# Development of a High-Sensitive qPCR-Based Molecular Diagnosis Method for Detection of Clonorchis sinensis in Fish Muscle and Environmental Water

## TL;DR

This study develops a highly sensitive qPCR method to detect the liver fluke Clonorchis sinensis in fish and water, improving monitoring in endemic areas.

## Contribution

A novel, species-specific qPCR method targeting the COI gene for sensitive detection of C. sinensis in fish and environmental samples.

## Key findings

- The qPCR method achieved a detection limit of 101 copies per reaction and quantification limit of 102 copies.
- The method successfully detected C. sinensis DNA in spiked environmental water samples.
- The qPCR outperformed conventional PCR in sensitivity for fish-derived samples.

## Abstract

A liver fluke, Clonorchis sinensis is a representative fish-borne parasite infecting humans, and sensitive detection in fish hosts or aquatic environments is important for monitoring infection sources in endemic areas. Conventional diagnostic methods based on microscopy or conventional PCR often show limited sensitivity, particularly under low-parasite conditions. In this study, we developed a high-sensitive and species-specific molecular marker and established a real-time PCR (qPCR)-based diagnostic method targeting metacercariae isolated from freshwater fish, representing the transmission stage of C. sinensis. Primers and a hydrolysis probe targeting the mitochondrially encoded cytochrome c oxidase 1 (COI) gene were designed, and all primer combinations produced stable amplifications with single melt curves in C. sinensis-positive samples. Among them, one combination was finally selected as the optimal marker due to its high specificity, including validation against mixed trematode samples to confirm species-specific detection. The qPCR assay showed excellent linearity (R2 = 0.998), with a detection limit of 101 copies per reaction and a quantification limit of 102 copies per reaction. In addition, the assay successfully detected C. sinensis DNA in environmental water samples spiked with metacercariae, demonstrating its applicability to aquatic samples for environmental surveillance purposes. Compared with conventional PCR, the developed qPCR method in this study exhibited markedly improved sensitivity in fish-derived samples. Overall, this qPCR assay provides a robust diagnostic tool for laboratory analysis and has potential utility for environmental DNA-based monitoring of clonorchiasis risk areas within a One Health framework.