【摘要】：作為大豆制品,腐乳有著悠久的歷史,其味道鮮美、營(yíng)養(yǎng)豐富、質(zhì)地細(xì)膩,越來越受人們喜愛。然而近幾年腐乳中蠟樣芽胞桿菌(Bacillus cereus)污染的報(bào)道越來越多,嚴(yán)重影響消費(fèi)者的健康。因此,對(duì)腐乳中Bacillus cereus進(jìn)行檢測(cè)、分型,并掌握Bacillus cereus分離菌株毒力基因攜帶情況及耐藥表型至關(guān)重要。本文建立基于基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜(MALDI-TOF-MS)技術(shù)針對(duì)Bacillus cereus的快速鑒定方法,并利用本研究建立的方法對(duì)腐乳中Bacillus cereus進(jìn)行檢測(cè)。同時(shí),針對(duì)Bacillus cereus分離菌株,運(yùn)用MALDI-TOF-MS進(jìn)行分型。最后對(duì)Bacillus cereus分離菌株的毒力基因及耐藥表型進(jìn)行檢測(cè)。具體研究?jī)?nèi)容和結(jié)果如下:(1)建立Bacillus cereus的MALDI-TOF-MS鑒定方法。以Bacillus cereus模式菌株為研究對(duì)象,優(yōu)化該菌樣品處理方法,并選取17株Bacillus cereus標(biāo)準(zhǔn)菌株補(bǔ)充MALDI-TOF-MS數(shù)據(jù)庫,考察MALDI-TOF-MS鑒定方法的靈敏度、穩(wěn)定性和準(zhǔn)確度。結(jié)果表明:Bacillus cereus最佳樣品處理方法是甲酸提取法,數(shù)據(jù)庫的補(bǔ)充提高了該菌鑒定的可信度,在此條件下MALDI-TOF-MS鑒定方法準(zhǔn)確度高、穩(wěn)定性好,靈敏度為104 cfu。研究的MALDI-TOF-MS鑒定方法準(zhǔn)確快速,為食源性Bacillus cereus快速鑒定和分型提供一定的技術(shù)支持。(2)腐乳中Bacillus cereus的分離鑒定及分型。選取北京市不同品牌、產(chǎn)地、種類或批次的腐乳樣品112份,運(yùn)用國(guó)家標(biāo)準(zhǔn)方法對(duì)腐乳中Bacillus cereus進(jìn)行分離純化、鑒定及計(jì)數(shù),并用MALDI-TOF-MS對(duì)分離菌株分型。結(jié)果表明:所有腐乳樣品均分離出Bacillus cereus,共分離Bacillus cereus 204株,且大部分腐乳中Bacillus cereus的MPN值大于103 MPN/g;在距離為0.9時(shí),MALDI-TOF-MS將204株Bacillus cereus分為4個(gè)型別,且同一品牌或產(chǎn)地腐乳中分離的Bacillus cereus具有一定的聚集性,這為Bacillus cereus的品牌及產(chǎn)地溯源提供依據(jù)。(3)Bacillus cereus分離菌株的毒力基因檢測(cè)。采用PCR、電泳等方法對(duì)Bacillus cereus的11個(gè)毒力基因hbl A、hbl C、hbl D、nhe A、nhe B、nhe C、ent FM、cyt K、bce T、ces、EM1進(jìn)行檢測(cè)。結(jié)果表明:11個(gè)毒力基因攜帶率依次為34.31%、25.98%、7.84%、100.00%、99.51%、99.02%、99.51%、35.29%、40.69%、4.90%、4.90%,說明非溶血素和腸毒素FM可能是腐乳中Bacillus cereus的主要毒力因子;基于毒力基因的攜帶情況將Bacillus cereus分為30個(gè)基因型,說明腐乳中分離的Bacillus cereus呈現(xiàn)多樣化;幾乎所有Bacillus cereus分離菌株攜帶4種及以上的毒力基因,說明腐乳中的Bacillus cereus有產(chǎn)生復(fù)合毒素的可能性。(4)Bacillus cereus分離菌株的耐藥表型檢測(cè)。采用紙片擴(kuò)散法檢測(cè)Bacillus cereus對(duì)青霉素、慶大霉素、四環(huán)素、紅霉素、氯霉素、環(huán)丙沙星、苯唑西林、克林霉素、利福平、阿米卡星、復(fù)方新諾明11種抗生素的藥敏性。結(jié)果表明:Bacillus cereus對(duì)11種抗生素的耐藥率依次為100.00%、0.00%、8.82%、0.00%、0.00%、0.00%、100.00%、0.00%、7.35%、0.00%、11.27%,說明青霉素、苯唑西林已經(jīng)不適用于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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS207.4

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