Development of a point-of-care detection method for the carcinogenic liver fluke Opisthorchis viverrini using a mitochondrial NADH dehydrogenase subunit 5 (nad5)-targeted isothermal DNA biosensor

Opisthorchis viverrini is a major cause of cholangiocarcinoma in Southeast Asia. Microscopic diagnosis has low sensitivity in light-intensity infections, delaying early intervention. We developed a loop-mediated isothermal amplification (LAMP) assay with a lateral-flow dipstick (LFD) DNA biosensor targeting the O. viverrini mitochondrial NADH dehydrogenase subunit 5 ( nad5 ) gene, using a fluorescein isothiocyanate (FITC)-labeled probe and biotinylated primers for visual readout. Temperature (59–65 °C), MgSO₄ and deoxynucleoside triphosphate (dNTP) concentrations were optimized. Optimal amplification was at 61 °C for 60 min, with visual detection on the dipstick within 5–10 min. The analytical detection limit under the tested conditions was 200 fg of O. viverrini DNA. No cross-reactivity was observed in a limited panel of six non-target intestinal parasite specimens (one per organism): echinostome, minute intestinal flukes, Taenia spp., Trichuris trichiura , Strongyloides stercoralis , and Blastocystis sp. In a preliminary evaluation of 51 samples (30 positive, 21 negative by the formalin-ethyl acetate concentration technique, FECT), sensitivity was 100% (30/30) and specificity 100% (21/21 negative samples, including 15 healthy controls) relative to the reference method. The positives comprised 15 culture-derived egg samples and 15 FECT-confirmed fecal samples, the latter all light infections. Overall, the LAMP–LFD detection strip requires no gel electrophoresis, thermal cycler, or optical reader, although DNA extraction currently still requires an autoclave and centrifuge. With an analytical detection limit of 200 fg DNA and visual readout, it is a candidate tool for point-of-care screening of O. viverrini infection, pending validation in larger multicenter field studies.