Establishment And Application Of Double Nested PCR And Double LAMP Detection Methods To Food-borne Staphylococcus Aureus And Salmonella

According to the data of China's Foodborne Disease Surveillance Network,we can find that the incidence of food poisoning caused by foodborne pathogens is the highest in all kinds of diseases,among which the proportion of food poisoning caused by Salmonella infection is the highest in all kinds of food poisoning.Food poisoning caused by Staphylococcus aureus(S.aureus)also occupies a large proportion.In order to strengthen the detection of food safety,it is necessary to establish a fast,specific and sensitive detection method.In this study,dual nested PCR dNTPs and dual LAMP methods were established for Staphylococcus aureus and Salmonella,and the results were as follows:1.Primers were designed for the highly conserved sequences of the heat-resistant nuclease gene nuc of Staphylococcus aureus and invA gene of Salmonella.Two sets of primers(common primers and nested primers)were designed for the same gene for nested PCR,and the optimal reaction concentration of the two sets of primers was explored respectively.At the same time,the annealing temperature,Mg2+concentration,enzyme concentration and concentration of the first and second expansion of nested PCR were explored.After determining the optimal reaction concentration of each reagent,the number of cycles of nested PCR was further explored,and a dual nested PCR reaction system with good amplification effect was finally established.The repeatability,specificity and sensitivity of the dual nested PCR established in this study were tested.The experimental results showed that the method had good repeatability and strong specificity for the detection of Staphylococcus aureus and Salmonella,and the detection sensitivity for food-borne Staphylococcus aureus was up to 157 fg/?L.The sensitivity to Salmonella was 1.57 pg/?L.2.Three groups of LAMP primers were designed for the conserved sequences of nuc and invA genes,and the most appropriate group was selected as the primer for the dual LAMP experiment.The dual LAMP reaction system and conditions were determined by exploring the concentration of internal and external primers,Mg2+ concentration,dNTPs concentration,betaine concentration,reaction temperature and reaction time.The method has good specificity and high sensitivity for the detection of Staphylococcus aureus and Salmonella,and the sensitivity for the mixed cDNA of Staphylococcus aureus and Salmonella is up to 157 fg/?L.In this study,the double LAMP reaction results could be obtained by visually observing the color changes of the reactants by adding hydroxynaphthol blue into the reaction system.The sensitivity of the visual double LAMP reaction system for the detection of the mixed cDNA of the two kinds of bacteria reached 15.7 fg/?L,which was about 10 times higher than that of the ordinary double LAMP reaction system.3.126 milk samples and 239 tissue samples were detected by traditional bacterial culture method,double nested PCR method and double lamp method.By comparing the detection results of milk and tissue samples by the two detection methods established in this experiment with the detection results of traditional bacterial culture method,it is proved that the two methods established in this experiment for Staphylococcus aureus and Salmonella can effectively detect the existence of these two food borne pathogens in milk and animal muscle tissue.