Abstract：ABSTRACT: To establish a rapid, sensitive and specific method to detect the number of cercariae in water and to evalute level of water stained by Schistosoma japonicum(S. japonicum) cercariae. Convenience PCR primer sequences were designed targeting pSjrH1.0(U92488.1), Sj18SrRNA(AY157226.1) and the clone G55A of the highly repetitive retrotransposon SjR2(AF412221.1) in S. japonicum genome, and sequence the PCR product. Based on conserved sequence of pSjrH1.0, Sj18SrRNA and the clone G55A of the highly repetitive retrotransposon SjR2 (G55A), design primers and flouresent quantitative real-time PCR essays were established, by which, the amplifying products are appromixately 150~170bp. Select the most sequence in S. japonicum genome and the best annealing temperature, by comparing their thresode cycle(Ct value). Then establish flouresent quantitative real-time PCR essays under the better annealing temprature, generate standard curve between the logarithm of gradient diluted DNA template and Ct value. Five DNA samples extracted from 1, 5, 10, 20 and 80 cercariae were used as quantitative template to generate standard curve between the logarithm of the number of S. japonicum cercariea and Ct value. Using the modified ROSE method, extract genome DNA from 200uL water samples containing cercariae and establish flouresent quantitative real-time PCR essays to detect the residue cercariea genome DNA in water containing S. japonicum cercariea after 24 hours. The results of the essays show that, pSjrH1.0 has higher sensitivity and lower Ct, in the same template concentration condition, comparing Sj18SrRNA and G55A, and could quantitatively detect as low as 2fg S. japonicum genome DNA in the study. The standard curve showed a fine linear relationship between the logarithm of gradient diluted DNA template and Ct value, and the correlation coefficient r2 was 0.9977; The standard curve showed a fine linear relationship between the logarithm of the number of S. japonicum cercariea and Ct value, and the correlation coefficient r2 was 0.9186. Repeated experiments prove that the method of quantitative detection of S. japonicum cercariea has high repetitiveness, which is a rapid, high sensitive and high repetitiveness method for evalution of the water containing S. japonicum cercariae.
2011, Vol. 27 
