猪繁殖与呼吸综合征病毒LAMP检测方法的建立  

Development of a LAMP Assay for Detection of Porcine Reproductive and Respiratory Syndrome Virus

作　　者：尹斐斐[1,2] 吴家强[2,3] 刘少宁[2,4] 李坤[1,2] 杜以军[2,3] 孙文博[2,3] 丛晓燕[2,3] 王可[2,3] 张玉玉[2,3] 时建立[2,3] 李俊[2,3] 周顺[1] 王金宝[1,2,3] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]山东省畜禽疫病防治与繁育重点实验室,山东济南250100 [3]山东省农业科学院畜牧兽医研究所,山东济南250100 [4]山东农业大学动物科技学院,山东泰安271018

出　　处：《家畜生态学报》2011年第2期50-54,共5页Journal of Domestic Animal Ecology

基　　金：农业科技成果转化资金项目(2010GB2C600259);农业科技跨越计划(2009农跨18);山东省自主创新成果转化重大专项(2010ZHZX1A0417);山东省农业重大应用技术创新课题(鲁财农便〔2010〕5号)

摘　　要：研究针对美洲型猪繁殖与呼吸综合征病毒(PRRSV)保守的N蛋白基因设计了4条LAMP的检测引物,通过优化反应条件,建立了一种检测PRRSV的LAMP诊断方法。特异性和敏感性试验结果显示,该方法能够特异地检测美洲型PRRSV,其最低检测限为1×101个拷贝,而常规PCR的最低检出限为1×105个拷贝。判定检测结果时不需要借助昂贵仪器设备,仅需加入1μL橙红色核酸染料就可以进行判断,颜色变绿判定为阳性,仍保持为橙红色判定为阴性。LAMP可以快速、直观、准确地检测PRRSV,是一种适合猪场现地应用的检测方法。Four pairs of primers were designed based on conserved N protein gene of porcine reproductive and respiratory syndrome virus（PRRSV） American type in this study.Through optimization of various reaction conditions,a loop-mediated isothermal amplification（LAMP） assay was established for the detection of PRRSV.It was shown that the LAMP assay had a good specifity and achieved theoretically a sensitivity of 1×101 copies,whereas routine PCR reached 1×105 copies.Without the help of expensive machines,the detection result of LAMP could be observed by adding 1 μL orange-red nucleic acid dyes.The positive samples showed green color,while the negative ones kept orange-red color.In summary,the constructed LAMP is a rapid,intuitive and accurate method,which is especially suitable for the detection of PRRSV in the pig farms.

关 键 词：猪繁殖与呼吸综合征病毒 LAMP 核酸染料 现地检测 

分 类 号：S811.5[农业科学—畜牧学]