The Isolation And PCR-based Detection Of S'aureus

Food-borne diseases had received more and more attentions these years,pathogens were considered to be one of the most important reasons in causing food-poisoning agents and the rapid detection research had been studied detaily recent years. Staphylococcus aureus(S.aureus) played an important role in food poisoning accidents and bovine mastic(BM),and the rapid detection methods had been studied detailly these years.Due to the superiorities such as high specity, sensitivity and simple processes,the polymerase chain reaction(PCR) had been used frequently in a lot of areas including pathogens'rapid detection.In our experiments, firstly we isolated some strains of S.aureus from raw milk samples according to their resistence to high salt concentrations and cultured in selective medium Baird-Parker+RPF produced by Biomerieux. Then we amplified the target gene nuc in all isolated strains,positive control strain ATCC25923,negetive strains such as E.coli,Salmonella and the expected amplified band of 279bp was considered to be the standand of speciality, furthermore we compared the product's sequence with the V01281 in Genebank and the result indicated 99% identification, which suguested great speciality of our amplification. In order to choose a relative superior approach for the extraction of DNA, we compared two pre-processed methods incluing 75% ethanol and acetone,then the results showed that acetone was more suitable in extracting DNA; Then we chose four major factors in PCR including Mg2+,primers'concentration,dNTP,Taq concentration and did a few of tests with single factor and multiplex factors orthogonal experiments, at last we confirmed the most superior conditions for PCR detection which consisted of Mg2+2ul,each primer 2ul,dNTP 3ul,Taq 1.5U。With such conditions our experiments suggested the detection limition was 105cfu/ml strains and 19.95ngDNA. Meanwhile the interfering experiments showed that the E.coli and Salmonolar played little role on our detection limitition. Enlarged culture experiments showed that we could detect positive results in 4h,6h and 12h respectively in accordance with the original strains'concentrations 1.5×104,103,102cfu/ml.To find the best approach for extracting DNA form S.aureus in milk samples, we compared three different processing methods including brine washing,PBS buffer washing and organic reagents treatment,the results confirmed that organic reagents were more effective ; The detection limitition achieved 3.2×105cfu/ml and 25.4ngDNA, which appeared the same level as what we have done before in pure cultures; A few of experiments were done to compare the different effects of milk fat and Ca2+ on PCR, and the results suggested that milk fat didn't affect our...