Rapid Detection Of E. Coli By Loop-Mediated Isothermal Amplification

Food safety is concerned by all over the world seriously.Escherichia coli is as a pathogenic bacteria in microbial contamination of foods. Escherichia coli is the indicator of polluted foods and water and the pollution level by feces is also evaluated and judged by it.There are many traditional methods to detect it, however, most of these methods are long-time and complicatel to operate. rapid detection technique of Escherichia coli has high application value to researchLoop-mediated isothermal amplification(LAMP)technology is a new nucleic acid amplification technique to instead of PCR, which is simple, quick and high specificity. The gene can be amplificated through identificating 6 specific areas by 6 specific primers.In this research,5 different groups of LAMP primers are designed by LAMP on-line base on the gene array of specificity β-glycuronidase. One group is choosed through primer specificity screening. The best reaction system are:0.2μM 1μL of F3 and B3 respectively, 1.6μM 1μL of FIP and BIP respectively,0.2μM 1μL of LF and LB,2.5μM 1μL of dNTPs,4U of Bst DNA polyase,2.5μL of 1×Thermopol Buffer,1.2M 5μL of Betaine,5μL of ddH2O, 64℃ of amplification temperature and 60min of reaction time. LAMP amplification sensitivity is researched by using DNA models with different dilution factors as the detection models under the optimizational condition. The results show:the amplification bands are detected under 10-7 of dilution factor, and the accurate detecetion results can be gained through precipitation and colour reactions by LAMP.