应用环介导等温扩增技术（LAMP）快速检测家蚕核型多角体病毒的研究
Prompt Diagnosis of Bombyx mori Nucleopoyhedrosis Virus by Loop-mediated Isothermal Amplification
唐芬芬，杨伟克，邵榆岚，谢道燕，廖鹏飞，杨海，白兴荣
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作者单位：云南省农科院蚕桑蜜蜂研究所
中文关键词：家蚕核型多角体病毒，环介导等温扩增技术，早期诊断，蚕业
英文关键词：Bombyx mori nucleopoyhedrosis virus (BmNPV), Loop-mediated isothermal amplification(LAMP), Diognosis, Sericulture
中文摘要：由家蚕核型多角体病毒 ( Bombyx mori nucleopoyhedrosis virus, BmNPV )侵染家蚕（Bombyx mori）引起的家蚕核型多角体病 (血液型脓病 )在养蚕生产中发生较为普遍,对蚕业生产造成重大的经济损失。本研究建立了快捷有效的环介导等温扩增技术（Loop-mediated isothermal amplification，LAMP)用于快速检测BmNPV，为蚕业生产提供了一个有效检测BmNPV和进行早期诊断的技术。该方法是针对BmNPV的pe38基因的六个区段设计的六条引物用于扩增检测，整个反应在恒温条件63℃下进行25分钟，扩增产物用电泳法和可视法检测（用SYBR Green I染色）。结果显示，LAMP 检测方法的灵敏度是常规PCR方法的100倍，能检测的范围为21个拷贝。另外，用4.86×108 OBs/mL感染4龄起蚕，提取血淋巴DNA为模板，PCR在感染36h后检出病毒DNA，LAMP法能检测感染12h后的样品。
英文摘要：Bombyx mori nucleopoyhedrosis virus (BmNPV) infects silkworm severely and leads to economic damage in sericulture. In this study, a robust and simple Loop-mediated isothermal amplification （LAMP） assay for rapid detection of BmNPV was developed，which is the first suitable for potential diagnosis and helping to monitor BmNPV infections in the sericulture. A set of six specific primers was designed to recognize six distinct genomic sequences of pe38 from BmNPV. The entire assay duration was recorded at 25min under isothermal condition at 63℃.The amplified products were analyzed by electrophoresis and visual judgment by the SYBR Green I dyeing. The results showed that LAMP assay was 100-fold more sensitive than the routine PCR assay, with a detection limit of 21 copies per reaction. The amount of viral DNA in hemolymph sampled from the larvae infected at different time points was also invested by PCR and LAMP