关键词: 日本血吸虫, 荧光定量PCR, TaqMan探针, 18S小亚基单位核糖体核酸

Abstract: Objective To establish a sensitive and specific fluorescent quantitative real-time PCR method for the detection of Schistosoma japonicum. Methods Based on 18SrRNA sequence of S. japonicum, a PCR assay was established. The 1 450 bp fragment was amplified and cloned into T vector which was subsequently transformed into E . coli DH5α. Following extraction and identification, the positive recombinant plasmid was used as quantitative template to generate standard curve. Reproducibility and specificity of the assay was determined as well. Results The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle （Ct） and template concentration, and the correlation coefficient was 0.998 7. Using the coefficient of variation （CV） value to evaluate the reproducibility, at the template concentration of 1.05×107-1.05×103 copies per reaction， the average Ct values were 17.55，20.93，24.32，27.59，30.95，and the CV values were 1.31%，1.53%，0.90%，1.85% and 0.90% respectively. In the evaluation of the reproducibility， the mean interassay CV was 1.27% and no unspecific amplification was observed. The real-time PCR assay could quantitatively detect as low as 6.15 pg S. japonicum genome in the study（Ct≤30.95）， and the detection should be done in 3 hours. Conclusion A fluorescent quantitative real-time PCR for the detection of S. japonicum is developed, which is rapid， sensitive and specific for pathogen detection.

Key words: Schistosoma japonicum, Real-time PCR, TaqMan probe, 18SrRNA