Application Study On Analysis Of Phenolic Compounds And DNA By Capillary Electrophoresis

Objective:In order to provide a rapid, sensitive and reliable technique for determination of phenolic pollutants in environment and monitoring foodborne pathogenic bacteria and DNA damage induced by chemical carcinogens from environment, capillary electrophoresis methods for chemical pollutants such as phenol and m-chlorophenol, multiple PCR products in terms of invA gene in salmonella, ipaH gene in Shigella, and uidA gene in E.coli .O157:H7 and DNA damage of N-ras gene in humanproto-oncogene were developed, respectively. Methods:Optimization for pretreatment of water sample and detectioncondition of Phenol, m-chlorophenol and those DNA moleculars were studied, CE methods for them were developed as follows:1. Phenol and m-chlorophenol in environmental water were cleaned up with large-size mesh resin D101, and then measured by capillary electrophoresis with chemiluminescence detector (CE-CL).2. A multiple PCR was performed to simultaneously amplify invA gene in salmonella, ipaH gene in Shigella , and uidA gene in E.coli.O157:H7. A RDPCR (Randomized terminal linker-dependent polymerase chain reaction) was applied to amplify N-ras gene in human proto-oncogene. And then simple and sencetive analytical methods of capillary electrophoresis with laser-induced fluorescence detection(CE-LIF) were respectively developed and applied to the rapid analysis of the above PCR products. Results:1. Optimizations for conditions of CE-CL detection for phenol and /M-chlorophenol were investigated. The linear ranges were 0.50~200ug/L for phenol and 0.35 ~ 200ug/L for /w-chlorophenol with correlation coefficients greater than 0.99. The detection limits of the method were 0.50ug/L for phenol and 0.35ug/L for m-chlorophenol, respectively. Migration-time and CL intensity of the between-day precisions were 1.2%~1.3% and 5.2%~5.9%, that of the within-day precisions were 1.0%~1.6% and 3.5%~5.7%. The target chemicals in tap water, hospital sewage and Jinjiang river water samples were determined and the recoveries for the spiked samples ranged from 85.0% to 95.5%.Compared with HPLC, the relative error of the results was 4.9%.2. The proposed method was able to simultaneously detect the PCR products of three genes existing in three kinds of pathogenic bacteria under the optimization conditions of PCR and capillary electrophoresis within 25 minutes. Migration time of the.within-day precisions were 0.51%~0.82%, those of the between-day precisions were 1.35%—1.92% The result wasconsistent with that obtained by gels electrophoresis.The proposed method(CE-FIL) was able to detect the RDPCR product of N-ras gene in proto-oncogene under the optimization detection conditions within 20 minutes. Migration time of the within-day precision was 0.63%, that of. the between-day precision was 3.17%. Compared with the Sourthern blotting, the distinguish ratio of CE-FIL is higher. Conclusions:1. The CE-CL method was applied to determine phenol and m-chlorophenol in environmental water samples. The proposed method is characterized by capillary electrophoresis as the separation mode and highly sensitive chemiluminescence as the detector and can finish the analysis within 10 minutes. Compared with the HPLC method, the proposed method was cheap in terms of separation column and used a small amount of solvent, thereby benefiting to environment-protecting. The method was simple, sensitive and rapid, and it was suitable for the determination of phenol and m-chlorophenol in environmental water samples.2. The CE-LIF method was developed to detect the multiple PCR products of Salmonella, Shigella and E.coli.O\57:H7. The proposed method is characterized by non-gel sieving capillary electrophoresis as the separation mode and the laser-induced fluorescence as the detection means and can simultaneously detect three kinds of foodborne pathogenic bacteria. The proposed method can be applied to detect various DNA moleculars. In comparison with traditional gels electrophoresis, the proposed method is rapid, sensitive, accurate, and isn't reported in China.In addition, the CE-LIF method was applied to detect DNA damage of N-ras gene. The method can be applied to evaluate the influence of environmental chemicals on human health and ecosystem. Compared with Southern hybridization, the proposed method is rapid and shows high resolution. The proposed method is important to protecting food safty, dealing with public health emergency, estimating the influence of environmental pollutants on human health and enviroment.