肠出血性大肠杆菌O157：H7的PCR与免疫学检测技术的研究

发布时间：2018-08-20 15:05

【摘要】：大肠杆菌O157:H7(Escherichia coli O157:H7)为一种危害严重的食源性致病菌,近20年来给世界各国带来了巨大影响。本文对其进行了微生物鉴定、定性和定量聚合酶链式反应(PCR)以及免疫学方法的检测技术的研究。 首先对其进行微生物学培养和检测,熟悉大肠杆菌O157:H7的基本生物学特性,并掌握用山梨醇麦康凯培养基(SMAC)初步鉴定O157:H7的方法。 其次,针对其特异性致病基因eae,stx等设计5对引物,进行常规的PCR检测,结果表明5对引物皆可以有效扩增且具有良好的特异性。通过优化试验确定其最适退火温度为58～60℃,反应的最佳引物浓度为2μmol/l。常规PCR检测大肠杆菌O157:H7的最低检测限为10~3cfu/ml。并主要对其中的引物a进行基于SYBRGreen Ⅰ染料的实时荧光定量(Real-time)PCR反应,结果也取得良好的扩增,最低检测限提高到lcfu/ml。在对Real-Time PCR进行了引物及模板浓度等的优化反应后,得出Real-time PCR标准曲线方程。 第三,通过动物试验得到大肠杆菌O157:H7的多克隆抗体,效价达到1:25000,建立的酶联免疫吸附试验(ELISA)最低检测限达到每个反应孔4.17x10~3cfu,且与其他大肠杆菌无交叉反应。
[Abstract]:Escherichia coli O157:H7 (Escherichia coli) is a serious foodborne pathogen, which has had a great impact on many countries in the world in recent 20 years. In this paper, microbial identification, qualitative and quantitative polymerase chain reaction (PCR) and immunological methods were studied. Firstly, microbiological culture and detection were carried out, and the basic biological characteristics of Escherichia coli O157:H7 were familiar, and the method of identifying O157:H7 with sorbitol wheat Kang Kai medium (SMAC) was mastered. Secondly, five pairs of primers were designed for the specific pathogenicity gene eaestrx. The results showed that all the primers could be amplified effectively and had good specificity. The optimum annealing temperature was 58 ~ 60 鈩，

本文编号：2194070