【摘要】：大腸桿菌O157:H7(Escherichia coli O157:H7)為一種危害嚴重的食源性致病菌,近20年來給世界各國帶來了巨大影響。本文對其進行了微生物鑒定、定性和定量聚合酶鏈式反應(PCR)以及免疫學方法的檢測技術的研究。 首先對其進行微生物學培養(yǎng)和檢測,熟悉大腸桿菌O157:H7的基本生物學特性,并掌握用山梨醇麥康凱培養(yǎng)基(SMAC)初步鑒定O157:H7的方法。 其次,針對其特異性致病基因eae,stx等設計5對引物,進行常規(guī)的PCR檢測,結果表明5對引物皆可以有效擴增且具有良好的特異性。通過優(yōu)化試驗確定其最適退火溫度為58～60℃,反應的最佳引物濃度為2μmol/l。常規(guī)PCR檢測大腸桿菌O157:H7的最低檢測限為10~3cfu/ml。并主要對其中的引物a進行基于SYBRGreen Ⅰ染料的實時熒光定量(Real-time)PCR反應,結果也取得良好的擴增,最低檢測限提高到lcfu/ml。在對Real-Time PCR進行了引物及模板濃度等的優(yōu)化反應后,得出Real-time PCR標準曲線方程。 第三,通過動物試驗得到大腸桿菌O157:H7的多克隆抗體,效價達到1:25000,建立的酶聯(lián)免疫吸附試驗(ELISA)最低檢測限達到每個反應孔4.17x10~3cfu,且與其他大腸桿菌無交叉反應。
[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，

本文編號：2194070