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6个区段设计的6条引物用于扩增检测,整个反应在恒温条件63℃下进行25 min,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4.86×108OBs/mL感染4龄起蚕,提取血淋巴DNA为模板,PCR在感染36 h后检出病毒DNA,LAMP法能检测感染12h后的样品。
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 ？rst suitable for potential diagnosis and helping to monitor BmNPV infections in the sericulture. A set of six speci？c 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 ampli？ed 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.
出处
《应用昆虫学报》
CAS
CSCD
北大核心
2013年第4期1013-1019,共7页
Chinese Journal of Applied Entomology
基金
云南省现代农业产业技术体系建设专项(蚕桑)
云南省农业科学院蚕桑蜜蜂研究所青年基金项目(OC2012002)
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
Bombyx mori nucleopoyhedrosis virus （BmNPV）, loop-mediated isothermal amplification （LAMP）,diognosis, sericuhure
