| Summary: | <p>Abstract</p> <p>Background</p> <p>Infections with the opisthorchid liver flukes <it>Clonorchis sinensis</it>, <it>Opisthorchis viverrini</it>, and <it>O. felineus </it>cause severe health problems globally, particularly in Southeast Asia. Early identification of the infection is essential to provide timely and appropriate chemotherapy to patients.</p> <p>Results</p> <p>In this study we evaluate a PCR-based molecular identification method, Multiplex Ligation-dependent Probe Amplification (MLPA), which allows rapid and specific detection of single nucleotide acid differences between <it>Clonorchis sinensis</it>, <it>Opisthorchis viverrini </it>and <it>O. felineus</it>. Three probe pairs were derived from the Internally Transcribed Spacer 1 (ITS1) of three opisthorchid liver flukes using a systematic phylogenetic analysis. Specific loci were detected in all three species, yielding three amplicons with 198,172 and 152 bp, respectively, while no cross reactions were observed. A panel of 66 <it>C. sinensis </it>isolates was screened using MLPA. All species were positively identified, and no inhibition was observed. The detection limit was 10<sup>3 </sup>copies of the ITS gene for the three liver flukes, or about 60 pg genomic DNA for <it>Clonorchis sinensis</it>. Amplification products can be detected by electrophoresis on agarose gel or in a capillary sequencer. In addition, genomic DNA of <it>Clonorchis sinensis </it>in fecal samples of infected rats was positively amplified by MLPA.</p> <p>Conclusion</p> <p>The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.</p>
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