Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium
Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium
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DOI：
10.1186/1471-2180-10-296
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发表时间：
2010-11-19
期刊：
影响因子：
4.2
通讯作者：
Sugimoto, Chihiro
中科院分区：
文献类型：
--
作者：
Nakao, Ryo;Stromdahl, Ellen Y.;Sugimoto, Chihiro
工作背景：立克次体细菌反刍埃里希体是心水病的病原体，心水病是一种由钝眼蜱属蜱传播的反刍动物潜在的人畜共患疾病。这种疾病分布在几乎所有的撒哈拉以南非洲和加勒比海的一些岛屿，从那里威胁到美国大陆。本报告描述了两种不同的环介导等温扩增（LAMP）检测灵敏度和特异性检测大肠杆菌的发展。结果：根据pCS20和sodB基因设计了两对LAMP引物。每种检测方法的检测限为pCS 20 10个拷贝和sodB 5个拷贝，这比传统的pCS 20 PCR检测方法高至少10倍。DNA扩增在60 min内完成。来自不同地理国家的反刍动物以及两种减毒疫苗分离株。未观察到与遗传相关的立克次体（包括来自美国的人畜共患埃里希体属）的交叉反应。LAMP检测到更多的阳性样品比传统的PCR，但低于实时PCR，当测试与现场样品收集在撒哈拉以南countries.Conclusions：由于其简单性和特异性，LAMP有可能用于资源贫乏的设置，也为E.反刍动物在心脏水流行地区和有感染该疾病风险的地区。
Background: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium.Results: Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries.Conclusions: Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.