The Establishment Of Simultaneous And Quick Detection Method For Salmonella Enteritidis, Staphylococcus Aureus And Listeria Monocytogens

Food-borne disease is one of the most important food security problems, which includes food poisoning, intestinal infectious disease, zoonosis, gut-derived virus infection and so on. In recent ten years, the food-borne disease which caused by Salmonella and Staphylococcus aureus are account for 17.9% and 8.9% respectively, so they are the most common and important incident. In some statistical information, 70% to 80% of the bacterial food poisoning was caused by Salmonella. Therefore, finding out rapid and high effective inspection and control methods to detect Salmonella, Staphylococcus aureus, and Listeria monocytogens is essential for ensuring the food security.According to the universal enrichment broth and selective enrichment broth, we formulated a broth which allows simultaneous growth of Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogens. We selected suitable additive agents by single factor experiment, and optimized the selective enrichment broth through response surface methodology. The formula is as follows: 17 g of tryptone, 3 g of peptone, 15 g of sodium chloride, 2.5 g of disodium hydrogen phosphate, 2.5 g of glucose, 2.0g lithium chloride, 1mg potassium tellurite, 10mg nalidixic acid, 2.5g Sodium pyruvate, 5g mannitol, 1L distilled water. It was proved proved that the SSL broth could support the similar growth of three target pathogens, which could yield cell densities of 107-108 CFU/mL after 24 h incubation at 37℃, 150r/min. Non-target pathogens were inhibited well.Three pairs of specific primers and three probes were designed according to the invA gene of Salmonella, hly gene of Listeria monocytogens and sa442 gene of Staphylococcus aureus. The three target fragments were simultaneously amplified by the multiplex PCR. At last, we detected the multiplex PCR product by the xMAP liquid chip. Through optimizing hybridization condition, probes and PCR products were hybridized at 52℃for 20min. The detection sensitivity could reach 100CFU/mL. The effect of the xMAP liquid chip was proved by detecting the artificial inoculation samples and naturally contaminated samples. The whole detection process could be finished within 24h, the detection sensitivity can reach 10CFU/mL.The xMAP liquid chip detection system we constructed is feasible in three target pathogens rapid detection. The results showed its good repeatability, specificity, sensitivity, and better than national standards detection methods.