Molecular Occurrence And PCR Detection

Adult worms of Metorchis orientalis mainly parasitize in the bile ducts and gallbladder ofducks, chikckens, wild birds and other poultry animals，which can cause pathological conditions,including gallbladder enlargement, thickening or hardening of the wall of gallbladder and bileducts, biliary obstruction, and breakdown in the bile drainage. In this case, these animalsexperience decreased appetite, weight loss, emaciation, anemia, diarrhea, and even cause death,causing significant economic losses to the livestock industries. Meanwhile, M.orientalis can alsoinfect human, but rarely reported, as M.orientalis and Clonorchis sinensis have the sameintermediate hosts, epidemic areas and clinical symptoms, therefore, M.orientalis is probably anignored zoonotic parasite.In recent years, researches on Metorchis orientalis is relatively little, mainly focus on themorphology and structure, life cycle, epidemiology, diagnosis and prevention. However, thecomplete mitochondrial genome of Metorchis orientalis has not been published exceptresearches on ribosome sequence. And mitochondria are the important gene markers ofmolecular method in taxonomy, population genetics and phylogenetic studies.Therefore, thisstudy aimed to to amplify the complete genome of M.orientalis, sequence and analyses of mtgenomes, and the phylogenetic trees were also re-constructed based on the protein-coding genesequences of mt genomes to study the phylogenetic relationships between the M.orientalis withother members of trematodes. The results showed that the mitochondrial genome of M.orientaliswas circular, and that of mtDNA sequence was13,834bp in size. The mt genome comprises36genes, including12protein-coding genes,22transfer RNA genes,2ribosomal RNA genes and2AT-rich. The gene content and arrangement were identical to other Opisthorchiidae trematodes.All genes were transcribed in the same direction. Phylogenetic relationships were reconstructedusing the concatenated amino acid sequences of12protein-coding by three methods, namely BI,MP and ML. Gyrodactylus salaries as the outgroup. The topological structures of trees weresimilar with different methods. In this tree, Opisthorchiidae, Fasciolidae, Heterophyidae,Paragonimidae, Paramphistomidae, Dicrocoeliidae, Schistosomatidae form monophyletic groups,respectively. However, the genus of Opisthorchis included two kinds of representative trematode(O.viverrini and O.felineus), which were not in the same clade, M.orientalis and O.viverrni were clustered together with high statistical support, M.orientalis was more closely related toO.viverrni than to O.felineus and C.sinensis.So far, the main diagnostic methods of M.orientalis infection are mainly to detect the eggs inthe feces, but to some similar trematode eggs, it is usually misjudged into each other. Therefore,developing a rapid detection method is very necessary. So this study was trying to do it.Specifically, according to the data of M.orientalis on the GenBank, two specific primers weresynthesized to amplification the DNA in the eggs by optimizing PCR conditions, followed toevaluate sensitivity, specificity and repeatability. Finally, we set up a way to detecet M.orientalis.The experiments above mentioned by PCR assay showed that the obtained specific band was260bp, and under the same constraints, PCR assay was negative to Clonorchis sinensis,Echinostoma revolutum, Notocotylus attenuatus, Echinostoma revolutum in red-crowned crane.The method can detect0.03125minimum eggs DNA per gram faeces. And6positive sampleswere detected based on87clinical samples, so the positive rate was6.9%. The results indicatedthat the poultry farm exist the epidemic of M.orientalis surrounding Daqing, The PCR detectionmethod had high sensitivity and specificity, which could be used to diagnose M.orientalis inducks and other poultry.According to the research, the complete mitochondrial genomes of M.orientalis was obtained,which enrich the mitochondrial genome data. And genetic relationships and evolutionary statusof M.orientalis and other trematodes was determined by evolution analysis. In the meantime, weestablished a PCR method with well sensitive, species-specificity, repeatability to detectM.orientalis, which laid the foundation for the epidemiological investigation of M.orientalis.