【摘要】：大腸桿菌O157:H7(Escherichia coli O157:H7)為一種危害嚴(yán)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(duì)其進(jìn)行了微生物鑒定、定性和定量聚合酶鏈?zhǔn)椒磻?yīng)(PCR)以及免疫學(xué)方法的檢測(cè)技術(shù)的研究。 首先對(duì)其進(jìn)行微生物學(xué)培養(yǎng)和檢測(cè),熟悉大腸桿菌O157:H7的基本生物學(xué)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(duì)其特異性致病基因eae,stx等設(shè)計(jì)5對(duì)引物,進(jìn)行常規(guī)的PCR檢測(cè),結(jié)果表明5對(duì)引物皆可以有效擴(kuò)增且具有良好的特異性。通過優(yōu)化試驗(yàn)確定其最適退火溫度為58～60℃,反應(yīng)的最佳引物濃度為2μmol/l。常規(guī)PCR檢測(cè)大腸桿菌O157:H7的最低檢測(cè)限為10~3cfu/ml。并主要對(duì)其中的引物a進(jìn)行基于SYBRGreen Ⅰ染料的實(shí)時(shí)熒光定量(Real-time)PCR反應(yīng),結(jié)果也取得良好的擴(kuò)增,最低檢測(cè)限提高到lcfu/ml。在對(duì)Real-Time PCR進(jìn)行了引物及模板濃度等的優(yōu)化反應(yīng)后,得出Real-time PCR標(biāo)準(zhǔn)曲線方程。 第三,通過動(dòng)物試驗(yàn)得到大腸桿菌O157:H7的多克隆抗體,效價(jià)達(dá)到1:25000,建立的酶聯(lián)免疫吸附試驗(yàn)(ELISA)最低檢測(cè)限達(dá)到每個(gè)反應(yīng)孔4.17x10~3cfu,且與其他大腸桿菌無交叉反應(yīng)。
[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(hào)：2194070