鼻气管鸟疫杆菌环介导等温扩增(LAMP)检测方法的建立与应用  

Establishment and adhibition of LAMP system for rapid detection of Ornithobacterium rhinotracheale

作　　者：王丽荣[1] 刁有祥[1] 唐熠[1] 鞠小军[1] 宋晓娜[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2013年第9期1364-1368,共5页Chinese Journal of Veterinary Science

基　　金：农业产业技术体系资助项目(CARS-43-34)

摘　　要：根据GenBank发表的鼻气管鸟疫杆菌的16SRNA(登录号:JF810495.1)序列,利用Primer Explorer V4在线软件设计了1套环介导等温核酸扩增技术(LAMP)引物,并对反应体系和反应条件进行优化,同时评价该方法的灵敏性和特异性。结果显示,建立的LAMP检测方法能够在63℃1h内实现对目的片段的大量扩增,检测结果可直接用肉眼判断。特异性试验结果显示,该方法只能检测到鼻气管鸟疫杆菌,与其他细菌如鸡白痢沙门菌、大肠杆菌、金黄色葡萄球菌、多杀性巴氏杆菌、副鸡嗜血杆菌的核酸无交叉反应,特异性强。敏感性试验表明,该方法可检测到1×101拷贝/μL的质粒标准品,比普通PCR高104倍。利用该方法对分离的阳性菌株进行检测,检出率为83.33%。结果表明,建立的鼻气管鸟疫杆菌LAMP检测方法,具有快速、简便、特异性强、灵敏度高的特点,可用于鼻气管鸟疫杆菌的临床检测。A suit of special primers was designed for ORT with Primer Explorer V4.The reaction system and conditions were established and optimized,meanwhile its specificity and sensitivity were assessed.The Ornithobacterium rhinotracheale（ORT）DNA could be amplified by incubation at 63℃ within 1h and the amplification products could be observed by naked-eye.Specificity test showed that the target bacterium could be specially detected by the method,while the amplification results of Salmonella pullorum,Escherichia coli,Staphylococcusaureus,Pasteurella multocida and Haemophilus paragallinarum were negative.Sensitivity test showed that the detection limit of the system was 1×100 DNA sample,which was 104-fold higher than that of the traditional PCR.The detection rate was 100% by using this newly developed LAMP methed to test positive for the separation of the strains.The established LAMP method appeared to be fast,accurate,sensitive and specific for the detection of this bacterium.

关 键 词：鼻气管鸟疫杆菌 环介导等温核酸扩增 快速检测 

分 类 号：S852.61[农业科学—基础兽医学]