Western Yunnan HIV-positive People Infected With Toxoplasma SAG2Gene Site Studied

Toxoplasma gondii is an obligate intracellular protozoan parasite that can infecthumans, including almost vertebrate and spread worldwide, causing zoonoticparasites while gondii is an opportunistic pathogenic protozoa,people with normalimmune function after infection often has a latent infection, Toxoplasma gondii is oneof the main reasons which can caused a fatal disease in AIDS, cancer, long-term useof immunosuppressive therapy in patients with severely impaired immune function, orother person. Toxoplasmosis is the most common AIDS opportunistic infections, isone cause central nervous system symptoms of focal occupying the most commoncause. With the domestic HIV incidence increased year by year, Toxoplasma gondiiinfection incidence of related diseases also increase and prevalence in many parts ofthe world too popular to enormous human and economic health threat serious losses,which has been increasing attention.Different regions or different hosts of Toxoplasma gondii in same area havedifferent genotypes. Conventional PCR (polymerase chain reaction PCR) technologyand its derivative technology is widely used in gene cloning, gene sequencing,genotyping, and other basic medical and clinical research. by using of PCRtechnology to study gene of Toxoplasma gondii, toxoplasmosis genotype can bedivided into type I, ⅡandⅢ, type is recognized as virulent strains，the main cause ofcongenital toxoplasmosis. The other two types are weak, Human infection is morecommon in type Ⅱ, Ⅲ type common in animal infections. Toxoplasma gondii strainsof different minor differences between genomes, which may lead to different insectvirulence between strains, appeal, etc. reasons for the differences, different genotypes of Toxoplasma gondii strains is sensitivity to drugs differently. Toxoplasma gondiistrains by gene identification and genotyping of T. gondii population biology,epidemiology, genetic status and disease patterns between the genotype and thepotential correlation studies provide an important basis. In this study, HIV-positivepersons in Yunnan by collecting blood for T. gondii IgG and IgM positive wholeblood samples for genetic extraction, using nested PCR and RFLP analysis techniques,and Toxoplasma gondii strains compared to international standards, Yunnan ProvinceHIV positive blood separate Toxoplasma SAG2gene (241bp、221bp) analysis ofgenetic markers to explore Yunnan Toxoplasma gondii infection among HIV-positivegenotype for the immune pathogenesis and infection-depth research and clinicaltherapy.1. Objective:Learn western of Yunnan Toxoplasma gondii infection in HIV-positive person ’sbasic situation, preliminary analysis and to determine the western of YunnanToxoplasma gondii infection in HIV-positive subjects major genotypes, so as towestern Yunnan HIV positive prevention and epidemiology of toxoplasmosis studyprovides an important theoretical basis.2. Method:2.1Sample CollectionCollection Yunnan Longling, Dali, Lincang City AIDS agencies HIV positiveblood samples were confirmed by Western blot (WB) test positive confirmation.2.2Toxoplasma antibody detectionCollected serum samples from HIV-positive by enzyme-linked immunosorbentassay (ELISA) for detection of Toxoplasma gondii IgG and IgM antibodies screenedor Toxoplasma IgG and IgM positive specimens.2.3Extraction and Toxoplasma gene amplification, recovery, purificationUse of whole blood gene extraction kit HIV positive Toxoplasma IgG and IgMpositive blood samples were extracted from reference gene; mature internationalprimer and nested PCR of Toxoplasma gondii SAG2gene was amplified by tworounds of amplification, electrophoresis, analysis of automatic gel imaging analysis system. The SAG2gene of Toxoplasma gondii amplification, gel Recovery Kit forrecovery, purification.2.4DigestedEndonuclease Sau3AI and HhaI positive for Toxoplasma SAG2geneamplification products were digested with restriction enzyme after electrophoresisresults were compared with standard strains of Toxoplasma gondii, identification,identify genotypes.2.5SequencingThe Toxoplasma gondii SAG2gene positive for gene amplification productswere recovered after purification of selected seven (six HIV-positive blood samples, aparts of purified Toxoplasma gondii,) to Shanghai Li Fei Limited sequencing resultswith the strain of Toxoplasma gondii registered on Genbank gene sequences wereanalyzed.3.Results3.1Serum antibodiesThe testing whole blood samples of392, of which114were positive forToxoplasma IgG antibody positive rate was29%; Toxoplasma IgM antibodies13, thepositive rate was3.3%; both together total positive rate was32.3%. The detection ofToxoplasma pick out127copies of HIV-positive antibody-positive whole bloodsamples.3.2Gene amplification and restriction digestionThis experiment for SAG2gene amplification of120whole blood samples forenzyme, after electrophoretic image seen a size of about241bp,221bp bands weredigested by the two bands seen after the target gene with the international standardstrain of Toxoplasma gondii gene I type strain (RH strain) were compared.since thelack of laboratory type Ⅱ and type Ⅲ strains of Toxoplasma gondii standard,references were analyzed according to the results show119pieces SAG2genedigested with the international standard strain gene type I (RH strain) consistent, only1piece with the same type Ⅲ.3.3SequenceAnalysis In the west of Yunnan HIV positive people infected with Toxoplasma strainsSAG2gene sequencing six pieces from randomly selected enzyme samples.Althougheach gene of Toxoplasma gondii strains SAG2difference between rate is very small,but there are six pieces still some presence of Toxoplasma gondii strains base pairdeletion, insertion or mutation.The standard strain (RH), Yunnan strain of Toxoplasmagondii infection in HIV-positive subjects (sample) and Genbank registeredToxoplasma strains comparative analysis of gene sequences, base pair differencesbetween them are the following few:241bp difference: sample3,4,7at182base pairsof G is missing.221bp difference: sample4at31base pairs of nucleotides at the Gbecomes C,48base pairs at the G into A, at163base pair deletion of T; sample5at84base pairs at the G becomes T,163base pairs at the G into A; sample6at163basepairs at the G becomes C, C is inserted after the A; sample2,3,7at163base pairs inthe sample at the T becomes as C, at165base pair G changes A.Visible sample4basic group is changes more, account for the standard strain(RH) inconsistentgenotype, there may be other genotypes, while the sample4digestion resultsconfirmed for type Ⅲ, coincided with the sequencing results.Therefore, completelyeliminated sample4are notⅠtype, to be sure the sample4whether is further study oftype Ⅲ to be.4. Conclusion4.1In the west of Yunnan HIV positive average infection rate of Toxoplasma gondii ishigher than the normal population.4.2Preliminary determine the genotype of Toxoplasma gondii infection in Western ofYunnan HIV positive persons forⅠtype, Ⅲ type rare, noⅡtype and other gene type.5. The innovation of this experimental study5.1In this study, the western region of Yunnan Province by collecting agencies AIDSHIV positive blood samples for Toxoplasma SAG2gene extraction, amplification,restriction enzyme digestion and sequence analysis, further integration of Toxoplasmagondii strains B1gene, GRA6genetic research results were analyzed, initiallyidentified the western region of Yunnan Province Toxoplasma gondii infection inHIV-positive genotype. 5.2In this study, mainly in the following two aspects of innovation:5.2.1Directly extracted from western of Yunnan HIV-positive people whole bloodsamples and successfully amplified gene Toxoplasma gondii SAG2genes.5.2.2From multiple loci were analyzed to determine the western region of YunnanProvince of Toxoplasma gondii infection in HIV-positive genotype.