALV-J and its specificity, sensitivity,reproducibility and comparative clinical trials were tested. In results, the nested-PCR could specifically detect ALV-J, but not REV, MDV, AIV（H9）, NDV, IBDV, and EDSV. The nested-PCR had the sensitivity of 1 fg/μL of ALV-J DNA for first-round PCR and 0.1 fg/μL for second-round PCR. The latter was 10 times higher than the former. Using 32 clinical samples,the results showed that the nested-PCR had the same detection rate as that of the virus isolation detection method, which was higher than the reference PCR by 18. 7 %. The established nested- PCR had satisfying results on its sensitivity, specificity and repeatability for detection of ALV-J, which could be used for clinical ALV-J subgroup diagnosis.

关 键 词：禽白血病病毒 嵌套式PCR 检测 

分 类 号：S852.65[农业科学—基础兽医学]