猪繁殖与呼吸综合征病毒LAMP检测方法的建立  

Development of a LAMP Method for PRRSV

作　　者：罗忠永 王印[1] 杨泽晓[1] 

机构地区：[1]四川农业大学动物医学院动物检疫实验室,四川成都611130

出　　处：《动物医学进展》2016年第3期1-5,共5页Progress In Veterinary Medicine

基　　金："十二五"国家科技支持计划项目(2013BAD12B04)

摘　　要：应用通用反转录试剂盒和环介导等温核酸扩增技术(LAMP)建立了猪繁殖与呼吸综合征病毒(PRRSV)的快速检测方法,同时评价了该检测方法的特异性、稳定性和灵敏度。结果表明,根据猪繁殖与呼吸综合征病毒M蛋白基因保守区设计的LAMP引物能够在64.5℃60min内实现对目标核酸片段的大量扩增,检测结果可通过直接观察反应副产物(焦磷酸镁)进行判读,该检测系统具有很高的特异性,与猪瘟病毒、猪乙型脑炎病毒等均无交叉反应;通过PRRSV不同毒株疫苗、模拟样品和临床样品确定,该检测系统具有很好的稳定性;通过质粒确定该检测系统可以检测到10拷贝/μL的病毒核酸模板。结果表明成功建立了猪繁殖与呼吸综合征病毒LAMP快速检测方法,为临床提供了一种很好的检测手段。A rapid detection method for porcine reproductive and respiratory syndrome virus was established by using the universal reverse transcription kit and loop-mediated isothermal amplification technology,and the specificity,stability,sensitivity of the detection were evaluated.The results showed that the target nucleic acid fragment can be amplified in 64.5℃ within 60 min using the LAMP primers designed according to porcine reproductive and respiratory syndrome virus M protein gene conservative region,the test results can be directly observed by the naked eye through the reaction-products（magnesium pyrophosphate）,the detection system has high specificity,and viruses such as classical swine fever virus,porcine Japanese encephalitis virus showed no cross reaction;the good stability of the detection system was confirmed by different strains of vaccines,simulated samples and clinical samples;the plasmid-template which is just 10copies/μL in concentration was detected by the detection system.The test results proved that the LAMP method of rapid detection for porcine reproductive and respiratory syndrome virus was successfully established.

关 键 词：猪繁殖与呼吸综合征病毒 环介导等温核酸扩增 检测 

分 类 号：S852.659.6[农业科学—基础兽医学]