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首页> 外文学位 >Assessment of molecular virulence gene profiling and antibodies for rapid detection of pathogenic Escherichia coli isolates.
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Assessment of molecular virulence gene profiling and antibodies for rapid detection of pathogenic Escherichia coli isolates.

机译：评估分子毒力基因图谱和抗体以快速检测病原性大肠杆菌分离株。

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In recent years, there have been several fatal Escherichia coli O157:H7 outbreaks in the United States. Therefore, precise detection and identification of virulent foodborne pathogens such as E. coli O157:H7 are now a high priority for food companies and environmental agencies alike. Methods used for this purpose should ideally utilize molecular biological tools that are precise, selective, convenient, rapid and automated. The objectives of this study were: (1) to utilize the TagManRTM PCR system (Applied Biosystems) to assess the presence of virulence genes in both foodborne and environmental E. coli isolates, (2) to assess the presence of E. coli toxin genes (encoding STI, LTI and LTII) in the environment, and (3) to characterize monoclonal antibodies (3C4A11 and 9C9C4) to E. coli O157:H7. TagMan RTM pathogen detection kits (O157:H7, stxI and stxII) correctly identified 54 foodborne E. coli O157:H7 isolates. Ninety (15%) out of 604 environmental isolates gave positive amplification with an O157:H7-specific kit. TagManRTM PCR amplification products from these ninety environmental isolates were analyzed by agarose gel electrophoresis, and 90% (81/90) of these samples contained the expected PCR product. Serotyping and ribotyping data indicate TagManRTM pathogen detection kits were useful for identification of pre-screened foodborne E. coli O157:H7 isolates but may give false positive results with environmental isolates. These 90 isolates also were tested for the presence of ETEC-specific toxin genes STI, LTI and LTII. These genes were present in the environmental isolates at a frequency of 1.1% (STI and LTII) and 54.4% (LTI). Neither 3C4A11 nor 9C9C4 antibodies reacted preferentially with E. coli strains by ELISA. In Western blot experiments, 3C4A11 reacted with two protein bands of approximately 45 and 60 kDa in both 204P and non-pathogenic E. coli K12. The N-terminal sequence data from the 45kDa band indicated that this protein was either a putative E. coli flippase or BetU, a glycine betaine transporter. The molecular biological methods used in this study were indeed rapid and sensitive regarding their abilities to detect E. coli O157:H7 isolates, but should be used in conjunction with each other before a presumptive positive result is reported.
机译：近年来，美国发生了几起致命的大肠杆菌O157：H7疫情。因此，对于食品公司和环境机构来说，精确检测和鉴定有毒的食源性病原体（例如大肠杆菌O157：H7）已成为当务之急。理想地，用于此目的的方法应利用精确，选择性，方便，快速和自动化的分子生物学工具。这项研究的目的是：（1）利用TagManRTM PCR系统（Applied Biosystems）评估食源性和环境性大肠杆菌分离物中毒力基因的存在，（2）评估大肠杆菌毒素基因的存在（在环境中编码STI，LTI和LTII）（3）表征针对大肠杆菌O157：H7的单克隆抗体（3C4A11和9C9C4）。 TagMan RTM病原体检测试剂盒（O157：H7，stxI和stxII）正确鉴定了54种食源性大肠杆菌O157：H7分离株。使用O157：H7特异性试剂盒，在604种环境分离物中，有90种（占15％）得到了阳性扩增。通过琼脂糖凝胶电泳分析了来自这90个环境分离株的TagManRTM PCR扩增产物，其中90％（81/90）的样品含有预期的PCR产物。血清分型和核糖分型数据表明，TagManRTM病原体检测试剂盒可用于鉴定预先筛选的食源性大肠杆菌O157：H7分离株，但对于环境分离株可能给出假阳性结果。还测试了这90个分离株是否存在ETEC特异性毒素基因STI，LTI和LTII。这些基因在环境分离物中的出现频率为1.1％（STI和LTII）和54.4％（LTI）。 3C4A11和9C9C4抗体均未通过ELISA与大肠杆菌菌株优先反应。在蛋白质印迹实验中，3C4A11在204P和非致病性大肠杆菌K12中与两条分别约为45和60 kDa的蛋白带反应。来自45kDa条带的N端序列数据表明该蛋白是推定的大肠杆菌翻转酶或甘氨酸甜菜碱转运蛋白BetU。这项研究中使用的分子生物学方法对于检测大肠杆菌O157：H7分离物的能力确实是快速和敏感的，但是在报告推测阳性结果之前应相互结合使用。

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