环介导等温扩增技术检测钩端螺旋体的研究  

Leptospira interrogans DNA detection by loop-mediated isothermal amplification

作　　者：曹志强[1] 张翠彩[1] 李秀文[1] 蒋秀高[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《中国人兽共患病学报》2015年第4期293-297,310,共6页Chinese Journal of Zoonoses

基　　金：国家科技重大专项(No.2013ZX10004-101&No.2012ZX10004-215)~~

摘　　要：目的利用环介导等温扩增技术,建立检测钩端螺旋体的方法。方法选取钩体LipL32基因,设计了LAMP引物。对27株钩体、25株非钩体样本予以检测,然后进行灵敏度和模拟样本的研究。结果扩增27株钩体结果为阳性,扩增25株非钩体结果为阴性。灵敏度为200拷贝/μL,模拟样本检测下限为200条钩体/μL(煮沸法)和20条钩体/μL(试剂盒提取法)。结论 LAMP实验操作简单,对设备要求不高,可以作为钩体的快速检测方法。Loop-mediated isothermal amplification has been widely used in pathogenic bacteria inspection. There is a lack of diagnostic tests for leptospirosis in technology-restricted settings or in emergency assistance activities. We developed and evalu- ated a loop-mediated isothermal amplification （LAMP） method for the rapid detection of pathogenic Leptospira spp. through amplification of lipL32 gene coding for the outer membrane protein LipL32. Specificity was high as we observed positive reac- tions of 27 pathogenic strains but negative reactions for other bacterial species. The lower limit of detection was 200 genomic equivalents/μL, and the sensitivity of LAMP was 100 times higher than that of PCR. The method enables detection of 200 lep- tospiral cells/μL following boiling of specimens and-20 leptospiral cells/μL of DNA extraction kit means. The lamp assay is easy to perform and inexpensive, so might be applied in the rapid and specific diagnosis of Leptospira.

关 键 词：问号钩端螺旋体属 环介导等温扩增 检测 核酸扩增 

分 类 号：R377[医药卫生—病原生物学]