Establishment And Application Of SYBR-Green1 Fluorescent Quantitative PCR Method For Detecting Toxoplasma Gondii B1 Gene

Objective To estiblish a SYBR-Greenlfluorescent quantitative PCR(SYBR-Greenl qPCR) method to detect Toxoplasma gondii B1 gene and estimate it with lab technology. Methods We amplified the Bl gene which was highly conserved in RH strain of Toxoplasma gondii with traditional PCR, and cloned it into T vector which was transformed into E. coli JM109. After extracting and identifying the positive recombinant plasmid by PCR and sequenced was used as quantitative template to generate standard curve. Reproducibility of the assay was determined. The B36 strain and Guigong strain were tested. The S japonicum and plasmodium falciparum were detected, too. Furthermore, 30 samples of pregnant women and 200 samples of healthy blood donors were detected with this method. Results The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration and the correlation coefficient was 0.997. The mean interassay coefficient of variation (CV) was 0.81%. The detections of B36 strain and Guigong strain were positive. The detections of S japonicum and plasmodium falciparum were negative. The positive rate in pregnant women was 16.7 %.The mean copies were 1.05×10~5/μ l. The detections of blood donors were negative. Conclusion A SYBR-Greenl fluorescent quantitative PCR for detecting Bl gene of toxoplasma gondii is established successefully in this study. The method is more rapid, sensitive and accurate than traditional PCR and can be used for clinical application and screening for blood donors.