环介导等温扩增法检测棘颚口线虫方法的建立

Sensitive and Rapid Detection of Gnathostoma spinigerum by Loop-mediated Isothermal Amplification (LAMP)

摘要:

棘颚口线虫是引起人体颚口线虫病最主要的病原，其引起的病例呈世界性分布。由于其复杂的生活史，以及不同生长阶段形态的变化差异，传统的形态学鉴定方法很难鉴别。为了快速、灵敏和可靠的鉴定棘颚口线虫，本研究建立了一套检测棘颚口线虫的DNA环介导等温扩增（loop-mediated isothermal amplification，LAMP）方法。根据LAMP方法原理，针对棘颚口线虫的ITS2 rDNA设计了三套特异性引物特异性识别靶基因。进行了特异性、灵敏度、稳定性和实际样品的测试。结果表明，该方法对棘颚口线虫DNA能够特异性扩增，而比对虫体DNA均无扩增；对含有棘颚口线虫ITS2目的基因片段的质粒DNA的检测限为1 fg/μL，比传统的PCR方法灵敏度高100倍；对51批次实际样品的进行检测，LAMP方法与传统的PCR测序方法结果相符。本研究设计的LAMP检测方法适用于特异性检测棘颚口线虫。

Abstract:

Human gnathostomiasis is a worldwide disease that is caused primarily by the larval and immature stages of Gnathostoma spinigerum. The detection of G. spinigerum is difficult due to its complicated life cycle and morphological variety at different stages. Hence, traditional parasitological techniques are only reliable when conducted by experienced laboratory personnel. In this study, we established a loop-mediated isothermal amplification (LAMP) approach for the sensitive, rapid, and reliable detection of G. spinigerum DNA. Based on LAMP reaction, three sets of specific and sensitive primers were designed from the ITS2 rDNA of G. spinigerum. The specificity, sensitivity, and stability were assessed using actual samples. Specific amplification products were obtained with G. spinigerum DNA, while no amplification products were detected with DNA of related parasites, thus demonstrating the specificity of the assay. The detection limit of this method for plasmid DNA containing G. spinigerum gene fragments was 1 fg/μL, which indicates that the LAMP assay was 100 times more sensitive than a conventional PCR for the detection of G. spinigerum. Fifty-one samples from South East Asia and South Asia were tested using the LAMP and PCR methods. The results of the LAMP method were consistent with those by the PCR method and the LAMP method established in this study is suitable for the specific detection of G. spinigerum.