Dali HIV-positive Infection Toxoplasma Gondii B1、GRA6、SAG3Gene Site Studied

1Objective1.1In order to understand the basic situation of the HIV positive people infection withToxoplasma gondii in Yunnan province Dali Bai Autonomous Prefecture.1.2Discussion Yunnan Province Dali Bai Autonomous Prefecture Toxoplasma gondiiinfection in HIV-positive B1, GRA6, SAG3gene, a preliminary understanding of thegenetic variation in Yunnan Dali T.gondii strains geographical situation, to furtherstudy the T.gondii molecular genetics and toxoplasmosis the control to provideinformation.2Method2.1Samples collectionProvided by the Yunnan Dali Bai Autonomous Prefecture AIDS preventionorganization by Western-blotting experiments verify the patient’s HIV-positive bloodsamples.2.2Toxoplasma gondii antibody detectionCollection HIV-positive blood samples, using enzyme-linked immunosorbent assay(ELISA) to detect Toxoplasma gondii IgM、IgG antibody, in order to screeningspecimens Toxoplasma IgM and IgG antibodies to provide the next experiment test samples of materials.2.3Toxoplasma gondii gene extractionSelect Toxoplasma gondii IgM and IgG antibody test results positive samples wereextracted Toxoplasma gondii genes.2.4Toxoplasma gondii gene amplificationUsing nested PCR amplification.2.5Toxoplasma gondii gene recycling, purificationSelect B1、GRA6、 SAG3gene amplification positive product was subjectedrecovery and purification.2.6Enzyme digestion of the Toxoplasma gondii geneIn this study, B1genes select restriction enzymes Pm1I and XhoI, GRA6genesselect restriction enzymes MseI, SAG3genes select restriction enzymes NciI, Use topurified DNA sample is added to the corresponding restriction enzymes, digest for16hours and subjected to agarose gel electrophoresis image.2.7Gene sequencingNested PCR amplification positive product B1、GRA6、SAG3gene purified,selected6parts genetic samples, the above samples sent to Gene Corporationrecycling sequenced.3Results3.1This study from Yunnan Dali Bai Autonomous Prefecture317whole bloodsamples, HIV-positive blood samples infected293,24copies of the normal controlsubjects, detecting of HIV-positive infection person with Toxoplasma gondiiinfection was36.2%, of which97copies of Toxoplasma IgG antibody positive, thepositive rate was33.1%; Toxoplasma IgM there are9copies of positive, thepositive rate of3.1%, healthy IgG antibody positive rate is4.2%.3.2In this study, nested PCR successfully amplified B1gene47copies, GRA6gene29copies,42copies SAG3genes. Performed with the appropriate restrictionenzyme digestion and agarose gel electrophoresis showed，B1gene has a length of500bp band, GRA6gene600bp and200bp has two bands, SAG3gene has a length of200bp band.3.3Selecting amplified positive Toxoplasma gondii B1, GRA6, SAG3genesamples, sent to Shanghai Li Fei company gene sequencing, with a registeredGenbank Toxoplasma gondii B1, GRA6, SAG3gene (Registration numbers:AF17987.1, AJ635332.1, JX218225.1) sequences were compared, found that smalldifferences exist between the base and the lack of the gene described above.Meanwhile, in the Toxoplasma gondii gene sequences25bp were found NciIrestriction sites (CCGGG) of SAG3gene, in Toxoplasma gondii gene sequence146bp and690bp were found MseI restriction site (TTAA) of GRA6gene.4Conclusion4.1Preliminary Determination get Yunnan Dali Bai Autonomous Prefecture HIVpositive persons with Toxoplasma gondii infection higher than Toxoplasma gondiiantibody positive rate of Yunnan Dali normal population.4.2Succeed B1, GRA6, SAG3gene by the nested PCR amplification fromHIV-positive samples of Dali in Yunnan.4.3Dali Bai Autonomous Prefecture of Toxoplasma gondii B1, GRA6, SAG3geneenzyme digestion gel electrophoresis with Toxoplasma gondii RH same genotype,Preliminary judgment mainly popular for genetype I.4.4Dali Bai Autonomous Prefecture amplified positive Toxoplasma gondii B1,GRA6, SAG3gene samples to sequence, found with registration on Genbankbetween base pairs of genes with Toxoplasma gondii there is only a small number ofdifferences and lack. Preliminary judgment mainly popular of Yunnan DaliToxoplasma gondii infected genotype consistent with RH strain.4.5Preliminary determine the genotype of Toxoplasma gondii infection in Dali ofYunnan HIV positive persons forⅠtype, noⅡtype,Ⅲ type and other gene type.