MPN-PCR法检测鸡肉中空肠弯曲杆菌和产气荚膜梭菌

发布时间：2018-05-10 13:55

本文选题：空肠弯曲杆菌 + 产气荚膜梭菌　；参考：《安徽农业大学》2015年硕士论文

【摘要】：随着人类生活水平的不断提高,禽类产品在人类食物中所占的比重日益加大,然而空肠弯曲杆菌和产气荚膜梭菌等人畜共患病原菌可污染禽类产品,引起人类细菌性疾病,且其发生呈逐渐增长趋势。空肠弯曲杆菌和产气荚膜梭菌不仅严重影响养禽业的发展,造成经济损失,同时也是造成人类胃肠炎等疾病的主要病原菌,严重威胁人类健康。目前,对食品中食源性致病菌定量检测的方法主要为活菌平板计数,耗时费力,很难计算准确的数量;MPN法(最大可能数)需要进行生化鉴定,同样操作繁琐,耗时费力;而(Real-time)PCR检测则无法判断样品中的细菌是否为活菌。因此,研究旨在建立针对空肠弯曲杆菌和产气荚膜梭菌的MPN-PCR检测方法,并初步应用于鸡肉制品中这两种病原菌的快速定量检测。首先,通过查阅相关文献,根据空肠弯曲杆菌特有的马尿酸酶hipO基因和产气荚膜梭菌各基因型均编码的cpa基因设计特异性引物,将PCR技术与MPN法相结合,建立了MPN-PCR快速定量检测方法,用于定量检测鸡肉制品中的空肠弯曲杆菌和产气荚膜梭菌。MPN-PCR方法的整个检测过程包括:样品处理、增菌培养、模板制备、PCR扩增,实现了对食品中病原菌的快速定量检测。用PCR技术替代了生化试验,不仅可以进行定量检测,还可以避免了由于细菌进入VBNC(活的但非可培养)状态所造成的漏检,并且检测时间至少缩短了两天以上。其次,应用建立的MPN-PCR定量检测方法,对采集的186份鸡肉样品进行实际应用,其中鲜鸡肉66份,冻鸡肉120份。结果:空肠弯曲杆菌的阳性检出率为17.2%,鲜鸡肉的阳性检出率为28.79%,冻鸡肉的阳性检出率为10.83%,含菌量范围在3.6-92 MPN/g;产气荚膜梭菌的阳性检出率为15.59%,鲜鸡肉的阳性检出率为13.64%,冻鸡肉的阳性检出率为16.67%,含菌量范围在3.6-360 MPN/g。综上所述,试验建立的空肠弯曲杆菌和产气荚膜梭菌MPN-PCR检测方法,具有特异性强、敏感性高、相对快速、高效等特点,能为养禽业中疾病的防控和禽肉类加工销售过程的监管提供有效的工具,对食品公共卫生安全领域中食源性病原菌的检测提供一定的帮助。
[Abstract]:With the improvement of human living standards, poultry products account for an increasing proportion of human food. However, zoonotic pathogens such as Campylobacter jejuni and Clostridium perfringens can pollute poultry products and cause human bacterial diseases. And its occurrence shows a gradual increase trend. Campylobacter jejuni and Clostridium perfringens not only seriously affect the development of poultry industry, resulting in economic losses, but also are the main pathogens causing gastroenteritis and other diseases, which seriously threaten human health. At present, the main method for quantitative detection of foodborne pathogenic bacteria in food is the counting of live bacteria, which is time-consuming and laborious, so it is difficult to calculate the exact quantity of MPN (maximum possible number), which requires biochemical identification, and the same operation is cumbersome and time-consuming. Real-time PCR could not determine whether the bacteria in the sample were living bacteria. Therefore, the aim of this study was to establish a MPN-PCR method for the detection of Campylobacter jejuni and Clostridium perfringens, and to apply them to the rapid quantitative detection of these two pathogens in chicken products. Firstly, specific primers were designed according to the specific cpa gene encoding the hipO gene of Campylobacter jejuni and the cpa gene of Clostridium perfringens. The PCR technique was combined with the MPN method. A rapid and quantitative MPN-PCR method was established for quantitative detection of Campylobacter jejuni and Clostridium perfringens. The rapid quantitative detection of pathogenic bacteria in food was realized. The biochemical test can be replaced by PCR technique, which can not only detect quantitatively, but also avoid the missed detection caused by bacteria entering the state of VBNC (living but not culturable), and the detection time is shortened by more than two days. Secondly, 66 fresh chicken samples and 120 frozen chicken samples were tested by the established MPN-PCR quantitative detection method. Results: the positive rates of Campylobacter jejuni, fresh chicken and frozen chicken were 17.2, 28.79, 10.83, 3.6-92 MPN / g, 15.59 and 10.83, respectively. The positive rate of frozen chicken was 16.67 and the amount of bacteria was in the range of 3.6-360 MPN / g. In conclusion, the MPN-PCR method for detection of Campylobacter jejuni and Clostridium perfringens has the characteristics of high specificity, high sensitivity, relatively fast and high efficiency. It can provide effective tools for the prevention and control of diseases in poultry industry and the supervision of poultry meat processing and marketing process. It can also provide some help for the detection of foodborne pathogens in the field of food public health and safety.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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