A Loop-mediated Isothermal Amplification Method For Detection Of Staphylococcus Aureus

Food-borne diseases had received more and more attentions these years,pathogens were considered to be one of the most important reasons in causing food-poisoning agents and the rapid detection research had been studied detaily recent years. Staphylococcus aureus played an important role in food poisoning accidents,and the rapid detection methods had been studied detailly these years.Due to the superiorities such as high specity, sensitivity and simple processes, the loop-mediated isothermal amplification method (LAMP) had been used frequently in a lot of areas including pathogens'rapid detection.This subject confirmed the culture method and the cell counting method of S.aureus,the method of extraction and quantitative of the template DNA.Boiled suspension and the DNA extraction liquid for LAMP are prepared.LAMP is describled to detect the nuc of Staphylococcus aureus.In this subject, two LAMP primers were designed and their systems were perfected.The most superior system of LAMP is consisted of 2.5μL 10×buffer,1.2 mM dNTP,0.2μM outer primers F3 and B3,1.6μM inner primers FIP and BIP,0.8 M betaine,4mM Mgcl2,1μL Bst DNA polymerase,2μL the extracted template DNA.The reaction condition of the first LAMP primer is 65℃for 45min and the reaction condition of the second LAMP primer is 62℃for 45min.By primers of LAMP ,we amplified nuc gene in S.aureus ATCC 25923, E.coli Top10and B.sublitis 168.The result is that nuc gene of S.aures was detected by the LAMP assay from S.aureus,but not the E.coli and B.sublitis strains.So,the specificity of nuc for S.aure is proved.Detection limit of nuc using LAMP primers is compared with the detection limit of nuc using PCR .The detection limit of the first LAMP primer is 0.36 pg DNA or 880 CFU ;the detection limit of the second LAMP primer is 32 fg DNA or 53 CFU ; the detection limit of PCR is 3.2-3.6 pg DNA or 530-880 CFU.On the perfected system and reaction condition of LAMP, the nuc gene was amplificated with LAMP's primers and the specificity and detection limit is strong.It's proved that LAMP can be used to detect Staphylococcus aureus.