Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, 1800 Denison Avenue, Manhattan, KS 66506-5606, United States.
J Microbiol Methods. 2010 Jul;82(1):85-9. doi: 10.1016/j.mimet.2010.05.003. Epub 2010 May 16.
A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 10(4)CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.
建立了一种多重 PCR 方法，用于检测大肠杆菌 O157:H7 中的 6 种主要毒力基因 fliC、stx1、stx2、eae、rfbE 和 hlyA。对这 6 种主要毒力基因的可用序列和已发表的引物进行分析，使我们能够开发出 6 基因多重 PCR 方案，同时保持每个引物对的特异性。fliC、stx1、stx2、eae、rfbE 和 hlyA 的 6 个基因的产物大小分别为 949、655、477、375、296 和 199 bp，条带均匀且明显。该方法用 221 株大肠杆菌菌株进行了验证，包括 4 株 ATCC、84 株牛源和 57 株人源大肠杆菌 O157:H7 菌株，以及 76 株非 O157 牛源和人源大肠杆菌菌株。所有 221 株菌株的结果与涉及两种独立反应检测 5 种毒力基因（stx1、stx2、eae、fliC 和 hlyA）的建立的多重 PCR 方法的结果相似。O 抗原的特异性通过仅扩增 O157 而不是 O25、O26、O55、O78、O103、O111、O127 和 O145 型大肠杆菌来表示。特异性试验表明，该方法可从经粪便样本中提取的含有至少 10(4)CFU/g（10 个细胞/反应）大肠杆菌 O157 的样品中扩增基因。在对含有大肠杆菌 O157 的样品进行 6 小时富集后，可达到 10 CFU/g 的灵敏度水平。
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