Detection Of Staphylococcus Aureus In Milk Using Real-time Fluorescence Loop-mediated Isothermal Amplification

Staphylococcus aureus is a representative of the gram-positive bacteria. On the one hand, S.aureus can cause pyogenic inflammation, on the other hand, after contaminate food S.aureus can produce Staphylococcal enterotoxin cause food poisoning. It is recognized as one of the major causes of human food-borne diseases. The pollution probability of meat, eggs, milk and their products is bigger. In recent years, a growing number of S.aureus food poisoning has become a worldwide health problem. At present, the detection of S.aureus takes long time, is not sensitive enough, and is not for real-time monitoring of reaction process, thus to create a new kind of rapid and sensitive detection method of Staphylococcus aureus is necessary.The real-time loop-mediated isothermal amplification we used in this study is the improvement of loop-mediated isothermal amplification.Put fluorescent dye SYBR Green Ⅰinto LAMP system,SYBR Green Ⅰ can combine to double-stranded DNA, and the fluorescence signal will enhance thousands of time.Put the reaction system into realtime fluorescence detecting instrument for reaction,through the corresponding software observed reaction process.This method can terminate the experiment at any time to control the reaction process.Based on the nuc gene sequences of S. aureus in Gen Bank, according to Primer design principle, this study use online Primer design software Primer V4 designed and selected a pair of specific primers to match the nuc gene sequences of S. aureus. By adjusting the reaction conditions of this study, established the following reaction system:1.6μM FIP and BIP, 0.4μM F3 and B3, 2m M Mg SO4, 0.35 m M d NTPs, 2.5μL 10×Bst DNA polymerase buffer, 12 U Bst DNA polymerase, 0.5μL DNA template, 0.5μL 1:350 dilution SYBR Green fluorescent dye, 6.5μL sterilized distilled water. Set the reaction temperature is 61 ℃, reaction time is 90 min, by using fluorescent real-time monitor and combined with computer software for real-time monitoring the test process. According to the reaction process can terminate reaction at any time.This study detected 8 strains S.aureus and 16 strains other food-borne pathogens for specificity test,the research results indicate that only the test of 8 strains S.aureus is positive, the other 16 strains foodborne pathogenic bacteria test results are negative. The result shows that this method to detect S.aureus has a high specificity.This study also compares the sensitivity of this method and the ordinary LAMP, the result shows that the real-time fluorescent LAMP detection minimum bacteria liquid concentration is 8.0 CFU/m L, and ordinary LAMP method is 80 CFU/m L. Visibly the sensitivity of the real-time fluorescent LAMP is 10 times higher than ordinary LAMP. The detection limit of real-time fluorescent LAMP to test actual samples is up to 23 CFU/m L.Results show that the research of real-time fluorescent LAMP test method has strong specificity, high sensitivity, simple result determination.