Abstract
BackgroundInfections with the opisthorchid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and O. felineus cause severe health problems globally, particularly in Southeast Asia. Early identification of the infection is essential to provide timely and appropriate chemotherapy to patients.ResultsIn 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 Clonorchis sinensis, Opisthorchis viverrini and O. felineus. 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 C. sinensis isolates was screened using MLPA. All species were positively identified, and no inhibition was observed. The detection limit was 103 copies of the ITS gene for the three liver flukes, or about 60 pg genomic DNA for Clonorchis sinensis. Amplification products can be detected by electrophoresis on agarose gel or in a capillary sequencer. In addition, genomic DNA of Clonorchis sinensis in fecal samples of infected rats was positively amplified by MLPA.ConclusionThe flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.
Highlights
Infections with the opisthorchid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and O. felineus cause severe health problems globally, in Southeast Asia
We evaluate Multiplex Ligation-dependent Probe Amplification (MLPA) for the rapid identification of the opisthorchid liver flukes C. sinensis, O. viverrini and O. felineus, and establish specificity of the method to discriminate these three liver flukes in a single-tube reaction
Three specific loci were selected for designing species-specific pairs of oligonucleotide probes for MLPA (Table 1)
Summary
Infections with the opisthorchid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and O. felineus cause severe health problems globally, in Southeast Asia. Clonorchis sinensis (C. sinensis), Opisthorchis viverrini (O. viverrini) and Opisthorchis felineus (O. felineus) (Opisthorchiidae) are among the most frequent endemic food-borne liver flukes, causing severe clonorchiasis and opisthorchiasis. About 35 million people are infected with C. sinensis globally. In. China the estimated infection by C. sinensis is 15 million [2,3]. 9 million people are infected with O. viverrini in Thailand, Cambodia, and Laos [4,5,6]. In eastern Europe 1.2 to 1.5 million patients are infected with O. felineus [7]. Endemic areas of liver flukes are expanding to North America and Europe due to fish import and immigration [7,8,9]
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