To Study The Dependablity Factors Caused The Genetic Polymorphism Of The Gene Of Tvmarl In Trichomonas Vaginalis Isolates And Fd Gene Sequencing

BackgroundTrichomonas vaginalis is a protozoan parasite with flagellum that infects the human urogenital tract,is the causative agent of trichomoniasis.T.vaginalis,can also increase the possibility of infecting sexually transmitted diseases(STDs),like human immunodeficiency virus(HIV)and hepatitis B virus(HBV)and so on.According to the statistics by WHO,there were more than 170 million new cases of T.vaginalis infections worldwide each year.T.vaginalis does great harm to human bodies and is widespread.Mycoplasma hominis is also one of the pathogen that hosts in the urinary tract and genital tract,is said to be the smallest micro-organism that can live independently. M.hominis and T.vaginalis can cause the similar complication:infertilitas feminis, spontaneous abortion,low birth weight infant and so on.The scholars overseas ever had relevant report earlier on their connection,and the relevant report home has been comparatively few,in this case,aiming to know about the information of symbiosis between M.hominis and T.vaginalis in Henan,so we detect M.hominis in T. vaginalis by the means of PCR. With the analysis of the genetic polymorphism of T.vaginalis isolates that were gotten from T.vaginalis from 5 cities(Zhengzhou,Anyang,Luoyang,Xinxiang, Kaifeng)in Henan with the technology of mobile genetic element(MGE)-PCR,the auther is trying to confirm the blood relationship among them.According to the relevant elements:geographic origin,co-existing of M.hominis and T.vaginalis, patients with clinical signs and symptoms(including 3 groups:light,moderate,and severe),the auther hopes to analyze the relationship of these relevant elements and the genetic polymorphism of T.vaginalis isolates,with the purpose of proposing valuable datas for the research on the molecule epidemiology in Henan.As the most homotenous eucaryote,T.vaginalis has many hydrogenosomes with double membrane that is the major organ of metabolism,but without mitochondria.As the important medium of electron transport during metabolic process,ferredoxin play a critical role in the reductive activation of metronidazole. Recent year,more and more scholars begin to pay close attention to Fd because of the metronidazole resistance.The auther chose the gene of Fd by PCR was sequenced from part isolates of T.vaginalis,in order to talk about the possibility of M.hominis in T.vaginalis and localities of the isolates affecting the gene Fd.In the end to provide the theory to the clinical treatment.Material and MethodAll of the T.vaginalis were provided by the department of gynaecology and obstetrics or test in the affiliated hospital of Zhengzhou University,and all are from female patients,and recorded the information of clinical situation.The auther chosed 30 isolates to make an experiment.Collected the T.vaginalis in mid-log-phase and DNA was then extracted by the means of phenol-chloroform.In the end we detected the purity and density of DNA.Analyze the length and location polymorphism of gene of Tvmarl by the means of MGE-PCR among the T.vaginalis.Phylogenetic tree of 30 isolates of T.vaginalis constructed on the basis of MGE-PCR,by the neighbour-joining method and software MEGA3.1.Detected M.hominis and amplification of Fd gene by PCR using specific primers.The auther chosed some isolates of T.vaginalis including isolates M.hominis negative and isolates M.hominis positive and isolates from different cities(Zhengzhou,Anyang,Luoyang,Xinxiang, Kaifeng)sent them to the biology company to sequence the gene of Fd.Results1.The outcome of the PCR detection of the M.hominls in T.vaginalis showed that 25 of 30 T.vaginalis isolates had the special DNA fragment with the length of 334bp,the other 5 failed,with the positive rate of 83.33%.2.The result of MGE-PCR shows that the genetic difference is noticeable,and the genetic distance is between 0 and 100%with the average of 69.11%.Genetics change greatly in T.vaginalis,the similarity coefficient is just 30.89%.Phylogenetic tree of 30 isolates of T.vaginalis show that among 5 groups of T.vaginalis([Tv12,Tv29]; [Tv18,Tv20];[Tv14,Tv26];[Tv10,Tv25];[Tv15,Tv21])the genetic relationship is the closest,and the genetic distance is 0.3.The outcome of Fd gene showed that all 30 isolates of T.vaginalis had the special DNA fragment with the length of 300bp.By the software of Clustalx,as a result 10 isolates of T.vaginalis ferredoxin gene are homology.But compare and contrast the outcome with the Fd gene in GenBank(M33717),we found that there are three bases ctg in the place 200,coding leucine,but this insertion didn't change the following reading code frame.The homology of the gene is 99%.Conclusion1.The positive rate of M.hominis in T.vaginalis Henan isolates is 83.33%.It shows that the co-existing phenomenon of M.hominis and T.vaginalis is very common in Henan.2.The gene of Tvmar1 in 30 isolates of T.vaginalis have noticeable difference.These differences may are related to the clinical situation,but not to the co-existing M. hominis and the area difference.3.A new gene of Fd is discovered.Contrast with the gene of Fd in GenBank,the homology is 99%.The Fd gene of T.vaginalis in Henan isn't influenced by geographic origin and co-existing of M.hominis and T.vaginalis.