Development of a duplex loop-mediated isothermal amplification together with lateral flow dipstick assay for the detection and discrimination of parasitic infections in chickens between cestodes belonging to genus Raillietina and trematodes in family Echinostomatidae

Abstract

Most poultry farming that has been conducted by smallholders in Thailand are free-range and housing systems, which have risks of parasitic infection from the environment, particularly from tapeworms in the genus Raillietina and trematodes in the family Echinostomatidae. These have become important health problems in the poultry industry, causing low feed conversion ratios and leading to the loss of economic value. Our objective was to develop and validate a molecular technique based on duplex loop-mediated isothermal amplification (LAMP) together with lateral flow dipstick (LFD) assay for discriminating the infections between the genus Raillietina (R. echinobothrida, R. tetragona, and R. cesticillus) and the family Echinostomatidae (E. miyagawai, E. mekongi, E. macrorchis, and H. conoideum) in a single reaction. The developed assay was highly specific without cross-amplification with other poultry helminths and their hosts, at the optimized condition of 66 °C for 80 min. In addition, the results could be clearly visualized with the naked eye via LFD after incubating with probes at 66 °C for 10 min. The detection limit or analytical sensitivity of the Raillietina and Echinostomatidae groups were found to be 5 × 10⁻⁴ and 5 × 10⁻² ng/μL, respectively. In clinical tests, the developed assay successfully detected parasites in naturally infected faeces from chickens in Thailand; results from McNemar's tests revealed no significant difference when compared to standard microscopy methods. Therefore, our assay is a viable alternative technique for an accurate and convenient diagnosis; it can also be used as a tool to guide anthelmintic drugs decision-making for treatments and farm management.