我国市售大米中食源性致病菌—蜡样芽孢杆菌的检测和致病性分析

发布时间：2018-06-16 23:42

本文选题：食源性致病菌 + 蜡样芽孢杆菌　；参考：《武汉轻工大学》2013年硕士论文

【摘要】：蜡样芽孢杆菌是条件致病菌，广泛存在于自然界，通常引起呕吐和腹泻两种食物中毒。本研究，采用MPN法、鉴别培养基并结合ITS序列分析，检测、分离和鉴定了138份市售大米样品的蜡样芽孢杆菌检出情况。国内134份样品，109(81.3%)份检出蜡样芽孢杆菌群微生物；107份(79.9%)检出蜡样芽孢杆菌(含菌量为3.0～1100CFU/g)，均值39.1CFU/g；7(5.2%)份检出苏云金芽孢杆菌，含菌量均值3.8CFU/g；1份(0.7%)检出蕈状芽孢杆菌，含菌量7.2CFU/g。原料源于泰国的3份样品中，蜡样芽孢杆菌检出率100%，含菌量16.9CFU/g；苏云金芽孢杆菌检出率66.7%，，含菌量4.7CFU/g；而越南1份样品只检出苏云金芽孢杆菌，含菌量150.0CFU/g。共分离有285株蜡样芽孢杆菌群微生物，268株蜡样芽胞杆菌，16株苏云金芽孢杆菌，1株蕈状芽孢杆菌。不同气候区域、不同加工工艺的大米蜡样芽孢杆菌含量有显著差异(P0.05)。蜡样芽孢杆菌对有利生长繁殖的气候区域有一定的选择性。利用PCR检测呕吐毒素合成相关基因ces，细胞毒素K基因cytK1、cytK2，溶血素BL基因hblC；非溶血性肠毒素基因nheB。各地区蜡样芽孢杆菌毒素检出率不等，在不同的气候区域，蜡样芽孢杆菌毒素的分布差异也极为显著(P0.0001)。nheB(75.0%)在蜡样芽孢杆菌中普遍存在，其次是cytK2(55.2%)和hblC(43.3%)，cytK1检出率为零，90.3%(242/268)的菌株至少携带一种以上的肠毒素基因，即各地区蜡样芽孢杆菌普遍携带肠毒素基因。蜡样芽孢杆菌分离株在8℃～48℃可生长。18(6.7%)株在低温如10℃生长较好，111株(41.4%)可在48℃生长良好。大部分分离株(82.8%)具有β-溶血环或微弱局限溶血环,也有46株(17.2%)不溶血。 16株苏云金芽孢杆菌的ces和cytK1均为阴性，87.5%的苏云金芽孢杆菌同时携带有cytK2、nheB和hblC。蕈状芽孢杆菌毒素基因均为阴性，但具有β-溶血环。 37株(13.8%)蜡样芽孢杆菌含有ces基因，分布在15个省市地区，比国外的蜡样芽孢杆菌ces检出率高许多。这些ces阳性株携带肠毒素的情况是：cytK1和hblC均为阴性，只携带cytK2和/或nheB基因,，值得注意的是有35株(94.6%)ces和nheB同时存在，高于nheB在蜡样芽孢杆菌分离株中的检出率75%。多数可在10～48℃生长良好。经过HPLC-MS分析，凡含ces基因阳性的蜡样芽孢杆菌其呕吐毒素cereulide也能检出高峰值，反之，ces阴性菌株其cereulide未检出。化学定量分析和PCR结果能互相印证。
[Abstract]:Bacillus cereus, a conditional pathogen, widely exists in nature and usually causes two kinds of food poisoning of vomiting and diarrhea. In this study, the detection, isolation and identification of Bacillus cereus in 138 market rice samples were detected by MPN method, identification medium and ITS sequence analysis. 134 samples in China and 109 (81.3%) samples were detected in wax samples. Bacillus spore microbes; 107 (79.9%) detected Bacillus cereus (3 ~ 1100CFU/g), mean value of 39.1CFU/g; 7 (5.2%) detected Bacillus thuringiensis, the mean value of Bacillus thuringiensis, 1 (0.7%) detection of Bacillus fungoides, the source of bacteria containing bacteria in 3 samples of Thailand, Bacillus cereus detection rate 100%, containing 100% The detection rate of Bacillus thuringiensis was 66.7%, the amount of bacteria was 4.7CFU/g, while that of 1 samples from Vietnam was only detected by Bacillus thuringiensis, which contained 150.0CFU/g. 16.9CFU/g
There were 285 strains of Bacillus cereus, 268 strains of Bacillus cereus, 16 Bacillus thuringiensis and 1 Bacillus mycosis. There were significant differences in the content of Bacillus cereus in different climatic regions (P0.05). Bacillus cereus had a certain selectivity for the climate regions with good growth and reproduction.
PCR was used to detect the gene CES, cytotoxin K gene cytK1, cytK2, and the hblC of the haemolysin BL gene, and the detection rate of Bacillus cereus toxin in each region of non hemolytic enterotoxin gene nheB. was different. In different climate regions, the distribution difference of Bacillus cereus toxin was also very significant (P0.0001).NheB (75%) in the wax like bud. Spores are common, followed by cytK2 (55.2%) and hblC (43.3%), cytK1 detection rate is zero, and 90.3% (242/268) strains carry at least one more enterotoxin gene. That is, Bacillus cereus generally carries the enterotoxin gene. Bacillus cereus isolates can grow.18 (6.7%) at 8 to 48 centigrade and grow better at low temperature, such as 10. 111 strains (41.4%) grew well at 48 C. Most of the isolates (82.8%) had beta hemolytic rings or weak hemolytic rings, and 46 (17.2%) did not hemolysis.
The CES and cytK1 of 16 Bacillus thuringiensis were negative, and 87.5% of Bacillus thuringiensis carried cytK2, nheB and hblC. of Bacillus funcidus were negative, but had beta hemolytic ring.
37 strains (13.8%) of Bacillus cereus contain CES gene, which are distributed in 15 provinces and regions, which are much higher than those of Bacillus cereus CES abroad. These CES positive strains carry enterotoxin, both cytK1 and hblC are negative, only cytK2 and / or nheB genes are carried, and 35 (94.6%) ces and nheB exist simultaneously, higher than nheB. The detection rate of 75%. in Bacillus cereus isolates most can grow well at 10~48 C. After HPLC-MS analysis, the high peak value of the vomit toxin cereulide of Bacillus cereus containing CES gene positive can also be detected. On the contrary, the cereulide of the CES negative strain is not detected. Chemical quantitative analysis and PCR results can confirm each other.
【学位授予单位】：武汉轻工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R155.5

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