| Toxoplasma gondii is a wide range of opportunities for the distribution of pathogenic protozoa, that parasitic on warm-blooded animals, including humans, almost all of the nucleated cells. It is a widely distributed Apicomplexan parasitic protozoa and zoonotic parasites epidemiced in the world-wide, and has a wide range of host group, which distributed worldwide, and many animals can be infected, suffering Zoonotic toxoplasmosis. The T. gondii is a kind of opportunistic protozoan. Toxoplasma gondii infection of pregnant women and immunocompromised can cause serious harm. In recent years, as the pet breeders, cancer patients, AIDS patients gra(?)ally increased, endangering Toxoplasma (?) increasingly prominent. It has a complex life history, morphology and diverse, it can be divided into two stages involved in sexual reproduction and asexual reproduction, which tachyzoites gentle tachyzoites (cysts) is the main transmission and pathogenicity stage in its life history. The tachyzoites in the asexual reproduction is more serious stage. When tachyzoites went into the host brain will form cysts and cause chronic infection, the chronic infection of Toxoplasma accounted for the vast majority of humans infected with Toxoplasma gondii, It is the most important manifestation too. chronic Toxoplasma infection can cause central nervous system damage.Currently, only one kind of Toxoplasma has been known, but there are different genotypes. In addition to the typical type Ⅰ, type Ⅱ and type Ⅲ, but also have at least 138 atypical genotypes. The distribution of Toxoplasma gondii genotypes has Territorial. Toxoplasma gondii will be divided into three major lineages By polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP), multilocus enzyme electrophoresis (MLEE), microsatellite analysis techniques. Respectively, type Ⅰ, type Ⅱ and type Ⅲ. In addition there are some special non-Ⅰ, Ⅱ, Ⅲ genotype is defined as atypical genotypes. They are only a small difference of 1% to 2% of the DNA sequence, but there is a large difference in virulence. I belong to the virulent strain type (eg RH strain), highly pathogenic, infused one tachyzoite can cause death in mice, resulting in a high degree of parasitaemia, with the acute phase of infection. Ⅱ, Ⅲ-type strains belonging to weak (eg.CEP strain), lethal dose in mice than 1000, Type Ⅱ belong to chronic infection. Toxoplasma gondii isolates can be found different genetic diversity, different genotypes of the parasite’s virulence and cause differences in susceptibility to drugs in different regions through genetic analysis. It can provide the basis for the diagnosis and treatment of toxoplasmosis by genes analyzed. This study collected the blood of HIV positive people in Dali of Yunnan province, Extract all the samples, analysis SAG1、c22-8 gene by the nested PCR and RFLP, Then consist With the standard virulent strain. Preliminary analysis SAG1、c22-8 gene of T. gondii in HIV positive people in Yunnan province, Provide the basis for the pathogenesis and infection of immune-depth research and clinical treatment.1. Objective1.1 In order to understand the basic situation of the HIV positive people infection with T. gondii in Yunnan province.1.2 Probe SAG1、c22-8 gene of T. gondii in HⅣ positive people in Yunnan province, Preliminary analysis SAG1、c22-8 gene of T. gondii in HⅣ positive people in Yunnan province, provides important reference for prevention of toxoplasmosis in HⅣ positive people in Yunnan province and its Molecular Genetics study.2. Methods2.1 Sample collectionCollect the HⅣ positive people blood samples, the blood sample from Dali bai autonomous prefecture areas of HⅣ/AIDS prevention and control institution in Yunnan province, and all were confirmed by protein imprinting (WB) test positive.2.2 gene extraction and amplification, recycling, purification of ToxoplasmaExtracted the genomic DNA from the blood samples that HⅣ-positive infection Toxoplasma gondii by the blood genomic DNA extraction kit, amplification T. gondii SAG1、 c22-8 gene reference on the international mature primers and nested PCR technology, electrophoresis after two rounds of amplification, automatic gel imaging analysis system analysis. T. gondii SAG1、c22-8 gene success amplification will been recycle and purify use agarose gel extraction kit.2.3 Enzyme digestionUsing restriction enzymes Sau96I and HaeⅡ to enzyme SAG1 gene amplification products, and Using restriction enzymes BsmAⅠ and MboⅡ to enzyme c22-8 gene amplification products. compared the electrophoresis results between standard strain and local strain. Preliminary analysis genotype of T. gondii in HIV positive people in Yunnan province2.4 sequencingRecycle and purify the amplification positive product of T. gondii SAG1、 c22-8 gene, select More samples to set up Life sequencing co. LTD in Shanghai and Baoshengwu sequencing co, LTD in Dalian, sequencing results were analyzed with the T. gondii strains gene sequences that register of on Genbank.3. Results3.1 A total of 291 blood samples of HIV have been testing,64 samples successful amplification SAG1 gene,32 sampls successful amplification c22-8 gene; SAG1 gene positive rate was 22.0% by molecular biology testing, c22-8 gene positive rate was 11.0% by molecular biology testing.3.2 A total of 291 blood samples of HⅣ have been collected, Extracted the genomic DNA and amplification T. gondii SAG1、c22-8 gene by the nested PCR technology, and agarose gel electrophoresis showed,64 samples successful amplification SAG1 gene, The purpose of the proposed strip fragment was amplified 390bp.32 samples successful amplification c22-8 gene, The purpose of the proposed strip fragment was amplified 521bp.3.3 64 blood sample SAG1 gene enzyme digestion is about 350 bp and 50 bp band, respectively in two bands by using Sau96Ⅰ、 HaeⅡ.32 blood sample c22-8 gene enzyme digestion is about 120 bp、240bp and 20 bp band, respectively in three bands by using BsmAⅠ、MboⅡ, There have same band and consist With the standard virulent strain.3.4 Selecting amplified positive Toxoplasma gondii SAG1, c22-8 gene samples, sent to Shanghai Li Fei company gene sequencing, with a registered Genbank Toxoplasma gondii SAG1, c22-8 (Registration numbers:GQ253073.1、AM055943.1) sequences were compared, found that small change exist between the base and the lack of the gene described above.4. Conclusion4.1 64 samples successful amplification SAG1 gene, SAG1 gene positive rate was 22.0%; 32 samples successful amplification c22-8 gene, c22-8 gene positive rate was 11.0%b y the nested PCR amplification from HIV-positive samples of T. gondii in Dali of Yunnan, and SAG1 loci detection was higher than c22-8.4.2 T. gondii strains that HIV positive people infected with in Dali of Yunnan is the same as the international standard strains of type I (RH strain) after enzyme results.4.3 After sequencing the amplified positive Toxoplasma gondii SAG1, c22-8 gene samples, found that small change exist between the base and the lack of the gene described above.4.4 Whether there are difference from RH strain of toxoplasma for HIV infection in Dali of Yunnan, it remains to be further research.5. The innovation points of experimentalIn this study, there are three aspects of innovation:5.1 This study choose Dali of Yunnan, three are the vast territory and abundant resources, a pleasant climate and is Bai-based multi-ethnic enclave, Preliminary understanding of characteristics of genetic polymorphism to the HIV positive people infection with 1:gondii in Yunnan province. It has significant local characteristics.5.2 Choosing Toxoplasma gondii-risk populations (HIV positive people in Yunnan province.) for the study, Preliminary analysis SAG1、c22-8 gene of T. gondii in HIV positive people in Yunnan province.5.3 Extracted HIV-positive blood samples from Dali of Yunnan directly and amplified SAG1, c22-8 gene of Toxoplasma gondii successful. |
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