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Clonorchis sinensis  and  Opisthorchis viverrini  are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of  C. sinensis  and  O. viverrini . Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of  C. sinensis . Moreover,  C. sinensis  and  O. viverrini  were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of  C. sinensis  and  O. viverrini .

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Rapid Detection and Differentiation of<i>Clonorchis sinensis</i>and<i>Opisthorchis viverrini</i>Using Real-Time PCR and High Resolution Melting Analysis

Rapid Detection and Differentiation ofClonorchis sinensisandOpisthorchis viverriniUsing Real-Time PCR and High Resolution Melting Analysis