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With the growing population and human needs, the growing concern about food safety. Foodborne microorganisms as a food safety hazard sources, threatening human health. Salmonella as the most common foodborne pathogenic microorganisms, the public concern, however, traditional detection methods are time-consuming and low detection efficiency, and no longer able to meet the needs of the high demand for food safety. So, We need to develop a highly efficient and rapid means of detection, monitoring Salmonella, ensure human health. Loop-mediated thermostat nucleic acid amplification method is to create an ideal means of PCR based on the detected overcome inherent in conventional PCR detection of a long time, easy to pollution and disadvantages of testing costs, at the same time and quick detection method than the traditional culture Convenient, significant savings in staff time. In addition, the technical requirements of this detection method to detect personnel is low, the actual operation is very simple, does not require special reagents and equipment, to the establishment of a low-cost rapid screening system, absolutely can establish the total cost of detection system . The method is suitable for field detection and a large number of samples of high-throughput detection has broad application prospects in the field of disease diagnosis, gene chip development and quality of food hygiene inspection. The situ heated nucleic acid amplification method is to improve an isothermal nucleic acid amplification method based on the theory directly in the intact cell, the new method of nucleic acid amplification. Its response characteristics, and does not need to be of bacterial proliferation, and visual inspection can be performed also on the different life cycle of the same kind of cells were observed. The technique does not require crushing to extract the nucleic acid of a cell or tissue, capable of a gene in cells or tissues for positioning, and also has a high sensitivity. In addition, the technology techniques using only a constant and a lower reaction temperature, thus reducing the damage to the cells and favor the use of fluorescent-labeled antibody to cells were identified, a large number of repeat of the target gene can be prevented from leaking extracellular affect the result of gene amplification, and that the The technology used in the low molecular weight nucleic acid polymerase is more easily enter cells. Salmonella as the object of study, mainly for Salmonella conserved genes, specific primers were designed thermostat nucleic acid amplification system to achieve the specific detection of Salmonella, at the same time, take advantage of the specific primers combined for Salmonella cell permeability the research, development thermostat Salmonella situ nucleic acid amplification method and applied to the detection of the artificially contaminated food samples to nucleic acid amplification series of methods used in food safety testing provide theoretical foundation and technical support in the field of in-depth development of the thermostat. The main research content and results are as follows: (1) In accordance with the Salmonella spp own virulence genes conserved the inva gene target sequence to design specific thermostat Salmonella spp nucleic acid amplification primers, to establish a new type of loop-mediated thermostat nucleic acid amplification method, rapid detection of Salmonella. The results showed that: a new type of the loop mediated thermostat nucleic acid amplification method in hour 65 ℃ under isothermal conditions to complete the detection of Salmonella. Detection process found that the amplification is very specific, and is not interested in the nucleic acid of other gram-negative bacteria for amplification; and the method has a high sensitivity, can reach 10CFU/reaction, PCR sensitivity 50 times; by LAMP Turbidimeter real-time observations of the entire amplification process, and be able to add a fluorescent dye SYBR Green I, so that the positive result of the green obviously distinguished from negative results orange, enables fast and convenient observation of test results. (2) for the bacteria under conditions of low temperature and nutrient poor not proliferate causing undetected problems create situ thermostat nucleic acid amplification method, to achieve the target sequence in a single cell in situ under conditions of constant temperature of 65 ℃ amplified. The results show that: the lower reaction temperatures reduce the damage to the cell structure, cell integrity fixed on slides, no peeling strong contrast in the obtained image background; detecting the mixed bacteria Salmonella and E. coli, through the green excitation, labeled with the fluorescence signal of the Salmonella specific detection; the artificial simulated polluted samples is detected, the detection result is not subject to the influence of impurities, can be subject to individual cells under inverted fluorescence microscope; by thermostat nucleic acid with the PCR method and the loop-mediated expansion increasing method for comparison can be found, the sensitivity of the method is higher than the PCR method, and considerable thermostat with the loop-mediated nucleic acid amplification method, however, its detection limits can be achieved individual cells and the observation can be more intuitive to the cells in the presence status, and do not nucleic acid extraction, significantly omitted human operator error.
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