title: Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA).
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 10(3) 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.
  
  
  CONCLUSIONS
  The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Key Laboratory for Tropical Diseases Control, Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong, China.
authors.forename: Jiufeng
authors.initials: J
authors.lastname: Sun
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.forename: Pei
authors.initials: P
authors.lastname: Liang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Mao
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Lv
authors.forename: Chuanhuan
authors.initials: C
authors.lastname: Deng
authors.forename: Chi
authors.initials: C
authors.lastname: Liang
authors.forename: G S
authors.initials: GS
authors.lastname: de Hoog
authors.forename: Xinbing
authors.initials: X
authors.lastname: Yu
mesh_terms: D000818:Animals; D003003:Clonorchiasis / Q000175:diagnosis* / Q000469:parasitology; D003004:Clonorchis sinensis / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D006801:Humans; D008107:Liver Diseases / Q000175:diagnosis / Q000469:parasitology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D009889:Opisthorchiasis / Q000175:diagnosis* / Q000473:pathology; D009891:Opisthorchis / Q000235:genetics / Q000302:isolation & purification*; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer; D015345:Oligonucleotide Probes