A MULTIPLEX PCR ASSAY FOR THE DIFFERENTIAL DETECTION OF OPISTHORCHIS FELINEUS AND METORCHIS BILIS.

Opisthorchis felineus and Metorchis bilis are two common small worms that parasitize in the gallbladder and bile ducts of the liver of humans and carnivores. These parasites have a severe impact on health and are considered pathogens of serious diseases worldwide, such as cholangiocarcinoma. However, there are still no commercially available molecular diagnostic kits capable of simultaneously detecting these parasites in humans. Therefore, the study aimed to develop a multiplex PCR analysis that will differentially determine these two opisthorchiasis infections in one reaction. Two specific primer pairs for a multiplex polymerase chain reaction (PCR) were designed based on corresponding mitochondrial genome sequences. The multiplex assay detection limit was assessed by serial dilutions of the genomic DNAs of trematode worms examined. Naturally, infected samples of human bile and feces were tested using the developed assay. A multiplex PCR assay was developed based on mitochondrial DNA that accurately and simultaneously identifies two trematode species in one reaction using specific fragment sizes of 307 and 252 bp for O. felineus and M. bilis, respectively. The optimal reaction conditions, specificity, and sensitivity of the multiplex PCR assay were investigated. The lowest DNA concentration detected was 100 pg for M. bilis and O. felineus in a 25μl reaction system. This study provides an efficient tool for the simultaneous detection of O. felineus and M. bilis. The proposed multiplex PCR assay will be potentially useful in epidemiological studies, diagnosis, and treatment of this mixed opisthorchiasis infection.