A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli
A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli
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DOI：
10.1016/j.mcp.2011.08.004
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发表时间：
2011-10-01
影响因子：
3.3
通讯作者：
Doepfer, Doerte
中科院分区：
文献类型：
--
作者：
Gonzales, Tina K.;Kulow, Megan;Doepfer, Doerte
肠出血性大肠杆菌（EHEC），包括O157和非O157血清型是重要的食源性病原体，需要敏感和区分的方法来检测和表征。有许多基于pcr的方法用于检测肠出血性大肠杆菌的毒力因子，但大规模筛查工作和人群水平分析所涉及的时间和成本限制了研究的规模和范围。最近的技术进步将微阵列的高通量性能与实时qPCR的特异性和敏感性相结合，使大规模筛选工作既省时又经济。本研究鉴定和评估了28个遗传标记，包括已知的毒力和调控基因、o抗原基因，并选择了O157和非O157肠出血性大肠杆菌的噬菌体前体区域，这些区域可用于高通量PCR对大量分离株进行病毒分型、血清分型和初步分型。使用多种提取方法和PCR平台，对目标基因的PCR分析显示是稳健的。初步定量PCR结果显示，可检出10(4)CFU/ml以下的肠出血性大肠杆菌，检测线性范围在5 ~ 6个数量级。由28个靶基因组成的小组有可能成为肠出血性大肠杆菌暴发、环境和遗传调查中不可或缺的工具。(C) 2011出版的爱思唯尔有限公司
Enterohemorrhagic Escherichia coli (EHEC), including O157 and non-O157 serotypes are significant foodborne pathogens that require sensitive and discriminatory methods for detection and characterization. There are numerous PCR-based methods for the detection of EHEC virulence factors, but the time and cost involved with large-scale screening efforts and population level analyses have limited the size and scope of studies. Recent technological advancements have combined the high-throughput performance of the microarray with the specificity and sensitivity of real-time qPCR to make large-scale screening efforts both time- and cost-effective. This study identified and evaluated a panel of 28 genetic markers including known virulence and regulatory genes, O-antigen genes, and select prophage regions of O157 and non-O157 EHEC that can be used with high-throughput PCR to virulotype, serotype, and preliminarily subtype large numbers of isolates. The PCR assays for the target genes were shown to be robust using multiple extraction methods and PCR platforms. Preliminary quantitative PCR showed that an EHEC concentration of 10(4) CFU/ml or lower could be detected, with a linear range of detection over five to six orders of magnitude. The panel of 28 target genes has the potential to become an integral tool in outbreak, environmental, and genetic investigations of EHEC. (C) 2011 Published by Elsevier Ltd.