Application Of Loop-Mediated Isothermal Amplification (LAMP) In Rapid Detection Of Proteus

Pathogenic microorganisms and food contamination constitute a large and growing public health problem worldwide. Monitoring network of food-borne disease statistics: over the past decade, caused by the pathogenic microorganisms and foodborne disease events involving the largest number of 46.4%, which accounted for 11.3% of Proteus, Proteus vulgaris and Proteus mirabilis Is the major cause of food poisoning Proteus. Therefore, Proteus vulgaris and Proteus mirabilis in the detection, identification, and prevention and control technology has been of great concern. Proteus detection method has been used in traditional culture, but have been unable to meet the modern requirements of rapid detection.Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, referred to as LAMP) is equal to 2000, the Japanese scholar Notomi invented a new method of nucleic acid amplification. The technology used to identify the target sequence sites on the 6 4 specially designed primers and a DNA with strand displacement activity of polymerase, at a constant temperature, specific and efficient, rapid amplification of nucleic acids. In amplification efficiency can be achieved within 1h 10⁹-10¹0 orders of magnitude, is a series of inverted repeat amplification target sequence stem-loop structure and composition of multi-ring structure of cauliflower-like mixture of DNA fragments, after gel electrophoresis showing zones of different sizes consisting of stepped patterns. In addition, to add specific ring in the reaction primers (loop primer) able to amplify shorter by half, so that the reaction rate greatly improved. LAMP technology in recent years for its specificity, so temperature sensitive, simple, easy to test the advantages of the product has been used in the field of food safety in many areas.In this study, Proteus atpD gene sequence (AX10⁹601) for analysis, design, six primers (two inner primers, two outer primers, two loop primers), and Proteus vulgaris and Proteus mirabilis were detected, optimal reaction conditions to verify the specificity and sensitivity of detection, applied to the direct detection of food samples, and using restriction enzyme digested PCR products Psp1406â… observed fragment size, the correctness of authentication methods, the establishment of the LAMP detection of Proteus methods. To establish the optimal reaction conditions we LAMP reaction were optimized. Experiments show that the maximum temperature is the LAMP factors, while the concentration of magnesium ions and the ratio of primer have little effect on the reaction. Informed of the outer ring to inner primer concentration ratio of 1:3:6, Mg²⁺ concentration was 4 mmol/L, reaction temperature is 61℃, reaction time was 50 min when the most sensitive test. To evaluate the specificity of the technology, we also on a Proteus vulgaris, a Proteus mirabilis and 13 strains of other pathogenic E.coli-specific test. The results showed that Proteus only in the DNA amplification products after electrophoresis specific laddering, the PCR products were digested further, the results are in line with expectations.This shows that the LAMP technology Proteus is a specific DNA amplification. Our serial dilution of the original template to test the sensitivity and experimental results show that the detection of LAMP technology can stabilize the lower limit of Proteus DNA 5.4 CFU/mL. Application of LAMP method Proteus pork DNA samples were detected, and the results compared with the normal PCR wears. The results showed that, LAMP detected Proteus pork detection limit of 15 CFU/mL, control PCR detection of pork in the detection limit of Proteus 150 CFU/mL. LAMP method than the PCR detection limit 10 times higher, and time is reduced by half.Therefore, our experiments show that the LAMP technology for the food Proteus DNA detection with high specificity and sensitivity. The LAMP is simple and rapid, easy to judge and does not require sophisticated technology and equipment is low-cost advantages of PCR can match. If further optimization, improvement and promotion, we believe that the technology will be infected with Proteus in food play an important role in prevention.