Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157

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Journal of Clinical Microbiology, February 1998, p. 598-602, Vol. 36, No. 2
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stx₁, stx₂, eaeA, Enterohemorrhagic E. coli hlyA, rfb_O111, and rfb_O157

Adrienne W. Paton and James C. Paton^*

Molecular Microbiology Unit, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia

Received 16 June 1997/Returned for modification 21 October 1997/Accepted 18 November 1997

Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of organisms capable of causing severe gastrointestinaldisease in humans. Within the STEC family, certain strains appearto be of greater virulence for humans, for example, those belongingto serogroups O111 and O157 and those with particular combinationsof other putative virulence traits. We have developed two multiplexPCR assays for the detection and genetic characterization of STECin cultures of feces or foodstuffs. Assay 1 utilizes four PCRprimer pairs and detects the presence of stx₁, stx₂ (includingvariants of stx₂), eaeA, and enterohemorrhagic E. coli hlyA, generatingamplification products of 180, 255, 384, and 534 bp, respectively.Assay 2 uses two primer pairs specific for portions of the rfb(O-antigen-encoding) regions of E. coli serotypes O157 and O111,generating PCR products of 259 and 406 bp, respectively. The twoassays were validated by testing 52 previously characterized STECstrains and observing 100% agreement with previous results. Moreover,assay 2 did not give a false-positive O157 reaction with enteropathogenicE. coli strains belonging to clonally related serogroup O55. Assays1 and 2 detected STEC of the appropriate genotype in primary fecalcultures from five patients with hemolytic-uremic syndrome andthree with bloody diarrhea. Thirty-one other primary fecal culturesfrom patients without evidence of STEC infection were negative.

^* Corresponding author. Mailing address: Molecular Microbiology Unit, Women's and Children's Hospital, North Adelaide, S.A.5006, Australia. Phone: 61-8-8204 6302. Fax: 61-8-8204 6051. E-mail:patonj{at}wch.sa.gov.au.

Journal of Clinical Microbiology, February 1998, p. 598-602, Vol. 36, No. 2
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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