猪呼吸与繁殖综合症病毒RT-LAMP检测方法的建立  

Development of a Novel RT-LAMP Assay for Porcine Reproductive and Respiratory Syndrome Virus

作　　者：鑫婷[1] 侯绍华[1] 贾红[1] 郭晓宇[1] 丁家波[2] 李延鹏[1] 丁敏[1] 朱鸿飞[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100081 [2]中国兽医药品监察所,北京100081

出　　处：《中国农业科学》2010年第1期185-191,共7页Scientia Agricultura Sinica

基　　金：国家科技支撑项目(2006BAD06A04)

摘　　要：【目的】建立一种适用于猪繁殖与呼吸综合症病毒的快速、灵敏、特异性检测方法,即一步反转录环介导等温扩增技术(RT-LAMP)。【方法】设计3对针对PRRSVN基因的8个位点的特异性引物,用优化后的反应体系检测RT-LAMP的特异性、灵敏性并对临床样本进行检测。【结果】RT-LAMP检测方法从核酸抽提到检测仅需要70min,肉眼即可观察检测结果,该方法具有良好的特异性,灵敏度是RT-PCR的10000倍,在对50份猪血液样本RNA的检测中,该方法与传统的RT-PCR检测方法具有很好的统一性(κ=0.83)。【结论】RT-LAMP检测方法可快速、灵敏、特异的检测PRRSV,并适用基层和现场检测。【Objective】 The objective of the experiment is to develop a one-step reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay for rapid and sensitive detection of porcine reproductive and respiratory syndrome virus （PRRSV）. 【Method】 Three pairs of primers were designed to identify 8 positions in N gene,the sensitivity and specificity of RT-LAMP were determined and clinical test was performed under optimized amplification condition.【 Result】 A RT-LAMP assay was developed for the rapid detection of PRRSV. The process of assay was conducted with one step amplification within 70 minutes and amplification results were visualized. The sensitivity of RT-LAMP assay was 10 000 times higher than RT-PCR. In the detection of 50 porcine serum samples,this assay showed excellent agreement with the standard RT-PCR assay （κ=0.83）. 【Conclusion】 Therefore,the rapid and simple assay is a potential useful technique for PRRSV detection in field condition.

关 键 词：猪繁殖与呼吸综合征病毒 RT-LAMP 检测 

分 类 号：S858.28[农业科学—临床兽医学]