Real-time fluorescence resonance energy transfer PCR with melting curve analysis for the detection of Opisthorchis viverrini in fish intermediate hosts

A real-time fluorescence resonance energy transfer (FRET) PCR combined with a melting curve analysis was developed for the detection of Opisthorchis viverrini in its fish intermediate host, cyprinoid fishes. Real-time FRET PCR is based on a fluorescence melting curve analysis of a hybrid between an amplicon generated from a family of repeated DNA elements, the pOV-A6 specific probe sequence (Genbank Accession No. S80278), a 162 bp repeated sequence specific to O. viverrini, and specific fluorophore-labeled probes. The real-time FRET PCR could detect as little as a single metacercaria artificially inoculated in 30 fish samples. The O. viverrini infected fishes were distinguished from non-infected fishes and from the genomic DNA of other parasites by their melting temperature. Sensitivity and specificity of this method were both 100% in the laboratory setting and it outperformed the microscopic method on field-collected samples as well. Melting curve analysis is a rapid, accurate, and sensitive alternative for the specific detection of O. viverrini infected fishes. It allows a high throughput and can be performed on small samples. The assay has not only great potential for epidemiological surveys of fish intermediate hosts but it could also be adapted as screening tool for a range of foodborne parasites in freshwater fishes.