Study On Identification Of Clonorchis Sinensis And Haplorchis Taichui By Analysis Of Egg Its-2Sequence And The Application Of This Methodology

Objectives To establish an identification method basis on molecularbiology techniques for Clonorchis sinensis and Haplorchis taichui,and assessthe infection of Haplorchis taichui in Guangxi Province.Methods Adult flukes of C. sinensis and H. taichui were collectedfrom Clonorchiasis endemic areas and the eggs were separated from the wormsunder stereomicroscope. First, the species were determined in accordance withthe electrophoresis of PCR products. DNA of each group was extracted andamplified ITS-2sequence, and amplified products were sequenced andhomology analyzed by DNAstar to ensure that the products were the targetfragments. Second, depending on the number of eggs,eggs were divided intodifferent experimental groups.Then the DNA extraction and ITS-2sequenceamplification, and amplification products electrophoresis and homologyanalysis,were repeated. The experiment method repeatability of different number of eggs was judged, according to whether the target banding wouldappear after electrophoresis.Then,four common eggs of Heligmosomoidespolygyrus：Roundworm, Whipworm, Hookworm and Pinworm，were used ascontrolled trials. Repeat the experiment steps, the species of C. sinensis and H.taichui were determined in accordance with the electrophoresis of PCR products.Finally, stool, whose inspection results were positive checked by Kato-Katz inFusui County, was used as the experimental material. The mixture of eggs anddung slag was got by cleaning and subsiding fecal samples, and then be used torepeat the previous experimental procedure. According to the results ofelectrophoresis of PCR products,which species of trematodes infectted wasinferred, for who was diagnosed as “Clonorchis sinensis infections”, and theproportion of H. taichui infections in C. sinensis endemic areas was evaluated.Results The electrophoresis of PCR products of C.sinensis and H.taichui display a distinct band, respectively at around400bp,500bp.Electropherogram with mixture DNA PCR products of two species eggs showedtow obvious bands at around400bp and500bp. After nucleotide sequencing andBlast comparison, both of sequences are highly homologous with C. sinensisand H. taichui respectively in GenBank. In the groups of classificated by eggsnumber from10to50, electropherogram with the experimental group of C.sinensis always showed obvious bands at around400bp,and most of H. taichuisamples showed bands at around500bp.There were no obvious band in thesample which used the any kind of Heligmosomoides polygyrus eggs DNA as atemplate. The PCR products mixed the four kinds of Heligmosomoidespolygyrus with C.sinensis eggs and H. taichui eggs had no other bands exceptthe objective strips. In the methodology application experiment, the results showed that positive rate of experiment samples was95.93%. ThePCR-determined community prevalences of C.sinensis and H. taichui infectionswere49.70%and46.06%，respectively，and mixture infection was4.24%.Negative rate was4.07%.Conclusion1. This method can identify the egg of C. sinensis andH. taichui effectively. This method can be used as identification for two speciesof fluke.2. Diagnosis of C. sinensis was misdiagnosed for nearly half of thecounty population surveys in Fusui County. The assess rate of C. sinensis in thiscounty was inaccurate.