| Staphylococcus aureus (SA) is recognized as one of the important food-bornepathogens in the world, Its widely exist in nature word, which can grow in the food,produce enterotoxin, cause food poisoning, when the contaminated food. At present,detection of Staphylococcus aureus mainly uses the traditional isolation, culture andbiochemical identification method in our country. It Can no longer meet the needs of therapid detection of pathogens in food because of the complicated operation and timeconsuming. It’s urgently needs to establish a new method for rapid detection ofpathogenic bacteria.Standard strains of Staphylococcus aureus and the strains screened are the theresearch object In this study to construct the Fingerprint chromatography ofStaphylococcus aureus, And use the chromatography detecting staphylococcus aureuscontaminated food. Using high performance liquid chromatography (HPLC) methodtesting34strains of staphylococcus aureus and20strains of non-staphylococcus aureusto establish fingerprint, determined the characteristic peak of Staphylococcus aureus bythe comparison. Look SPA as internal standard (standard peak), the relative retentiontime of the characteristics peak and the standard peak is the basic data ofstaphylococcus aureus fingerprint to construct, determine the threshold by comparativeanalysis the distribution of correlation coefficient of two types of fingerprint, buildingup the calculation method of fingerprint to Identify the staphylococcus aureus.Optimized the accumulation of cell coextracted content,extraction and its liquidchromatographic conditions, and then evaluate and applicat the method ofStaphylococcus aureus fingerprint. Optimization of intracellular coextracted contentconditions were determined:103CFU/mL of Staphylococcus aureus were cultured in200mL nutrient broth access, shake cultivation12h, the bacterial suspension wasprepared by centrifuged for6min at6654g, washed with sterilizated water two times,Add10mL of methanol made bacteria suspension,and then broken10min in theultrasonic cell disruptor ultrasonic. Rerunning centrifuged for6min at6654g, rotaryevaporation to1~2mL, Through organic membrane filter. This condition can guaranteethe intracellular coextracted content of staphylococcus aureus can be detected at least 15common peaks that are completely separated. The fingerprint of Staphylococcusaureus was established by the grope of HPLC conditions: Aminopropyl XB-NH (4.6mm×25mm,5μm); mobile phase A:0.05%phosphoric acid solution; B: acetonitrile.Gradient program (min/B%) is:0-15min to90%-80%,15-20min to80%-65%,20-35minto65%-5%,35-40min to5%-0%. UV detector wavelength detection:280nm, flow rate:0.8mL/min, column temperature:30℃, injection volume:20μL. From the method ofextraction of intracellular material to the report the results, time-consuming2h.Thereare14h from nutrient broth culture to the identification results report. Testing thefeasibility and accuracy of the method of identify staphylococcus aureus by the mixedbacteria identification and double blind experiment. Experimental evidence that themethod of staphylococcus aureus fingerprint can identify staphylococcus aureus fromthe mixed bacteria cultures.The results of double blind experiment are consistent withthe initial record of pollution, Verified further the correctness and the feasibility of themethod of rapid detection of staphylococcus aureus.research shows that, HPLC method has certain feasibility in the identification ofstaphylococcus aureus, The method is simple in operation, less time, wellspecificity.this study that we had developed a new and rapid method for detection ofStaphylococcus aureus Strains, the rapid detection of foodborne pathogens build atechnology platform. |
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