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