【作者】 李洋洋；

【导师】 张伟；

【作者基本信息】 河北农业大学 ， 微生物学， 2012， 硕士

【摘要】 食源性疾病是食品安全的主要问题,时刻威胁着人们的健康和生命。在食源性疾病中,细菌性食物中毒居首位。近年来,阪崎肠杆菌、金黄色葡萄球菌、蜡样芽胞杆菌等病原菌引起的奶粉中毒事件时有发生。因此,食源性病原菌的检测是食品卫生安全检测中一个重要的部分。目前,对食品中病原菌的检测方法主要有传统的分离鉴定方法、免疫学方法和分子生物学方法。其中,传统检测方法操作繁琐,检测时间较长,灵敏度较低;免疫学方法的特异性和灵敏度不是很理想;分子生物学方法特异性较强、检测时间较短、灵敏度较高。三重PCR是一种在同一反应体系中加入三对特异性引物,同时扩增出三条目的DNA片段的扩增方法。本文根据阪崎肠杆菌的外膜蛋白基因ompA、金黄色葡萄球菌的耐热核酸酶基因nuc、蜡样芽胞杆菌的溶血素基因hblA设计了3对特异性引物,进行三重PCR反应,可实现对阪崎肠杆菌、金黄色葡萄球菌、蜡样芽胞杆菌三种食源性病原菌的同时检测。对15株菌进行检测,结果4株阪崎肠杆菌、2株金黄色葡萄球菌、1株蜡样芽胞杆菌均扩增出了特异性的目的条带,而另外的8株其它病原菌均无扩增条带。将阪崎肠杆菌、金黄色葡萄球菌、蜡样芽胞杆菌的扩增产物测序序列在GeneBank上进行BLAST比对,同源性均达到99%以上。试验过程中通过对退火温度、3对引物、Mg²⁺、dNTPs、EasyTaq DNA Polymerase等参数的优化,确定了三重PCR的反应体系和反应程序,其反应体系为:10×Easy Taq Buffer(-Mg²⁺)2.5μL,2.5 mM dNTPs 2.5μL,50 mM MgSO4 1.5μL,10μM ompA上下游引物各0.6μL、10μM nuc上下游引物各1.0μL、10μM hblA上下游引物各1.0μL,模板DNA各2μL,5 U/μL EasyTaq DNA Polymerase 0.3μL,ddH2O补足25μL;反应程序为:95℃预变性5 min;按照94℃45 s,61℃45 s,72℃45 s进行30个循环;最后72℃延伸10 min。将阪崎肠杆菌、金黄色葡萄球菌、蜡样芽胞杆菌三种病原菌随机组合,均能扩增出特异性的目的条带。在最佳试验条件下,该三重PCR方法同时检测阪崎肠杆菌、金黄色葡萄球菌、蜡样芽胞杆菌三种病原菌纯培养的灵敏度是103 CFU/mL。采用无水乙醇、氨水、石油醚与试剂盒相结合的方法提取DNA,该三重PCR方法同时检测阪崎肠杆菌、金黄色葡萄球菌、蜡样芽胞杆菌三种病原菌在婴幼儿奶粉中的检出限是104 CFU/g。对实际样品进行检测,并将三重PCR方法与国标方法进行比较,三重PCR方法对阪崎肠杆菌、金黄色葡萄球菌、蜡样芽胞杆菌三种病原菌的敏感性均为100%;特异性分别为96.4%、96.6%、100%;符合率分别为96.7%、96.7%、100%。本研究以婴幼儿奶粉中出现过的阪崎肠杆菌、金黄色葡萄球菌和蜡样芽胞杆菌为对象,建立了检测这三种病原菌的三重PCR方法,为同时检测食品中的阪崎肠杆菌、金黄色葡萄球菌、蜡样芽胞杆菌提供了新方法。更多还原

【Abstract】 Food-borne diseases constantly threatening people’s health and lives is a major public health problem in food security. Especially, bacterial food poisoning is the main factor which causes the food poisoning. At present, the contaminated infant milk powder scandal owing to Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus happens occasionally. So the detection of food-borne pathogenic bacteria is a great of part in food and health security.At present, the detection of food-borne pathogenic bacteria mainly uses the traditional isolation and biochemical identification method, the immunological methods and the methods in molecular biology. It is complicated operation, time consuming and low-sensitive for the traditional isolation and biochemical identification method. The specificity and sensibility of immunological methods are unsatisfactory. The methods in molecular biology are higher-specific, shorter-time and higher-sensitive.Triplex PCR is an amplification method that can amplify three target sequences through adding to three pairs of specific primers. In this study, three pairs of specific primers were designed according to Enterobacter sakazakii ompA gene, Staphylococcus aureus nuc gene and Bacillus cereus hblA gene and triplex PCR was established to simultaneous diagnose three kinds of food-borne pathogens. The detecting results of 15 strains of bacteria are that 4 strains of Enterobacter sakazakii, 2 strains of Staphylococcus aureus and 1 strain of Bacillus cereus had amplified specific target channels, and another 8 strains of bacteria could not have amplified channels. The sequencing results of Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus were compared with the target sequences at GenBank and homologies of three pathogenic bacteria were all above 99%. The reaction system and reaction procedure of triplex PCR were determined through optimizing annealing temperature, three pairs of primers, Mg²⁺, dNTPs and EasyTaq DNA Polymerase. The reaction system was 25μL: 10×Easy Taq Buffer(-Mg²⁺) 2.5μL, 2.5 mM dNTPs 2.5μL, 50 mM MgSO4 1.5μL, each 10μM ompA primer 0.6μL, each 10μM nuc primer 1.0μL, each 10μM hblA primer 1.0μL, each template DNA 2μL, 5 U/μL EasyTaq DNA Polymerase 0.3μL, ddH20 7μL. The reaction procedure was pre-degenerated at 95℃for 5 min, degenerated at 94℃for 45 s, annealing at 61℃for 45 s, extention at 72℃for 45 s, run 30 cycles, final extention at 72℃for 10 min.Specific target channels could be amplified with random combinations of Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus. Under the above optimum experiment condition, the sensitivities of the triplex PCR for Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus were 103 CFU/mL. Extracting genomic DNA with absolute ethyl alcohol, ammonia water, petroleum ether and kit extraction, the detection limits of the triplex PCR for Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus in infant milk powder were 104 CFU/g. Compared with the national standard method, the sensitivities of triplex PCR to detect the actual samples for the three pathogenic bacteria were all 100%; the specificities of triplex PCR to detect the actual samples for the three pathogenic bacteria were 96.4%, 96.6%, 100%, respectively; the coincidence rate of triplex PCR to detect the actual samples for the three pathogenic bacteria were 96.7%, 96.7%, 100%, respectively.In the study, triplex PCR assay had been established according to the food-borne pathogenic bacteria appeared in infant milk powder and it would provide a new method to simultaneously detect Enterobacter sakazakii, Staphylococcus aureus and Bacillus cereus.更多还原