Development of a PCR assay and pyrosequencing for identification of important human fish-borne trematodes and its potential use for detection in fecal specimens

BACKGROUND: Small liver and minute intestinal flukes are highly prevalent in Southeast Asia. Definitive diagnosis of parasite infection is usually achieved parasitologically by finding the fluke eggs in feces. However, their eggs are difficult to differentiate morphologically in fecal samples, even for experienced technicians. The present study developed a PCR assay coupled with DNA pyrosequencing for identification of the fish-borne trematodes (FBT), Opisthorchis viverrini, Clonorchis sinensis, Haplorchis taichui, H. pumilio and Stellantchasmus falcatus, and to evaluate potential detection in fecal specimens, and identification and differentiation of cercarial and metacercarial stages. METHODS: Primers targeting the partial 28S large subunit ribosomal RNA gene were designed and about 46–47 nucleotides were selected as the target region for species identification by a PCR assay coupled with a pyrosequencing technique. RESULTS: The nucleotide variations at 24 positions, which is sufficient for the identification of the five species of FBT were selected. The method could identify O. viverrini and C. sinensis eggs in feces, cercarial and metacercarial stages of O. viverrini, and metacercarial stage of H. pumilio and H. taichui. The detection limit was as little as a single O. viverrini or C. sinensis egg artificially inoculated in 100 mg of non-infected fecal sample (equivalent to 10 eggs per gram), indicating highly sensitivity. The method was found to be superior to the traditional microscopy method and was more rapid than Sanger DNA sequencing. CONCLUSIONS: DNA pyrosequencing-based identification is a valuable tool for differentiating O. viverrini and other Opisthorchis-like eggs, and can be applied to epidemiological studies and for molecular taxonomic investigation of FBT in endemic areas.