Produce Safety and Microbiology Research Unit, Western Regional Research Center, U.S. Department of Agriculture/Agricultural Research Service Albany, CA, USA.
Front Cell Infect Microbiol. 2012 May 11;2:61. doi: 10.3389/fcimb.2012.00061. eCollection 2012.
Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne illness worldwide. The present study developed the use of DNA microarrays with the ampliPHOX colorimetric method to rapidly detect and genotype STEC strains. A low-density 30-mer oligonucleotide DNA microarray was designed to target O-antigen gene clusters of 11 E. coli serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157) that have been associated with the majority of STEC infections. In addition, the DNA microarray targeted 11 virulence genes, encoding adhesins, cytotoxins, proteases, and receptor proteins, which have been implicated in conferring increased ability to cause disease for STEC. Results from the validation experiments demonstrated that this microarray-based colorimetric method allowed for a rapid and accurate genotyping of STEC reference strains from environmental and clinical sources and from distinct geographical locations. Positive hybridization signals were detected only for probes targeting serotype and virulence genes known to be present in the STEC reference strains. Quantification analysis indicated that the mean pixel intensities of the signal for probes targeting O-antigen or virulence genes were at least three times higher when compared to the background. Furthermore, this microarray-based colorimetric method was then employed to genotype a group of E. coli isolates from watershed sediment and animal fecal samples that were collected from an important region for leafy-vegetable production in the central coast of California. The results indicated an accurate identification of O-type and virulence genes in the tested isolates and confirmed that the ampliPHOX colorimetric method with low-density DNA microarrays enabled a fast assessment of the virulence potential of STEC using low-cost reagents and instrumentation.
产志贺毒素大肠杆菌（STEC）是全球食源性疾病的主要致病原因。本研究开发了使用 DNA 微阵列和 ampliPHOX 比色法快速检测和基因分型 STEC 菌株。设计了低密 30-mer 寡核苷酸 DNA 微阵列，以针对与大多数 STEC 感染相关的 11 种大肠杆菌血清群（O26、O45、O91、O103、O104、O111、O113、O121、O128、O145 和 O157）的 O 抗原基因簇。此外，DNA 微阵列还针对编码黏附素、细胞毒素、蛋白酶和受体蛋白的 11 种毒力基因，这些基因与 STEC 引起疾病的能力增加有关。验证实验的结果表明，这种基于微阵列的比色法可快速准确地对来自环境和临床来源以及不同地理位置的 STEC 参考菌株进行基因分型。只有针对已知存在于 STEC 参考菌株中的血清型和毒力基因的探针才能检测到阳性杂交信号。定量分析表明，与背景相比，针对 O 抗原或毒力基因的探针的信号平均像素强度至少高出三倍。此外，该基于微阵列的比色法随后用于对从加利福尼亚州中部海岸一个重要的叶菜生产区采集的流域沉积物和动物粪便样本中的一组大肠杆菌分离株进行基因分型。结果表明，在所测试的分离株中可以准确鉴定 O 型和毒力基因，并证实低密 DNA 微阵列的 ampliPHOX 比色法可使用低成本试剂和仪器快速评估 STEC 的毒力潜力。
