Rapid Detection Of Proteus By Loop-mediated Isothermal Amplification

Proteus.vulgaris is a kind of food-borne pathogenic bacteria which is generally distributed in the environment, such as in polluted water, soil and also in intestinal tract of human and animal. It is a conditional pathogenic bacteria, usually polluting the animal-derived food, such as meat and egg. It is leading to gastroenteritis, meningitis, septicemia etc. Traditional method for routine detection of Proteus.vulgaris is complex and time-consuming. It takes from 48 to 72 hours. This method can not fulfil the need for detecting food-borne pathogens rapidly and sensitive. So is need to establish method for detection proteus mirabilis, so as to provide a reference method for sanitary inspection.Loop-mediated isothermal amplification (LAMP) which stands for Loop-mediated Isothermal Amplification is a new nucleic acid amplification method solely developed by Eiken Chemical Co.Ltd in 2000. The LAMP method employs four primers which specifically recognize six distinct sequences on the target DNA and a bst DNA polymerase that has a strand displacemet activity. The target DNA can be amplified with high specificity, efficiency, andrapidity under isothermal conditions ranging from 60 to 65℃.LAMP can amplify a few copies of DNA to 10⁹ in less than an hour. The final products are stem-loop DNA with several inverted repeats of the target and cauliflower-like structures with multiple loops. The LAMP method will be replace PCR because of its simplicity, rapidity, specificity, and cost-effectiveness.Loop-mediated isothermal amplification(LAMP)was applied to quickly detect Proteus DNA. The specific LAMP primers of Proteus.vulgaris were designed on the basis of the published sequence of strain atpD with the LAMP primer design support software program, using primer explorer software. The reaction was carried out at 62℃for 1 hour and the products were separated by 2.0% agarose gel electrophoresis.The reaction conditions were optimized including Mg²⁺ concentration,temperature,time,BST concentration etc. The LAMP reaction was carried out in a 25μLtotal reaction mixture volume with containing 1.6μmol/L each of inner primers FIP and BIP, 0.2μmol/L each of outer primers F3 and B3, 1.6 mmol/L each deoxynucleoside triphosphate, 6.0mmol/L MgSO4, 2.5μL Bst DNA Polymerase Buffer(20 mM Tris-HCl (pH8.8, 25°C), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2mM MgSO₄, 0.1% Triton X-100), 0.4μL of Bst DNA polymerase,and 1.5μL of target DNA. The mixture was incubated at 62°C for 60 min in a heating block and then heated at 80°C for 10 min to terminate the reaction.There were 11 bacterial amplified by LAMP method in order to evaluate the specificity of primers. The result of only Proteus was positive and those of other strains were negative. The sensitivities of LAMP assays using the FTA filters as templates were 6.4×10²CFU/mL. The detection limits of LAMP assays obtained from artificially inoculated meet samples were 3.6×10³CFU/g. 45 samples were analyzed The detection rate of LAMP amplification was 91.1%, 100% for sensitivity and specificity of 75.0%, the according rate was 95.6%, the detection time was 2h.The results showed that the LAMP method developed in this experiment was with high specificity and sensitivity, the deveolped LAMP method can meet the demands of Rapid Detection of Proteus.