二温式多重PCR检测鉴别对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)的研究与应用  

Studies on development of two-temperature multiplex PCR for WSSV and IHHNV

作　　者：谢芝勋[1] 庞耀珊[1] 刘加波[1] 邓显文[1] 唐小飞[1] 

机构地区：[1]广西壮族自治区兽医研究所,广西南宁530001

出　　处：《海洋科学》2005年第12期9-12,24,共5页Marine Sciences

基　　金：广西科技攻关资助项目(桂科攻0322006-3A)

摘　　要：建立了一种同时检测对虾白斑综合征病毒(WSSV)、传染性皮下和造血器官坏死病毒(IHHNV)的二温式多重PCR技术。根据基因库中WSSV(AF369029)和IHHNV(NC002190)基因序列,设计了两对分别与WSSV和IHHNV某段保守基因序列互补的引物,用这两对引物对同一样品中的WSSV和IHHNV DNA模板进行多重PCR扩增,结果均同时得到了两条大小与实验设计相符的593 bp(WSSV)和356 bp(IHHNV)特异性多重PCR扩增条带,而对其他对虾疾病病原的PCR扩增结果均为阴性;敏感性测定结果表明,该多重PCR技术最低能检测到WSSV和IHHNV DNA各100 pg。用该多重PCR对400份临床病料进行检测,结果230份检出WSSV,阳性率57.5%;96份检出IHHNV,阳性率24%;56份同时检出WSSV和IHHNV,阳性率14%。18份为阴性,阴性率为4.5%。结果提示了WSSV和IHHNV广泛存在于中国南方的养殖对虾中,作者建立的二温式多重PCR可以用于这两种病毒的临床快速检测和鉴别诊断。A two-temperature multiplex polymerase chain reaction was optimized to simultaneously detect two pathogens including white spot syndrome virus （WSSV） and infectious hypodermal and hematopoietie necrosis virus （IHHNV） in this article. Two sets of specific primers were designed according to the sequences of WSSV（AF369029） and IHHNV （NC002190） in Genebank. It was shown that the all samples, which contained WSSV and IHHNV,could be amplified by the multiplex PCR using these two sets of primers, yielding two specific bands of WSSV 59a bp and IHHNV a56 bp, but no specific bands were amplified from other penaeid shrimp pathogenic virus and bacteria. As little as 100 pg of WSSV and 100 pg of IHHNV DNA were detected respectively using gel electrophoresis in this multiplex PCR. We used this multiplex PCR to detect 400 samples from some farms in south China. Results showed that WSSV could be detected in 230 samples, IHHNV could be detected in 96 samples and both WSSV and IHHNV could be simultaneously detected in 56 samples. The other 18 samples were negative to these two pathogens. It suggested that WSSV and IHHNV existed in the cultivated penaeid shrimps in south China and this multiplex PCR could be used as a sensitive tool to detect WSSV and IHHNV in clinical samples, simultaneously.

关 键 词：多重聚合酶链式反应 检测 对虾 白斑综合征病毒(WSSV) 传染性皮下和造血器官坏死病毒(IHHNV) 

分 类 号：S945.4[农业科学—水产养殖]