Open Access Research

Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA)

Jiufeng Sun1, Jin Xu1, Pei Liang1, Qiang Mao1, Yan Huang1, Xiaoli Lv1, Chuanhuan Deng1, Chi Liang1, G S de Hoog2 and Xinbing Yu1*

Author Affiliations

1 Department of Parasitology, Zhongshan School of Medicine; Key Laboratory for Tropical Diseases Control, Ministry of Education, Sun Yat-sen University. No 74, The Second Zhongshan RD, Guangzhou, Guangdong, 510080, China

2 Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, The Netherlands

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Parasites & Vectors 2011, 4:98 doi:10.1186/1756-3305-4-98

Published: 7 June 2011

Abstract

Background

Infections 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.

Results

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 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.

Conclusion

The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.