上海市食源性金黄色葡萄球菌肠毒素基因多样性及分型研究

发布时间：2018-08-17 16:35

【摘要】：本研究从上海市各农贸市场及超市采集各类食品样品,按GB4789.10-2010进行金黄色葡萄球菌的分离及鉴定工作,并用PCR方法进一步鉴定,以减少假阳性,最终获得金黄色葡萄球菌食品分离株;同时提取金黄色葡萄球菌食品分离株的DNA,PCR检测其肠毒素基因的多样性,统计分析各种肠毒素基因的分布情况;最后对金黄色葡萄球菌食品分离株进行多位点序列分型(MLST)研究。首先采集食品样品(生牛奶,生鲜肉,水产品、速冻食品、果蔬、豆制品)505份,按国标方法进行金黄色葡萄球菌的分离和鉴定,共检出被金黄色葡萄球菌污染的食品样品117份,分离金黄色葡萄球菌183株,总检出率为23.2%。在不同种类食品样品中,金黄色葡萄球菌的检出率存在明显差异,其中生鲜肉样品检出率最高,为32.9%;其次为生牛乳和速冻食品,检出率分别为26.3%和26.7%;其它食品中金黄色葡萄球菌的检出率相对较低。该实验结果基本上反映了上海市各类食品中金黄色葡萄球菌的污染状况,为监控金黄色葡萄球菌引起的食物中毒及追溯污染源提供了参考依据。其次,鉴于金黄色葡菌球菌肠毒素是金黄色葡萄球菌引起食物中毒的主要原因,本实验应用PCR技术检测金黄色葡萄球菌食品分离株肠毒素基因的分布特征。在183株金黄色葡萄球菌食品分离株中,有161株携带肠毒素基因,携带率为88.0%;22株金黄色葡萄球菌不携带所检测的18种肠毒素基因,占12.0%;携带率最高的肠毒素基因是seb和sem,均为48.1%;携带率最低的肠毒素基因为see,未检出;5种传统肠毒素基因的携带率(67.8%)低于新型肠毒素基因的携带率(84.7%);含有egc毒力基因簇(同时携带肠毒素基因seg、sei、sem、sen、seo或seg、sei、sem、sen、seu)的分离株有28株,占15.3%;同时携带2种及以上肠毒素基因的分离株有141株,占所有菌株的77.0%,占携带肠毒素基因菌株的87.0%;携带10种以上肠毒素基因的菌株有9株,占全部分离株的4.9%。最后,为鉴别菌株间的相关性,进一步确定菌株特征,进行金黄色葡萄球菌食品分离株的MLST分型研究。研究发现,183株金黄色葡萄球菌食品分离株中,共有22个序列型,其中数量最多的序列型为ST-188,有101株;其次为ST-2094和ST-7,分别有22株和20株;ST-97、ST-5、ST-1281、ST-398和ST-1等也比较常见;同时发现,不同种类食品中序列型的分布不同,如金黄色葡萄球菌生鲜肉分离株中:最主要的序列型为ST-7,有19株;其次为ST-188,有7株;ST-2094、ST-5等也比较常见;而在金黄色葡萄球菌生牛乳分离株中,最主要的序列型为ST-188,有67株;其次为ST-2094,有15株;ST-97、ST-965、ST-9等也比较常见。金黄色葡萄球菌果蔬及速冻食品分离株中,最多的序列型是ST-188。金黄色葡萄球菌豆制品分离株中,出现的序列型包括ST-15、ST-5、ST-512、ST-188和ST-2094。在金黄色葡萄球菌水产品分离株(共6株)中,序列型分别为ST-2094、ST-6、ST-72、ST-88和ST-50。
[Abstract]:In this study, all kinds of food samples were collected from farmers' markets and supermarkets in Shanghai. Staphylococcus aureus was isolated and identified according to GB4789.10-2010, and further identified by PCR method in order to reduce false positive. Finally, the Staphylococcus aureus food isolate was obtained, and the DNA polymerase chain reaction (DNA-PCR) of the Staphylococcus aureus food isolate was used to detect the diversity of the enterotoxin genes, and the distribution of the enterotoxin genes was analyzed statistically. Finally, (MLST) was used to study the multi-locus sequence typing of Staphylococcus aureus food isolates. First, 505 samples of food (raw milk, fresh meat, aquatic products, frozen food, fruits and vegetables, bean products) were collected. Staphylococcus aureus was isolated and identified according to the national standard method. A total of 117 samples of food contaminated by Staphylococcus aureus were detected. 183 strains of Staphylococcus aureus were isolated and the total detection rate was 23.2%. The detection rate of Staphylococcus aureus was significantly different in different kinds of food samples, among which the detection rate of fresh meat was the highest (32.9). The detection rates of Staphylococcus aureus in other foods were 26.3% and 26.7%, respectively. The results of the experiment basically reflect the contamination of Staphylococcus aureus in various kinds of food in Shanghai and provide a reference basis for monitoring the food poisoning caused by Staphylococcus aureus and tracing the pollution sources of Staphylococcus aureus. Secondly, in view of Staphylococcus aureus enterotoxin is the main cause of food poisoning caused by Staphylococcus aureus, the distribution of enterotoxin gene of Staphylococcus aureus food isolate was detected by PCR technique. Of 183 Staphylococcus aureus food isolates, 161 were enterotoxin genes, with a carrying rate of 88.0 and 22 Staphylococcus aureus strains, which did not carry 18 enterotoxin genes detected. The enterotoxin genes with the highest carrying rate were seb and sem48.1, and the lowest enterotoxin groups were see. the carrying rate of the five traditional enterotoxin genes (67.8%) was lower than that of the new enterotoxin genes (84.7%), and the egc virulence group was the lowest. 28 strains were isolated from the cluster (which also carried the enterotoxin gene segseianseo or segsei semsenseu). There were 141 isolates carrying two or more enterotoxin genes at the same time, accounting for 77.0% of all strains and 87.0% of the strains carrying enterotoxin genes, and 9 strains carrying more than 10 enterotoxin genes, accounting for 4.9% of the total isolates. Finally, MLST typing of Staphylococcus aureus food isolates was carried out in order to identify the correlation between strains and further determine the characteristics of the strains. Among the 183 Staphylococcus aureus food isolates, there were 22 sequence types, of which ST-1888 was the most abundant, followed by ST-2094 and ST-7, with 22 and 20 strains of ST-97T5 ST-1281, ST-398 and ST-1, respectively. The distribution of sequence type in different kinds of food is different, such as Staphylococcus aureus fresh meat isolate: the most important sequence type is ST-7, there are 19 strains, followed by ST-188, there are 7 strains ST-2094 ST-5 and so on, but in Staphylococcus aureus raw milk isolate, The main sequence type was ST-188 (67 strains), followed by ST-2094 (15 strains ST-97 ST-965ST-9). Among the isolates of Staphylococcus aureus fruits, vegetables and frozen foods, ST-188 was the most frequent. The sequence types of Staphylococcus aureus soybean products isolates include ST-15, ST-5, ST-512ST-188 and ST-2094. The sequence of Staphylococcus aureus aquatic product isolates (6 strains) were ST-2094 ST-72N ST-88 and ST-50, respectively.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5

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