Establishment And Application Of Rapid Molecular Detection Technology For Staphylococcus Aureus In Ready-to-eat Fruits And Vegetables

Staphylococcus aureus is one of the common foodborne pathogens,a rapid growth in consumption of ready-to-eat fruits and vegetables has showed up in the last decade.New method to detect pathogens in these kind of food must be developed quickly.The combination of high-resolution melting curve(HRM)and real-time PCR has provided a new method of molecular detection.In the last few years,HRM has been applied widely in medical domain,however,with the detection of foodborne pathogen,it still had huge progress to be made.Therefore,this study was aimed to develop a new selective enrichment broth to cultivate the staphylococcus aureus in ready-to-eat fruits and vegetables rapidly,then develop a real-time PCR method combined with HRM for the detection of Staphylococcus aureus and enterotoxin type A and enterotoxin seu,in the end,assemble the components into a kit for commercial sale.1.The study of selective enrichment broth for Staphylococcus aureusFor the new Staphylococcus aureus selective enrichment broth(SSA),glucose and sodium pyruvate were added into Luria-Bertani as promoters,while nalidixic acid and phenethyl alcohol as inhibitors.Response surface methodology was used to optimize four chosen factors,the optimized concentration were 1.73 g/L of Glucose,12.52 g/L of sodium pyruvate,3.82 mg/L of nalidixic acid and 1.60 m L/L of phenethyl alcohol.The growth of S.aureus in SSA was superior to that in LB and faster than TSB with 7.5% Na Cl.SSA also showed good inhibition ability against 8 strains which were easy to find in ready-to-eat fruits and vegetables.Further,SSA gave a better recovery environment to heat-stressed pathogen cells than LB and 7.5% Na Cl broth.A dosage of 102 CFU/m L of S.aureus in fruits and vegetables samples could be detected positive by PCR method after culture of 6 hours by SSA.2.The establishment of real-time PCR method to detect S.aureus and its enterotoxinThe detection method was established with HRM and real-time PCR.The target gene of Staphylococcus aureus filtrated in our laboratory,enterotoxin type A and enterotoxin seu were used to design specific primers.This study used Syto9 Green with the concentration of 1× as the dye of detection system,which could monitor the signal immediately.The concentration of Ex Taq Hs enzyme and d NTP mixture were 0.015 U/μL and 0.10 m M.Three pairs of primers to amplify three specific resolution peak had concentration of 0.05 μM,0.10 μM and 0.20 μM.Genome DNA with the concentration of 4.1 fg/μl could be detected with the result of three specific resolution peak,for colony concentration,it came to 1.19×103 CFU/m L.After culture of 6 hours in SSA and 18 hours in 7.5% Na Cl broth,102 CFU/m L of S.aureus in fruits and vegetables samples were detected by real-time PCR totally and 90% respectively.With baird-parker agar,it only had 90% and 40%.3.Assemble and application of S.aureus real-time PCR kitThe detection system described above was assembled as a kit and verified to accomplish commercial application.The intra-group and inter-group repeatability of the kit in detection of S.aureus with different concentration were all 100%.The result of kit did not have significant difference after freezing and thawing for 60 times,storing in styrofoam box for 72 hours,90 days in-20℃ and 45 days in 4℃.Moreover,the kit showed good specificity and capacity of resisting disturbance,no false-positive or false-negative result have been found in detection of 31 S.aureus strains and 15 other strains.In a coenrichment with other pathogens of 106 CFU/m L and S.aureus of 102 CFU/m L,there were no significant difference showed up.The same result was achieved in detection of template DNA with 100 ng/μL and S.aureus template DNA with 1 ng/μL.In summary,real-time PCR was demonstrated as a promising rapid kit for the detection of S.aureus and its enterotoxins combined with SSA in a total assay time of 10 h.