【作者】 王频佳；

【导师】 张德纯；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2005， 硕士

【摘要】 目的: 弓形虫病(Toxoplasmosis)是由刚地弓形虫(Toxoplasma gondii,Tox)引起的一种重要的机会感染性寄生虫病。由于该疾病对人类特别是孕妇和胎儿具有严重的危害性,因此对弓形虫的检测已成为产前诊断常规项目之一。本课题研究的目的就是建立实时荧光定量 PCR 检测弓形虫的方法,并将此方法应用于临床标本的检测,以期为弓形虫的早期诊断和治疗疗效的监测提供可靠的实验室依据。 方法: 1. 根据弓形虫RH株529bp重复片断段,设计并合成引物和TaqMan-MGB探针;将 PCR 扩增的特异片段克隆入 T 载体,并转化入大肠杆菌DH5α 中。 2. 重组质粒经筛选、鉴定后,作为阳性模板用于 Real-time PCR 的建立,包括 PCR 反应条件的优化,标准曲线的制备以及敏感性、特异性、重复性的方法学评价。 3. 将建立的 Real-time PCR 用于临床妇女血液标本弓形虫的检测,其结果与巢式 PCR 和 ELISA 的结果相比较。 结果: 1. 确立了 Real-time PCR 最佳反应条件:MgCl2 1.5mmol/L、引物 0.5 mol/L、探针 1.0mmol/L,退火温度 60℃。 2. 应用定量阳性重组质粒制作的标准曲线其循环阈值与起始模板拷贝数的对数值之间具有良好的线形关系,相关系数大于 99%,试验内和试验间变异系数均小于 5%,无非特异性扩增,与普通 PCR 相更多还原

【Abstract】 Objective: Toxoplasmosis is an important opportunistic infectious disease caused by parasite protozoan toxoplasma gondii. This disease may cause severe damages to pregnant women and fetuses, so detection of T. gondii has become one of targets of prenatal diagnosis. In this study, we developed a Real-time PCR for detection of T. gondii and applied it in the clinical samples in order to provide accurate laboratory data for early diagnosis and inspection of curative effect. Methods: 1. According to T. gondii RH strain 529bp repeat element, primers and TaqMan-MGB probe were designed and synthesized. The fragment of T. gondii was cloned into T vector and transformed into DH5α. 2. The positive recombinant plasmid was used as standard quantitative template to develop Real-time PCR, including optimization of PCR system, establishment of standard curve and evaluation to sensitivity, specificity and reproducibility. 3. Blood samples from 54 women with histories of abnormal pregnancy and 200 healthy women were detected. The results of Real-time PCR were compared with those of nested PCR and ELISA. Results: 1. Optimized the condition of Real-time PCR: Concentration of MgCl2, primers and probe was 1.5 mmol/L, 0.5 mmol/L, and 1.0 mmol/L, respectively. Annealing temperature was 60℃ . 2. The standard curve obtained by plotting Ct values as a linear function of base 10 logarithm of the initial copies of template DNA. The correlation coefficient was more than 0.99. The inter-assay and intra-assay coefficient of variation were both less than 5%. Not any nonspecific amplification was observed either. Compared with conventional PCR, Real-time PCR was 10-fold higher sensitive. 3. In the clinical samples, the results of Real-time PCR were consistent with those obtained through the nested PCR. The difference in the positive ratio between Real-time PCR and ELISA was statistically significant(P<0.05). Conclusion: We developed a sensitive, specific, simple and quick Real-time PCR for detection of T. gondii, which is suitable for clinical laboratory.更多还原