Isolation, Identification And Rapid Detection Of Pathogenic Microbe In Eggs By LAMP Technology

Food safety is one of the shared concern all over the world. Foodborne pathogen is themajor cause of food contamination, such as salmonella, Enterohemorrhagic E.coli,Staphylococcus aureus, Bacillus cereus. Egg which is established as nutritional food has highper-capita consumption in our city. However, in recent years, the safety problem of egg hadarisen, especially the microbial contamination of pathogenic bacteria cause the loss of healthand wealth. Loop-mediated isothermal amplification uses a set of2primers, which canidentify the purpose gene sequence of six specific areas. Next, it has gene amplification underthe condition of constant temperature (60-80℃).Loop-mediated isothermalamplification(LAMP)technology which is simple, quick and high specificity, is a new nucleicacid amplification technique and has high value in application.The experiment isolated4bacterial strains from the surface of eggs, through themorphological characteristics, physiology and biochemistry and the sequence analysis of16SrRNA, one bacterial strain was identified Staphylococcus aureus, others were Bacilluscereus.According to the heat-stable nuclease gene of Staphylococcus aureus,5different groupsof primers were designed and one group was chosen as specific primer. The test of specificityand sensitivity showed these primers could apply to the LAMP detection of Staphylococcusaureus.The substrate, Bst DNA polymerase, temperature and time had an effect on theamplification except for the primers. Though the single-factor experiments, the best reactionsystem was primer0.5μl, Bst DNA polymerase1μl, dNTPs1μl，Betaine5μl, reactivetemperature64℃, reactive time60min, total volume25μl.The limit of detection of Staphylococcus aureus was102cfu/ml by LAMP in this assay,there was no amplification for Concentration gradient of101cfu/ml and100cfu/ml. Referto the related literature of sensitivity of PCR detection, the sensitivity of PCR detection wasbetween104cfu/ml to l02cfu/ml. In conclusion, the method of LAMP detection ofStaphylococcus aureus was high specificity and sensitivity, the method could be applied toresearch the kit of rapid detection of Staphylococcus aureus.