Establishment Of Loop-mediated Isothermal Amplification Assay For Detection Of Salmonella

Today, the world economy rapid development, gradually improve the people’s material life, also will increase the consumption of products of animal origin, followed by, is into people’s horizons, more and more people focus on food safety problems caused by food of animal origin. According to the national foodborne disease surveillance network according to statistics, foodborne pathogenic bacteria is one of the leading causes of foodborne illness. In food of animal origin, bacterial food poisoning is the most common, and because the Salmonella poisoning caused by food poisoning cases worldwide, is at the forefront.At present, the biochemical experimental test and serological identification of salmonella traditional detection methods, such as need longer experimental cycle, reagent and more complex, has certain limitations in terms of rapid and specific detection, and all kinds of PCR detection method is sensitive, rapid, large instruments and equipment, but expensive will be too complicated electrophoresis process, etc., thus make the PCR detection in inspection work is limited by conditions, it is hard to get universal application.Loop-Mediated isothermal Amplification was invented by Notomi, etc in2000a new isothermal nucleic acid Amplification techniques, this technology overcomes the traditional detection method and PCR detection method of limitations, has many advantages, the advantages of low cost, fast response is suitable for application in high flux testing a large number of samples, the advantages of easy operation, the results can be directly observed is advantageous for the on-site rapid detection, in the diagnosis of disease and the detection of food safety prospect is extremely broad.Announced the experiment based on GenBank salmonella highly conservative enterotoxin gene sequence specific primer design, which in turn will LAMP reaction system of reaction time, reaction temperature, concentration of Mg-T, inside and outside the primer concentration is optimized, the specificity, sensitivity, stability, and a series of experiments, and the LAMP compared with conventional PCR, in order to establish the most appropriate system of Loop-Mediated isothermal Amplification, realize specificity of detection of salmonella.The result shows:the LAMP method for detection of salmonella in65℃under the condition of constant temperature40min can be completed, only salmonella amplification, and won’t amplify other gram-negative bacteria, can check out the lowest concentration of101cfu/mL, high concentrations of the minimum detection than the conventional PCR, one order of magnitude by adding a fluorescent dye SYBR Green I observation of can easily and quickly detect Green positive results is very obvious differences in orange negative result.