| 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. |
