禽白血病病毒多重PCR检测法的建立及初步应用  

Establishment and application of multiple PCR for avian leukosis virus

作　　者：管宏伟[1] 吴润[1,2] 赵春林[1] 刁小龙[1] 罗鹏[1] 何轶群[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2012年第5期12-17,共6页Journal of Gansu Agricultural University

基　　金：国家自然科学基金项目(31160510);甘肃省自然科学研究基金计划项目(1107RJZA198);家畜疫病病原生物学国家重点实验室开放基金项目(SKLVEB2009KFKT012)

摘　　要：根据GenBank中登录的禽白血病病毒A、B、J亚群的核苷酸序列设计合成了3对引物,各对引物可分别扩增出大小约为195、260、924bp的核苷酸片段;经优化建立禽白血病病毒多重PCR检测法,并进行其敏感性试验及对马立克病病毒(MDV)、火鸡疱疹病毒(HVT)、新城疫病毒(NDV)、传染性囊病病毒(IBDV)、传染性喉气管炎病毒(ILTV)、传染性支气管炎病毒(IBV)和DF-1细胞基因组的特异性试验;应用该方法对1 294份临床样品进行禽白血病病毒检测,对阳性检出样品抽样进行测序鉴定.结果表明:该方法的最小检出拷贝数为103,特异性试验均为阴性,检出临床样品中的核苷酸片段与参考毒株核苷酸序列的同源性达98%以上,符合率100%,具有良好的特异性、敏感性和符合率,可为禽白血病病毒感染的临床诊断及流行病学调查研究提供有效借鉴.According to the published gene sequences of subgroup of A,B and J avian leukosis virus in GenBank,three pairs of primers were designed and synthesized which respectively amplified nucleotide fragment with about the size of 195 bp,260 bp and 924 bp.A multiplex PCR assay specific for avian leukosis virus A,B and J subtypes were optimized and established,which amplified successfully the target fragments and obtained the very good effect on the sensitivity test and specificity test among the virus of MDV,HVT,NDV,IBDV,ILTV,IBV,and DF-1 cell.The assay was used to detect the 1 294 clinical samples and the positive samples were sampled and sequenced.Test showed that the minimum detectable copy numbers was 103,specificity test were respectively detected negative,the homology between the positive clinical samples nucleotide fragments and the reference strains virus＇ sequence were more than 98% and coincidence rate were 100%.Therefore the multiplex PCR assay could be an effective tool for specificity,sensitivity and high compliance rate detection and simultaneous subgroup of clinical diagnosis of avian leukosis virus and surveys of epidemiology.

关 键 词：禽白血病病毒 多重PCR检测 核苷酸 

分 类 号：Q78[生物学—分子生物学]