The Research Of Detecting Schistosoma Japonicum Cercariea In Water And Early Detection For Mice By Real-time PCR

Objective (1) To compare the abundance on the three Schistosoma japonicum repeated sequences, pSjrh1.0 (U92488.1), 18SrRNA(AY157226.1)and retrotransposon SjR2 clone G55A(AF412221.1)and select the optimum target gene and reaction condition by SYBR Green I fluorescence real-time quantitive PCR(RQ-PCR). (2) Establish the method for quantitively detecting the S. japonicum genomic DNA, by drawing the standard curve between the logarithms of DNA concentration and threshold cycle(Ct value), and evaluate the reliability of the method. (3) Quantitively detecting the number of S. japonicum cercariae, by drawing the standard curve between the logarithms of the cercariae number and Ct value, and estimate the reliability of this method. (4) Compare the efficiency of the two methods,RQ-PCR and ELISA.Select the better early diagnosis for assuring infection with S. japonicum cercariae.Methods (1) Design conventional PCR primers using softwares, Primer Premier 5 OLIGO6.55 and NCBI website sequence blast (http: // blast. Ncbi. Nlm. Nih. gov/ Blast.cgi). Establish temperature gradient PCR essays, sequence the PCR products and select conservative sequences. (2) Targetting to the conservative sequences of pSjrh1.0, 18SrRNA and G55A, design the RQ-PCR primers, establish RQ-PCR essays. Comparing the Ct valve, determine the optimum targetting sequence and the reaction condition. (3) Under the optimum reaction condition, establish S. japonicum DNA concentration gradient RQ-PCR, generating standard curve between the logarithms of S. japonicum DNA concentration and Ct value by SDS7500, and get correlation coefficient. (4) Detect the S. japonicum DNA concentration of different number of cercariae using RQ-PCR, drawing the standard curve between the logarithms of the cercariae number and Ct value, and get correlation coefficient. (5) 42mice were divided into three groups, 14 per group, respectively infected 5, 10 and 20 cercariae, and extract 20μl serum DNA in 2, 4, 6, 8 and 15days in every group,establish RQ-PCR essays to detect the S. japonicum DNA and using the method of ELISA to detect the mice IgG antibody to antigen of Schistosomula in serum of mice infected by S. japonicum cercariae after 1, 2, 3 and 4 weeks. Assure the earliest time when the two methods can detect the schistosomiasis.Result (1) The agarose gel electrical pulse of conventional PCR products showed that the optimum PCR reaction condition of pSjrh1.0 and 18SrRNA is annealing temperature 57.4℃, primer concentration 20pmol, and the optimum PCR reaction condition of G55A is annealing temperature 58.8℃, primer concentration 20pmol. The sequences of pSjrh1.0 and 18SrRNA are identical with the sequence published on NCBI web, and 20bp of G55A are different from the sequence published. (2) Design fluorescence RQ-PCR primers, the PCR products are approximately 160bp, the temperature gradient essays showed that the optimum reaction condition is annealing temperature 58℃, primer concentration 20pmol; There are more copies of pSjrh1.0 than 18SrRNA and G55A in S. japonicum genome, the difference is statistically significant (P<0.05). (3) The method of RQ-PCR targetting pSjrh1.0 sequence can detect 2fg S. japonicum genomic DNA reaction, generate the standard curve between the logarithms of S. japonicum DNA concentration 2fg~2×108fg and Ct value, by software SDS 7500, and the correlation coefficient is 0.9977. The essays of 0.2fg DNA and double steamed water are negative. Repeated experiments showed that the variation coefficient is less than 2%, which shows the reliability is good. (4) Extract the DNA from 1, 5, 10, 20 and 80 S. japonicum cercariae, establish the RQ-PCR essays, and establish standard curve between the logarithms of the cercariae number and Ct value. The correlation coefficient is 0.9186, and the correlation is good. The variation coefficient of repeated experiments and standard curve is less than 3%, which show the reliability is good. (5) Respectively extract the DNA from serum samples of mice infected by S. japonicum cercariae by using ROSE and the kit, and the results of PCR indicate that the effectiveness of ROSE is better than that of the kit. The results of mice infected by ten cercariae is positive in the second week. The results of mice infected by ten cercariae detected by ELISA is positive in the fourth week.Conclusion RQ-PCR is a rapid, high sensitive and high reliable method for detecting S. japonicum DNA; The fluorescent RQ-PCR method for targetting pSjrh1.0 can detect 2fg S. japonicum DNA, the logarithms of DNA concentration and Ct value have good linear relationship, and the logarithms of the cercariae number and Ct value have good linear relationship. This method has good reliability, and Ct value can correctly reflect the number of cercariae. And it can achieve the effectiveness of early diagnosis, and have good value of clinical applications.