Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1 pg of purified genomic DNA, 5 EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.

Amplification plot of fluorescence (y-axis) vs cycle numbers (x-axis) showing the analytical sensitivity of the real-time PCR for detecting Clonorchis sinensis. 1–6 represent 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, and 0.1 pg, respectively. 7 represents distilled water.Figure optionsDownload as PowerPoint slideResearch highlights
► Based on ITS-1 sequences, a TaqMan based real-time PCR was developed for detection of C. sinensis.
► The detection limit of the assay was one pg of genomic DNA, five EPG or one metacercaria per gram fish filet.
► The assay is useful for detecting C. sinensis in countries where O. viverrini and O. felineus are absent.