长春地区孕妇弓形虫感染虫株的基因分型

发布时间：2018-05-07 12:12

本文选题：刚地弓形虫 + 酶联免疫吸附试验　；参考：《吉林大学》2011年硕士论文

【摘要】：刚地弓形虫(Toxoplasma gondii)是一种专性细胞内的顶复门原虫,能感染所有的恒温脊椎动物,包括哺乳动物和鸟类。刚地弓形虫在人群中广泛分布,世界上1/3的人口呈慢性感染。根据不同的环境特点和饮食习惯,血清阳性率在不同地理区域中是不同的:在欧洲,育龄期女性血清阳性率在英国为10-18%,西班牙为19-29%,意大利为40%,法国为55%。通过食用未煮熟的或生的被感染的肉(例如猪肉,羊肉)或通过猫排泄的卵囊污染的食物,水,或未洗的蔬菜而感染弓形虫。感染弓形虫常见但通常无症状,但是却对免疫受损的患者如癌症病人、艾滋病、器官移植患者等产生威胁。因为孕期病原会通过胎盘传播给胎儿,所以孕期初次和再次感染尤其对胎儿来说是高危因素。先天性弓形虫病会导致许多各种不同的临床表现,从轻微症状,视觉缺陷或视网膜脉络膜炎的形成到严重的脑积水,小头畸形,智能缺陷,甚至死亡。在南美洲和欧洲,刚地弓形虫是高度克隆的,有三种主要的系谱组成(I型,II型和III型),然而,在南美洲,分布于人群中的一些虫株呈高度多样性,这些虫株在生物学和遗传方面不同于南美和欧洲的虫株,这些显示弓形虫种群整体的多样性很高。目前,关于亚洲刚地弓形虫多样性的数据很有限。在这项研究中,多基因位点聚合酶链反应-限制性片段长度多态性(PCR-RFLP)分析被用于确定与中国人弓形虫病相关的刚地弓形虫(I型、II型、III型)不同基因型的流行情况。首先,通过间接酶联免疫吸附试验(ELISA)检测血清样本中抗刚地弓形虫IgG抗体的水平。然后,从IgG抗体阳性孕妇外周血中提取DNA样本,通过聚合酶链反应(PCR)检测刚地弓形虫529bp重复序列。最后,用12个基因标记物包括SAG1, 5’SAG2, 3’SAG2, alt.SAG2,SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1和APICO通过多基因位点聚合酶链反应-限制性片段长度多态性(PCR-RFLP)进行刚地弓形虫分离株的基因鉴定。中国东北地区840份孕妇血清样本用间接ELISA检测抗刚地弓形虫IgG抗体水平, 80名孕妇抗刚地弓形虫IgG抗体为阳性的,血清阳性率为9.5%。从IgG抗体阳性孕妇的外周血中提取DNA样本通过聚合酶链反应用于检测529bp重复序列,33份样本为聚合酶链反应阳性。通过多基因位点聚合酶链反应-限制性片段长度多态性分析对阳性DNA样本进行基因分型,8个样本基因分型为II型。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a specific intracellular protozoa that infects all isothermal vertebrates, including mammals and birds. Toxoplasma gondii is widespread among people, with a third of the world's population chronically infected. According to different environmental characteristics and dietary habits, the positive rate of serum is different in different geographical regions: in Europe, the positive rate of seropositive in women of childbearing age is 10-18 in Britain, 19-29in Spain, 40 in Italy and 5555 in France. Toxoplasma gondii is infected by eating uncooked or raw infected meat (such as pork or mutton) or by excreting food, water, or unwashed vegetables. Infection with Toxoplasma gondii is common but usually asymptomatic, but poses a threat to immunocompromised patients such as cancer patients, AIDS, and organ transplant patients. As pathogens transmit to the fetus through the placenta during pregnancy, first and second infections during pregnancy are particularly high risk factors for the fetus. Congenital toxoplasmosis can lead to a wide variety of clinical manifestations ranging from mild symptoms visual defects or retinal choroiditis to severe hydrocephalus microcephaly mental retardation and even death. In South America and Europe, Toxoplasma gondii is highly cloned, with three major pedigree types, type I and III. However, in South America, some of the species of Toxoplasma gondii are highly diverse. These strains are biologically and genetically different from those in South America and Europe, indicating a high diversity of Toxoplasma gondii populations as a whole. Currently, data on the diversity of Toxoplasma gondii in Asia are limited. In this study, polygenic locus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to determine the prevalence of different genotypes of Toxoplasma gondii type I and type II associated with Toxoplasma gondii in Chinese. First, the level of IgG antibody against Toxoplasma gondii in serum samples was detected by indirect enzyme-linked immunosorbent assay (Elisa). Then, DNA samples were extracted from the peripheral blood of pregnant women with positive IgG antibody, and the 529bp repeats of Toxoplasma gondii were detected by polymerase chain reaction (PCR). Finally, 12 gene markers, including SAG1, SAG2, SAG2, alt.SAG2, BTUB, GRA6, c22-8, c29-2, L358, PK1 and APICO, were used to identify Toxoplasma gondii isolates by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Indirect ELISA was used to detect the level of IgG antibody against Toxoplasma gondii in 840 pregnant women in northeast China. The positive rate of IgG antibody against Toxoplasma gondii in 80 pregnant women was 9.5%. DNA samples were extracted from peripheral blood of pregnant women with positive IgG antibody. 33 samples of 529bp repeats were detected by polymerase chain reaction (PCR). The positive DNA samples were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R382.5

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