北京市腐乳中蜡样芽胞杆菌检测及毒力基因和耐药性研究

发布时间：2018-07-25 17:48

【摘要】：作为大豆制品,腐乳有着悠久的历史,其味道鲜美、营养丰富、质地细腻,越来越受人们喜爱。然而近几年腐乳中蜡样芽胞杆菌(Bacillus cereus)污染的报道越来越多,严重影响消费者的健康。因此,对腐乳中Bacillus cereus进行检测、分型,并掌握Bacillus cereus分离菌株毒力基因携带情况及耐药表型至关重要。本文建立基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术针对Bacillus cereus的快速鉴定方法,并利用本研究建立的方法对腐乳中Bacillus cereus进行检测。同时,针对Bacillus cereus分离菌株,运用MALDI-TOF-MS进行分型。最后对Bacillus cereus分离菌株的毒力基因及耐药表型进行检测。具体研究内容和结果如下:(1)建立Bacillus cereus的MALDI-TOF-MS鉴定方法。以Bacillus cereus模式菌株为研究对象,优化该菌样品处理方法,并选取17株Bacillus cereus标准菌株补充MALDI-TOF-MS数据库,考察MALDI-TOF-MS鉴定方法的灵敏度、稳定性和准确度。结果表明:Bacillus cereus最佳样品处理方法是甲酸提取法,数据库的补充提高了该菌鉴定的可信度,在此条件下MALDI-TOF-MS鉴定方法准确度高、稳定性好,灵敏度为104 cfu。研究的MALDI-TOF-MS鉴定方法准确快速,为食源性Bacillus cereus快速鉴定和分型提供一定的技术支持。(2)腐乳中Bacillus cereus的分离鉴定及分型。选取北京市不同品牌、产地、种类或批次的腐乳样品112份,运用国家标准方法对腐乳中Bacillus cereus进行分离纯化、鉴定及计数,并用MALDI-TOF-MS对分离菌株分型。结果表明:所有腐乳样品均分离出Bacillus cereus,共分离Bacillus cereus 204株,且大部分腐乳中Bacillus cereus的MPN值大于103 MPN/g;在距离为0.9时,MALDI-TOF-MS将204株Bacillus cereus分为4个型别,且同一品牌或产地腐乳中分离的Bacillus cereus具有一定的聚集性,这为Bacillus cereus的品牌及产地溯源提供依据。(3)Bacillus cereus分离菌株的毒力基因检测。采用PCR、电泳等方法对Bacillus cereus的11个毒力基因hbl A、hbl C、hbl D、nhe A、nhe B、nhe C、ent FM、cyt K、bce T、ces、EM1进行检测。结果表明:11个毒力基因携带率依次为34.31%、25.98%、7.84%、100.00%、99.51%、99.02%、99.51%、35.29%、40.69%、4.90%、4.90%,说明非溶血素和肠毒素FM可能是腐乳中Bacillus cereus的主要毒力因子;基于毒力基因的携带情况将Bacillus cereus分为30个基因型,说明腐乳中分离的Bacillus cereus呈现多样化;几乎所有Bacillus cereus分离菌株携带4种及以上的毒力基因,说明腐乳中的Bacillus cereus有产生复合毒素的可能性。(4)Bacillus cereus分离菌株的耐药表型检测。采用纸片扩散法检测Bacillus cereus对青霉素、庆大霉素、四环素、红霉素、氯霉素、环丙沙星、苯唑西林、克林霉素、利福平、阿米卡星、复方新诺明11种抗生素的药敏性。结果表明:Bacillus cereus对11种抗生素的耐药率依次为100.00%、0.00%、8.82%、0.00%、0.00%、0.00%、100.00%、0.00%、7.35%、0.00%、11.27%,说明青霉素、苯唑西林已经不适用于Bacillus cereus引起的食源性疾病。
[Abstract]:As soybean products, sufu has a long history, its taste delicious, nutritious, delicate texture, more and more popular. In recent years, however, more and more reports of (Bacillus cereus) contamination by Bacillus cereus in sufu have seriously affected the health of consumers. Therefore, it is very important to detect and type Bacillus cereus in sufu, and to master the virulence gene carrying and drug resistance phenotype of Bacillus cereus isolates. In this paper, a rapid identification method for Bacillus cereus based on matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was established, and the method was used to detect Bacillus cereus in sufu. At the same time, MALDI-TOF-MS was used to type the strains isolated from Bacillus cereus. Finally, the virulence genes and drug resistance phenotypes of Bacillus cereus isolates were detected. The main contents and results are as follows: (1) establish MALDI-TOF-MS identification method for Bacillus cereus. The sample processing method of Bacillus cereus model strain was optimized, and 17 Bacillus cereus standard strains were selected to supplement MALDI-TOF-MS database to investigate the sensitivity, stability and accuracy of MALDI-TOF-MS identification method. The results showed that the best sample processing method of MALDI-TOF-MS was formic acid extraction, and the reliability of identification was improved by the supplement of database. Under this condition, the accuracy and stability of MALDI-TOF-MS identification method was high, and the sensitivity was 104cfu. The method of MALDI-TOF-MS identification was accurate and rapid, which provided some technical support for fast identification and typing of foodborne Bacillus cereus. (2) isolation, identification and typing of Bacillus cereus in sufu. 112 samples of sufu from different brands, origin, species or batches in Beijing were selected. The Bacillus cereus in sufu was purified, identified and counted by national standard method. The isolated strains were classified by MALDI-TOF-MS. The results showed that Bacillus cereus was isolated from all sufu samples, 204 strains of Bacillus cereus were isolated, and the MPN value of Bacillus cereus in most sufu was more than 103MPN / g, and the Bacillus cereus was divided into four types by MALDI-TOF-MS when the distance was 0.9. Moreover, the Bacillus cereus isolated from the same brand or origin of sufu had a certain aggregation, which provided the basis for tracing the origin and brand of Bacillus cereus. (3) the virulence gene detection of the isolated strains of) Bacillus cereus. Eleven virulence genes of Bacillus cereus were detected by PCR and electrophoresis. The results showed that the carrying rate of 11 virulence genes was 34.31 / 25.987.987.84and 100.000.0099.51and 99.02and 99.02respectively, which indicated that non-hemolysin and enterotoxin FM might be the main virulence factors of Bacillus cereus in fermented milk, and Bacillus cereus was divided into 30 genotypes based on the carrying of virulence genes. The results indicated that the Bacillus cereus isolated from sufu was diversified, and almost all Bacillus cereus isolates carried four or more virulence genes, which indicated that Bacillus cereus in sufu could produce compound toxin. (4) the phenotypic detection of drug resistance of) Bacillus cereus isolates. The susceptibility of Bacillus cereus to 11 antibiotics including penicillin, gentamycin, tetracycline, erythromycin, chloramphenicol, ciprofloxacin, oxacillin, clindamycin, rifampicin, amikacin and compound sulfamide was determined by disk diffusion method. The results showed that the drug resistance rate of 10: Bacillus cereus to 11 antibiotics was 100.00000.000 and 8.82, and 0.0000and 0.00100.002, respectively, and 0.0000100.002. The amount of 0.000.00100.35 was about 0.007.35.The results showed that penicillin and oxacillin were no longer suitable for foodborne diseases caused by Bacillus cereus.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS207.4

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