A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7

被引:106
作者
Bai, Jianfa [1 ]
Shi, Xiaorong [1 ]
Nagaraja, T. G. [1 ]
机构
[1] Kansas State Univ, Coll Vet Med, Dept Diagnost Med Pathobiol, Manhattan, KS 66506 USA
关键词
Shiga toxigenic E. coli; E. coli O157:H7; Multiplex PCR; Virulence genes; POLYMERASE-CHAIN-REACTION; FECAL SAMPLES; BOVINE FECES; O157-H7; PREVALENCE; STRAINS; IDENTIFICATION; CONTAMINATION; FEEDLOT; HARVEST;
D O I
10.1016/j.mimet.2010.05.003
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 (six!, 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. (C) 2010 Elsevier B.V. All rights reserved.
引用
收藏
页码:85 / 89
页数:5
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