Detection of enterohemorrhagic Escherichia coli O157:H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins

Authors

Vijay K. Sharma, USDA-ARSFollow
Evelyn A. Dean-Nystrom, Pre-Harvest Food Safety and Enteric Diseases Research UnitFollow

Document Type

Article

Date of this Version

2003

Citation

Veterinary Microbiology 93 (2003) 247–260; doi:10.1016/S0378-1135(03)00039-7

Abstract

A multiplex real-time PCR (R-PCR) assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan) to detect enterohemorrhagic Escherichia coli (EHEC) O157:H7 in pure cultures, feces, and tissues. Three sets of primers and fluorogenic probes were used for amplification and real-time detection of a 106-bp region of the eae gene encoding EHEC O157:H7-specific intimin, and 150-bp and 200-bp segments of genes stx1 and stx2 encoding Shiga toxins 1 and 2, respectively. Analysis of 67 bacterial strains demonstrated that the R-PCR assay successfully distinguished EHEC O157:H7 serotype from non-O157 serotypes and provided accurate profiling of genes encoding intimin and Shiga toxins. Bacterial strains lacking these genes were not detected with this assay. The detection range of the R-PCR assay for the three genes was linear over DNA concentrations corresponding from 10³ to 10⁸ CFU/ml of EHEC O157:H7. The R-PCR allowed construction of standard curves that facilitated quantification of EHEC O157:H7 in feces and intestinal tissues. Detection sensitivity of the R-PCR assay ranged from 10⁴ to 10⁸ CFU/g of feces or tissues without enrichment. Enrichment of feces in a non-selective broth for 4 and 16 h resulted in the detection of levels (from 10⁰ to 10³ CFU/g of feces) considered sufficient for infection in humans. The R-PCR assay for eae_O157:H7, stx1, and stx2 proved to be a rapid test for detection of EHEC O157:H7 in complex biological matrices and could also potentially be used for quantification of EHEC O157:H7 in foods or fecal samples.