Abstract
We developed a combined conventional polymerase chain reaction (PCR) and real-time PCR (qPCR)-based assay for detecting and discriminating between Opisthorchis viverrini and Haplorchis taichui parasite infections. The first PCR amplifies the mitochondrial cytochrome c oxidase subunit I (COI) genes of parasites, and differential diagnosis is achieved by performing qPCR with specific primers and SYBR Green I. The detection limit of the assay was found to be 2.0 × 10(2) plasmid copies in a test in which a stool sample was spiked with a single egg, which is equivalent to 5 eggs per gram (EPG). The testing of 34 clinical stool samples that had been demonstrated to contain "Opisthorchis-like" eggs by microscopy showed that the novel assay exhibited a sensitivity of 100% for "Opisthorchis-like" parasitic infections, and 71% and 91% of these samples were found to be infected with O. viverrini and H. taichui, respectively. A further four parasitic infections were diagnosed in the 16 negative samples, and the microscopic findings of these samples were confirmed to be false negatives by sequencing analysis. The assay also displayed high specificity during the testing of 10 samples containing other common parasites. The fact that our qPCR SYBR Green I-based assay detected submicroscopic traces of parasitic DNA and was able to differentiate between parasites that produce eggs with similar morphologies indicates that it has a good potential for development of diagnostic application to use in areas where multiple parasites coexist.