Improved environmental DNA detection sensitivity of Opisthorchis viverrini using a multi-marker assay.

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, owing to 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 in implementing preventive measures in areas where fish are a significant component of the local diet. Using the environmental DNA (eDNA) approach to detect parasitic DNA in water samples, it is possible to understand the distribution of O. viverrini and the dynamics of infection with high accuracy. However, the sensitivity of environmental samples remains a bottleneck. In this study, we developed a highly sensitive method for detecting O. viverrini eDNA by designing a multi-marker assay targeting two mitochondrial and two nuclear genes. We compared its performance with single-marker assays using tissue-derived DNA. 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 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.