大肠菌群快速检测培养基的研制及评价

发布时间：2018-03-08 00:11

  本文选题：大肠菌群　切入点：显色培养基　出处：《郑州大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景及目的大肠菌群作为评价食品卫生质量的重要指标之一,已被广泛的应用于食品卫生领域,它不仅能反映被检测物是否被污染及受污染程度,而且在一定程度上也能反映出食品在生产、加工、运输及储存等过程中的卫生状况,其检出具有重要的微生物学意义。我国的食品卫生微生物学检验国家标准(GB4789)系列中,大肠菌群的测定常常作为必检项目。目前,对于大肠菌群的检测有很多类方法,最为广泛采用的仍是国标三步法检测,此法具有准确、可靠等优点,但检测周期长,操作繁琐,耗时费力,且不能满足快速检测的需求,所以寻求快速、准确、并且操作简单的大肠菌群检测方法,是食品安全部门亟待解决的问题。上世纪末法国科玛嘉公司发明的显色培养基技术从一定程度上解决了关于微生物常规检测中出现的难题,目前国内使用最多的是进口的大肠菌群显色培养基产品,价格昂贵,成本高,而且配制过程中对温度、实验环境等外在因素要求比较严格,因此不适用于现场检测工作。为了满足国内食品检验对大肠菌群快速检测培养基的实际需求,本课题利用微生物所具有的特异性酶及其相对应的显色底物研制出一种能快速检测大肠菌群的鉴别显色培养基,并对其实际应用价值进行评价,以期获得快速、准确、敏感度高、特异性强并且适用于实时监测工作的经济型检测产品。方法以初步筛选的普通营养琼脂平板为基础,添加促大肠菌群生长和抑制杂菌生长等物质,如琼脂、蛋白胨、酵母粉、乳糖、氯化钠、月桂基硫酸钠、磷酸二氢钾、磷酸氢二钾等,同时设计相应的酶显色底物及添加方式,通过两组正交优化实验,确定其最终配方,形成大肠菌群显色培养基平板(Coliform Chromogenic Medium)以下简称为CCM。观察培养基的显色效果,以大肠菌群标准菌株及其他食源性致病菌标准菌株、100份实际食物样品为对象；准确可靠的国标检测法为参考,从检测时间及成本、平板效率、特异性、敏感度和准确性等方面综合评价该显色培养基的性能。结果①该培养基在37℃培养18h就可直接观察菌落颜色来鉴别大肠菌群菌属,显色清晰,结果易于观察,并且检测成本明显低于现有的大肠菌群国标检测法。②该显色培养基与国标法中采用的鉴别培养基结晶紫中性红胆盐琼脂(VRBA)的出菌率即平板效率对比,差异无显著性(t=0.578,P0.05)。③该显色培养基的特异性和敏感度分别能达到100%和90%,证实具有较高的敏感度和特异性。④用实际食物样品进行评价,该方法与国标法的检测准确性比较,差异无显著性(Z2=0.125,P0.05)。结论自主研制的大肠菌群快速检测显色培养基具有快速、准确、灵敏度和特异性高等优点,可用于实际食品卫生微生物的初筛工作。
[Abstract]:Background and objective coliform bacteria, as one of the important indicators for evaluating food hygiene quality, have been widely used in the field of food hygiene, which can not only reflect whether the detected substances are contaminated or not, but also the degree of contamination. To a certain extent, it can also reflect the hygienic status of food in the process of production, processing, transportation and storage, and its detection has important microbiological significance. At present, there are many kinds of methods for coliform group detection, and the most widely used method is the national standard three-step method. This method has the advantages of accuracy and reliability, but the detection period is long and the operation is tedious. Time-consuming and difficult to meet the need for rapid detection, so seek fast, accurate, and simple operation of coliform detection methods, It is an urgent problem for food security department. In 0th century, Colour medium technology invented by Comarga Company of France solved the problem of microorganism routine detection to some extent. At present, the imported coliform coliform coliform medium products are used most in our country. They are expensive and expensive, and the external factors such as temperature and experimental environment are strictly required in the preparation process. Therefore, it is not suitable for field testing. In order to meet the actual demand of domestic food inspection for rapid detection of coliform bacteria, In this paper, the specific enzyme of microorganism and its corresponding chromogenic substrate were used to develop a medium for rapid detection of coliform bacteria, and its practical application value was evaluated in order to obtain fast and accurate coliform. Methods based on the preliminary screening of common nutrient Agar plate, we added some substances, such as promoting coliform group growth and inhibiting the growth of miscellaneous bacteria, such as Agar, peptone, etc. Yeast powder, lactose, sodium chloride, sodium lauryl sulfate, potassium dihydrogen phosphate, potassium hydrogen phosphate, and so on. At the same time, the corresponding enzyme chromogenic substrate and adding method were designed, and the final formula was determined by two groups of orthogonal optimization experiments. The formation of coliform Chromogenic medium was referred to as CCM.To observe the color effect of the medium, 100 samples of food samples from coliform standard strains and other standard foodborne pathogenic bacteria strains were taken as objects. Accurate and reliable national standard detection method for reference, from detection time and cost, plate efficiency, specificity, Results 1 the color of coliform bacteria could be directly observed at 37 鈩，

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