Detection of Opisthorchis viverrini in infected bithynid snails by real-time fluorescence resonance energy transfer PCR-based method and melting curve analysis.

A real-time fluorescence resonance energy transfer (FRET) PCR combined with melting curve analysis was developed for the detection of Opisthorchis viverrini in experimentally infected bithynid snails, its first intermediate hosts. The test is based on the fluorescence melting curve analysis of a hybrid between an amplicon from the pOV-A6-specific probe sequence, 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 cercaria artificially introduced in a pool of 30 non-infected snails. The O. viverrini-infected snails were discriminated from non-infected snails and from genomic DNA of other parasite DNAs by their melting temperatures. Sensitivity and specificity of this method were both 100%. Melting curve analysis is a sensitive alternative for the specific detection of O. viverrini-infected snails; it is rapid, allows a high throughput, and can be done on small samples. The assay not only has a high potential for epidemiological surveys of O. viverrini-infected bithynid snails, but also for the detection of cercariae infestations of natural waterways when monitoring transmission sites.