Abstract
Opisthorchiasis, caused by the liver fluke Opisthorchis viverrini, is endemic to Southeast Asian countries and constitutes a major health problem, as it increases the risk of cholangiocarcinoma. However, because of the complicated life cycle of O. viverrini, there is no rapid method for monitoring the risk of infection in the environment. The inability to identify water sources at risk of infection presents a challenge for implementing preventive measures in areas where fish are a significant component of the local diet. Using the environmental DNA (eDNA) approach to detect parasite DNA in water samples, it is possible to better understand the distribution of O. viverrini and the dynamics of infection with high accuracy. However, sensitivity remains a bottleneck in environmental samples. In this study, we developed an improved, highly sensitive method for the detection of O. viverrini eDNA. To increase the sensitivity, we designed a multi-marker assay comprising two mitochondrial and two nuclear genes as target regions and compared its sensitivity with single-marker assays using tissue-derived DNA samples. The multi-marker assay exhibited the highest detection sensitivity. We also collected data from 56 sites in Sakon Nakhon, Thailand, in October 2022 and tested them for the eDNA detection of O. viverrini. Eleven sites were positive in the multi-marker assay, whereas five sites were positive in the single-marker assay. This multi-marker assay can be used in the field to detect small organisms and rare species, other than parasites, that are expected to have low levels of eDNA.