تشخیص و افتراق سریع فاسیولا هپاتیکا و فاسیولا ژیگانتیکا با استفاده از روش loop-mediated Isothermal Amplification

Background and purpose: Fasciola hepatica and Fasciola gigantica are common liver flukes which affect both human and livestock worldwide. In this study we evaluated the loop-mediated isothermal amplification (LAMP) assay for detection and discrimination of Fasciola species. Materials and methods: Fifty adults of Fasciola worms were isolated from sheep and cattle liver form abattoirs in Mazandaran province. A total of 8 primer sets for LAMP was designed to amplify the 28S ribosomal RNA gene of Fasciola sp. Conventional LAMP was carried out in a 20µI reaction mixture under isothermal condition at 60°C for 90 minutes. Amplification result was observed by monitoring the turbidity by naked-eye, using fluorescent dye and gel electrophoresis. The specificity of LAMP method for detecting Fasciola sp. was tested by amplification of Dicrocoelium dendriticum, Trichostrongylus colubriformis, and Echinococcus granulosus DNA templates. To evaluate the detection limit of LAMP assay in detecting Fasciola genus, serial dilution of the extracted DNA was used. Results: A positive LAMP reaction by the specific primers of two species produced many bands of different sizes in 600C after 90 min. The optimal assay conditions were established with no reaction with other parasites’ DNA. The detection limit of this LAMP assay was 1 pg DNA/tube. The result of turbidity and fluorescent dye detection were consistent with agarose gel electrophoresis. Conclusion: Our results demonstrated that LAMP is a rapid, cost-effective, highly specific, easy, and reliable method for differentiation of Fasciola sp. in epidemiological and clinical researches on human and domestic animals in endemic regions of fasciolosis.