应用实时荧光定量PCR技术检测婴幼儿奶粉中阪崎肠杆菌（克罗诺杆菌属）的研究

发布时间：2018-07-17 00:46

【摘要】：阪崎肠杆菌(Enterbacter sakazakii),是寄生在人和动物肠道内的一种革兰氏阴性无芽孢杆菌,周身鞭毛、能运动、兼性厌氧。该菌能引起严重的脑膜炎、坏死性小肠结肠炎和菌血症等病症,可感染各年龄段免疫力低下的人群,尤其是新生儿、早产儿及出生时身体条件较差的婴儿,感染后婴儿死亡率高达50%以上,是一种重要的食源性条件致病菌。近年来,国内外越来越多的研究表明:婴幼儿配方奶粉是引起婴幼儿感染的主要传染源和传播媒介。目前,GB法检测阪崎肠杆菌主要是基于传统的微生物培养,然后综合菌落形态、生化特征等进行鉴定。此法操作繁琐、耗时耗力、且不易分辨结果,存在一定假阳性。所以近年来,采用分子生物学的方法检测食源性病菌得到越来越广泛的应用。本论文基于中华人民共和国出入境检验检疫行业标准(SN/T 1632.3-2013)中阪崎肠杆菌(克罗诺杆菌属)实时荧光定量PCR检验方法,首先将PCR反应各组分预混成Mix Buffer,大大简化了操作步骤,同时对各组分浓度及反应体系进行了优化,实现阪崎肠杆菌Real-time PCR的快速检测。其次,通过对比酚氯仿和加热裂解两种DNA提取方法,对加热裂解法进行了优化,并用贝因美奶粉样本和人工污染样本进行了验证。人工污染奶粉的阪崎肠杆菌的检出限可达4.7× 103CFU/ml。本文中所有实时荧光定量PCR实验都是在博日公司自主研发的实时荧光定量PCR仪器平台和博日专利产品“标准PCR实验室”内完成,从而初步建立了“试剂、仪器、软件、PCR污染防控”为一体的阪崎肠杆菌快速检测系统,为客户提供从硬件到软件,从实验操作到环境污染控制的整体解决方案,实现“稳定”的快速检测。
[Abstract]:Enterbacter sakazakii), is a gram-negative bacillus that is parasitic in human and animal intestines. The bacteria can cause severe meningitis, necrotizing enterocolitis and bacteremia, and can infect people of all ages with low immunity, especially newborns, premature infants and babies with poor physical conditions at birth. The infant mortality rate after infection is over 50%, which is an important food-borne conditional pathogen. In recent years, more and more studies at home and abroad show that infant formula is the main source of infection and transmission media. At present, the detection of Enterobacter sakazakii by GB method is mainly based on the traditional microbial culture, and then comprehensive colony morphology, biochemical characteristics and so on. This method is complicated, time-consuming, and difficult to distinguish the results, there is a certain false positive. Therefore, molecular biology has been used to detect foodborne bacteria more and more widely in recent years. Based on the real-time fluorescence quantitative PCR method of Enterobacter sakazakii (Crotonia) in the Entry-Exit Inspection and Quarantine Industry Standard of the people's Republic of China (SNR / T 1632.3-2013), the PCR reaction components are premixed into mix buffer, which greatly simplifies the operation procedure. At the same time, the concentration of each component and reaction system were optimized to realize the rapid detection of Enterobacter sakazakii Real-time PCR. Secondly, by comparing phenol chloroform and heating cracking two DNA extraction methods, the heating pyrolysis method was optimized and verified by Beimei milk powder samples and artificially contaminated samples. The detection limit of Enterobacter sakazakii in artificially contaminated milk powder was 4.7 脳 103 CFU / ml. In this paper, all the real-time fluorescent quantitative PCR experiments were carried out in the real-time fluorescent quantitative PCR instrument platform developed by Boji Company and the standard PCR laboratory, which is the patented product of Boji Company. Thus, the "reagent, instrument" was preliminarily established. The rapid detection system of Enterobacter sakazakii which is integrated with PCR pollution prevention and control software provides the whole solution from hardware to software, from experimental operation to environmental pollution control, and realizes the "stable" rapid detection of Enterobacter sakazakii.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS252.51;O657.3

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