【作者】 姜延龙；

【导师】 霍贵成；

【作者基本信息】 东北农业大学 ， 食品科学， 2006， 硕士

【摘要】 食源性疾病是近年来倍受关注的话题之一,对相关的食源性致病菌的检测研究受到了前所未有的重视,其中细菌性食物中毒又占据了重要地位。金黄色葡萄球菌(金葡菌)是引起食物中毒的主要病源菌之一,同时也是引起奶牛乳房炎及多种临床化脓性感染的主要病原菌,其快速检测技术一直是研究热点。PCR技术自问世以来由于其本身高特异性、灵敏性及操作简便等常规方法无法媲美的优点而被广泛应用于包括食品微生物检测在内的诸多领域。本实验首先利用金葡菌的高度耐盐性及法国梅里埃公司的选择性培养基(Baird-Parker+兔血浆)自行从乳中分离出金葡菌菌株;对阳性标准株ATCC25923、自行分离金葡菌株,以及大肠杆菌、沙门氏菌等阴性对照菌同时进行PCR扩增,以是否出现279bp条带为标准验证nuc基因对金葡菌的特异性,完成了PCR扩增产物的测序并与Genebank中标准序列V01281比对,结果显示两者有99%的同源性,进一步确定了扩增片段的特异性;通过比较75%乙醇、丙酮预处理后的扩增效果确定了丙酮处理对金葡菌细胞壁有更好的裂解作用;以影响PCR反应的主要因素:Mg2+浓度、引物浓度、dNTP浓度及Taq酶浓度为对象进行了单因素及多因素正交优化实验,并最终确定了各成分的最佳配比为:Mg2+2ul、引物2ul、dNTP3ul、Taq酶1.5U。在上述实验条件下确定了检测灵敏性为1.2×105cfu/ml菌或19.95ngDNA;干扰实验表明大肠杆菌和沙门氏菌对特异性扩增及灵敏性无明显影响;培养基增菌检测实验表明:初始菌浓度为1.5×104、103、102cfu/ml时PCR方法可分别在培养4h、6h及12h后检出。比较了生理盐水洗涤、PBS缓冲液洗涤和有机试剂处理三种方法对乳中金葡菌DNA提取的不同影响作用,确定了有机试剂处理对排除乳中背景基质的影响作用效果最好;灵敏性实验表明可检测出乳中存在的3.2×105cfu/ml金葡菌,或25.4ngDNA,这与培养基中的检测灵敏性接近;初步比较了不同乳脂含量、乳钙含量对PCR反应的影响作用,发现乳脂含量对扩增结果无明显影响,而乳钙含量则相对来说有更明显的影响作用;对典型的市售食品(羊肉片、豆腐干、肉肠、巴氏乳)进行了增菌检测实验,虽然由于各次实验中初始接菌量及增菌条件略有不同而导致检测结果出现了一定的差异,但从整体来看,初始菌量为10、102、103cfu/ml时各种食品中均可以在10h内检测出。本实验验证了nuc基因在金黄色葡萄球菌鉴定实验中的特异性,通过优化反应条件确定了PCR检测的灵敏性,并完成了几种常见市售食品的模拟检测实验,证明了以nuc为目的基因的PCR检测方法在实际病原菌检测时的可行性。更多还原

【Abstract】 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更多还原