Use Of Visual Loop-mediated Isothermal Nucleic Acid Amplification For Rapid Detection Of Listeria Monocytogenes In Raw Milk

Listeria monocytogenes is a Gram-positive bacterium, which can cause zoonotic diseases. Inrecent years, the food safety incidents caused by this bacterium are going up, so there are more andmore researchers paying more attention to this kind of bacteria. These detection methods, appliedto Listeria monocytogenes, have many shortcomings. In another words, they can not meet thepractical needs of the rapid development of the food industry. Therefore, a fast and convenientdetection method for Listeria monocytogenes is required.As a new type of nucleic acid amplification, loop-mediated isothermal amplification (LAMP)can identify the target gene by the use of the four primers. Due to the superiorities such as betterspecificity, sensitivity and faster detection speed, the LAMP method will be especially suitable forrapid detection of pathogenic microorganisms.In order to simplify the detection program of the LAMP’s product, rhodamine-based dualchemosensor has been developed. It can be added into the reaction system before the amplification.The color of negative (red) and positive (blue) samples contrast with each other obviously.Furthermore, the result is detected by2%agarose gel electrophoresis consistent with that of LAMPvery well.For the purpose to detect Listeria monocytogenes quickly, ethidium monoazide bromide(EMA)-LAMP was introduced as a diagnostic DNA-based method, the hemolysin gene (hlyA) waschosen as the target gene, and rhodamine-based dual chemosensor acted as the reaction indicator.At last, a fast, accurate loop-mediated isothermal nucleic acid amplification (LAMP) detectionsystem was established by optimizing the indicator, magnesiumion concentration etc.25μL LAMPdetection system:0.2μmol/L outer primers F3and B3,1.6μmol/LμM inner primers FIP and BIP,0.8μmol/L Loop primers LF and LB,0.6mol/L betaine,3mmol/L MgSO4,8U Bst DNA polymerase,2μL template,4.14×10-5g/mL rhodamine-based dual chemosensor. Time and temperatureconditions for amplification of Listeria monocytogenes were optimized to be60min at64°C andthen10min at80℃. The LAMP assay gave artificially contaminated raw milk samples detectionlimit level of1.08×101cfu/mL, while the detection level of conventional PCR was1.08×102cfu/mL.The former method was10-fold more sensitive than the later. These data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific, giving100%concordancewith the GB4789.30-2010reference method.In this study, Liquid LAMP reaction system (no BstDNA polymerases, no DNA template) wasdivided into two groups, one of which was added rhodamine-based dual chemosensor but the otherwithout addition. They were placed at room temperature, and tested until the reaction system couldnot been optimized. Then, the main factors were added into these reaction systems which had losttheir amplification activity, for the purpose to find which factor been needed. The results showedthat these reaction systems, with rhodamine-based dual chemosensor, were unstable. So they werebetter prepared when needed. These LAMP reaction system, without rhodamine-based dualchemosensor, coud been optimized before the8th day. In the reaction systems which had lost theiramplification activity, the primers were most needed, followed by dNTPs.LAMP reaction systems of the above two groups were pre-frozen at-20℃12h, thenfreeze-dried into powder, and then repeated the same operation. To achieve the purpose that whichfactors were required, these dry powders were explored at room temperature and tested timely.Atlast, we found that these LAMP reaction systems, with rhodamine-based dual chemosensor, coudnot been optimized. But the other kind could be used to detect Listeria monocytogenes until30days later. In these LAMP reaction systems which had lost their amplification activity, thethermopol reaction buffer was most needed.To avoid these errors which were caused by researchers, we have scanned these LAMPreaction systems, containing rhodamine-based dual chemosensor, heated for different time, byDU800Nueleic acid/Protein ANALYZER.The data indicated that these LAMP reaction systems,heated for60min, Abs been greater than0.72, were positive, otherwise it was negative.The conclusion was that the visual LAMP method was a powerful tool for detection ofListeria monocytogenes in raw milk samples.