Development And Evaluation Of A Loop-mediated Isothermal Amplification Assay For The Rapid Detection Of Staphylococcus Aureus In Food

Staphylococcus aureus (S. aureus) is one of the most common and important pathogenic bacterium. In the word, there are many cases that are cause by S. aureus. Alimentary toxicosis caused by S. aureus is now a very serious problem worldwide. To solve this problem, a fast and highly efficient method for detecting S. aureus should be established.Current standard detection methods require lengthy culture enrichment steps to increase target bacterial numbers before isolation and identification can be performed. These conventional food microbiological techniques often require several days to detect bacterial pathogens present in food at low levels.The sensitivity and specificity of immunological Methods are unsatisfactory. Recently, a number of new molecular biological methods for rapid detection of bacteria have been reported, including polymerase chain reaction (PCR). The PCR methods provide powerful tools for rapid, specific, and sensitive detection of food-borne pathogens and are considered reliable alternatives for traditional bacteriological methods. However, owing to the expensive systems required and complicate electrophoretic analysis, this application is still not very common in laboratories.Loop-mediated isothermal amplification (LAMP) is a new microbiological method devised by the Japanese scientist Notomi. Because of it can provide a sensitive , specific, simple and cost-effective test in detecting microbes and viruses。The loop-mediated isothermal amplification method (LAMP) had been used frequently in a lot of areas including pathogens'repid detection.We used the the heat-stable nuclease (nuc) gene of S. aureus in the GenBank database (GenBank accession V01281.1),which was the high specificity and conserved sequence, as target sequences and used Primer Explorer software, version 3(http://primerexplorer.jp/lamp3.0.0/index.html), to design LAMP primers. The reaction conditions were optimized including temperature,time and Mg²⁺ concentration etc. The LAMP mixture was made in 25μL of reaction mixture containing a 1.6μM concentration of each inner primer (FIP and BIP), a 0.4μM concentration of each outer primer (F3 and B3), a 0.8μM concentration of the loop primer(LB and LF), 4.0μM each deoxynucleoside triphosphate, 3.0μM MgSO₄,10×Bst DNA polymerase reaction buffer, 8 U of the Bst DNA polymerase large fragment, 2.5μL isolated DNA templates and sterilized double-distilled water. The mixture was incubated at 64℃for 20 min and then heated to 80℃for 10 min to terminate the reaction. We can obtain the results of detection when the white precipitate was observed by visual examination.For an assessment of the sensitivity and detection limit of artificial contamination of LAMP, PCR with the two outer primers (F3 and B3) of LAMP with two loop primers was performed. We extracted DNA using the same DNA kit method. The detection limit of pure bacterial culture was 1.25 CFU per reaction tube(2.5×10¹CFU/mL)and the detection limit of artificially contaminated food sample was 10.3 CFU per reaction tube(2.06×10²CFU/mL) with LAMP detection for two hours. In contrast, the detection limit of pure bacterial culture was 1.25×10² CFU per reaction tub(e2.5×10³CFU/mL)and the detection limit of artificially contaminated food sample was 1.03×10³ CFU per reaction tube(2.06×10⁴CFU/mL)with PCR detection for three hours. Our results showed this LAMP assay had a high specificity for the detection of Staphylococcus aureus by amplifying a fragment of nuc gene of one Staphylococcus aureus strain rather than other sixteen non- S. aureus strains.The effects of eight methods of DNA extraction from S. aureus in food sample on detetion of S. aureus were compared. The results showed that LAMP had fewer requirements for performing a successful detection. Sensitivity of the method in S. aureus detection was 100%, with 97.93% specificity and a 98.5% match ratio when compare using Chinese National standard GB/T4789.10-2010. The result indicated that the sensitivity of detection of S. aureus by LAMP was superior to those of other methods. Its specificity was also excellent when distinguishing S. aureus from other bacteria. Finally, the sensitivity, specificity, and matched route of LAMP were comparable to other methods. Given that LAMP is cheaper, more convenient, and faster, it will be readily accepted by many quarantine institutes requiring microbiological detection methods. The LAMP method is an easy and convenient testing method for improving food sanitation, maintaining food safety, as well as developing international trade.