柞蚕核型多角体病毒实时荧光LAMP检测方法的初步建立  

Establishment of Real-time Fluorescence LAMP for Detection of Antheraea pernyi Nucleopolyhedrovirus

作　　者：张洋 薛强 金英 李凤芹 闫伟[2] 

机构地区：[1]吉林省蚕业科学研究院,吉林吉林132112 [2]吉林省农业科学院,长春130124

出　　处：《蚕业科学》2017年第6期957-961,共5页ACTA SERICOLOGICA SINICA

基　　金：吉林省财政行业与基本课题项目(No.吉科发财[2016]40号)

摘　　要：柞蚕脓病是柞蚕生产上的主要病害之一,病原为柞蚕核型多角体病毒(Ap NPV)。建立快速而准确检测蚕体样品中Ap NPV的技术,将有助于对病害的早期诊断及流行病学调查和实施有效的防控措施。以柞蚕核型多角体病毒ORF142基因为靶基因,设计2组特异性环介导等温扩增(LAMP)引物,利用实时荧光PCR技术进行检测分析。结果表明,设计与合成的2组引物均能对柞蚕核型多角体病毒基因组扩增出特异性产物,其中引物组2在63℃开始孵育60 min,反应起始扩增时间为12 min,当反应时间达到40 min时,扩增曲线的最大相对荧光强度(RFU)值达6 000,相对于引物组1具有更好的及时性,可缩短一半反应起始时间。初步建立的Ap NPV实时荧光LAMP检测方法相对于传统PCR方法,检测效率提高4倍左右。Polyhedrosis is one of the major diseases in tussah silkworm（ Antheraea pernyi） production. Its pathogen is A.pernyi nucleopolyhedrovirus（ Ap NPV）. The establishment of a rapid and accurate technique for detection of Ap NPV in tussah silkworm body will be helpful for early diagnosis,epidemiological investigation and effective prevention of polyhedrosis. With ORF142 of Ap NPV as target gene,2 primer sets for specific loop-mediated isothermal amplification（ LAMP）were designed and used for analysis by real-time fluorescence PCR. The results showed that both primer sets could amplify specific product from Ap NPV genome,among which primer set 2 needed to be incubated at 63 ℃ for 60 min,and needed 12 min for initial amplification. When the reaction time reached 40 min,the maximum relative fluorescence intensity（ RFU） of the amplification curve reached 6 000,showing better promptness than primer set 1 and shortening the initial reaction time by half. The established real-time fluorescence LAMP improves efficiency of Ap NPV detection by around 4 times compared with the traditional PCR method.

关 键 词：柞蚕核型多角体病毒 环介导等温扩增技术 引物 反应条件 早期诊断 

分 类 号：S884.51[农业科学—特种经济动物饲养]