Development Of A New Method About PCR-FRET For Quick Detection E.coli

The problems of the drinking water threaten human life and health all the time. However, in natural environment, the sources of drinking water was vulnerable to infection of pathogenic microorganisms. A large number of pathogenic bacteria and intestinal virus always can be detected in polluted water, so many pathogenic microorganism not only caused serious disease to human health, but also greatly complicated detection complex. E.coli as a standard indicator of pathogenic bacteria in the water, is of great significance for monitoring water quality to ensure the safety. The National Standard of China about the hygiene standards for drinking water has strict rules:the limit of Escherichia coli in 100 mL water shall not be checked out. The standard detection of E.coli still is given priority to traditional detection methods, those detection methods is time consuming and cumbersome operation. How to accurately and quickly detect the E.coli in water attracts the attention of many researchers.In this paper, the research work centras on the rapid detection of E.coli. The PCR is high specificity and good sensitivity and FRET with accurate quantitative principle, both of that were utilized to achieve detection of E.coli.First of all, a normal PCR method was built. The specific and conservative gene of uidA in E.coli was chose as target DNA. The PCR experiments were performed after the primer F, R were been designed. The uidA gene was cloned to new plasmid, and the reaction system and reaction conditions of PCR were been optimized according to the plasmid.And then a FRET detection system was been built. The synthesis of CdTe quantum dots chose water reflux method which is mild reaction conditions and high efficiency, a series of good luminous efficiency QDs were obtained by regulating reflux time. The QDs which have emission peak at 602 nm and quantum yield is 53% was chose as the donator of FRET, after contrast the absorption with emission spectrum of quantum dots and the dye according to the principle of Forster, the cyanine dyes Cy5 has been chosen as receptors to FRET. A set of DNA aptamer has been designed according to objective DNA, by linking with QDs and Cy5, they can form sandwich structure with target DNA, and achieve the purpose of DNA testing by FRET.The uidA gene were tested successful after all possible influence factors was optimized, the limit of detection is 2 nmol/L, and it has a good linear relationship in the range from 2 nmol/L to 100 nmol/L, and the linear coefficient about 0.999.Lastly, the PCR-FRET was achieved, the detection can be finished within 3 h. The detection result of the uidA gene show that it LOD is 30 copies, is lower than ordinary PCR method detection and lower about an order of magnitude; For E.coli samples test, the results show that this method is suitable for detecting the water sample which the concentration of E.coli is range from 103 to 107 CFU/mL; the last of actual water samples were tested, and the testing result is consistent with the results of Plate Method and normal PCR method, but it remains to be further improve on accuracy.