A PCR test for detecting Escherichia coli O157:H7 based on the identification of the small inserted locus (SIL O157 ) S. Perelle, P. Fach, F. Dilasser and J. Grout Agence Franc ¸aise de Se´curite´ Sanitaire des Aliments (AFSSA), Laboratoire d’Etudes et de Recherches sur l’Hygie`ne et la Qualite´ des Aliments, Unite´: Atelier de Biotechnologie, Maisons-Alfort, France 321/10/01: received 19 October 2001, revised 22 February 2002 and accepted 10 April 2002 S. PERELLE, P. FACH, F. DILASSER AND J. GROUT. 2002. Aims: This paper provides identification of a DNA sequence derived from Shiga toxin- producing Escherichia coli (STEC) O157:H7 and information on its utilization for detecting STEC O157 by PCR. Methods and Results: Random Amplified Polymorphic DNA and DNA library were used to identify in STEC O157:H7 (strain EDL 933) a 2634-bp Small Inserted Locus, designated SIL O157 . Analysis of 211 bacterial strains showed that the PCR assays amplifying the SIL O157 region could be used to detect STEC O157 with a good specificity. Conclusions: Characterization of a novel locus in STEC O157 is attractive since the serotype O157:H7 of STEC is still by far the most important serotype associated with more serious diseases. This island encodes putative proteins and especially one that is predicted to be an outer membrane protein designated IHP1. Significance and Impact of the Study: Further investigations could now be developed to appreciate the role of the SIL O157 in pathogenicity. INTRODUCTION Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen, which has been associated with a number of outbreaks. STEC strains belonging to a very diverse range of serotypes cause several disease syndromes, including diarrhoea, haemorrhagic colitis, hae- molytic uraemic syndrome, thrombotic thrombocytopenic purpura and in some cases can be fatal (Griffin and Tauxe 1991; Nataro and Kaper 1998; Paton and Paton 1998a). The main pathogenic property of STEC strains is the production of Shiga toxins (Stx) (Calderwood et al. 1987), but other bacterial virulence factors may play a role in the pathological process. The chromosomal locus of enterocyte effacement (LEE) encodes proteins involved in bacterial adhesion and cytoskeletal damage of intestinal cells (Jerse et al. 1990; Beebakhee et al. 1992; McDaniel et al. 1995; McDaniel and Kaper 1997). Recently, efa1, which encodes a novel virulence-associated determinant of attaching-effacing E. coli, has been described (Nicholls et al. 2000). Moreover, a 90-kbp plasmid (Burland et al. 1998) encodes potential virulence factors, such as an enterohaemolysin (Schmidt et al. 1995a, 1996), a catalase-peroxidase (Brunder et al. 1996), a serine protease (Brunder et al. 1997) and a type II secretion pathway system (Schmidt et al. 1997). Among the STEC group, the serotype O157:H7 is a dominant serotype in many parts of the world. Different methods have been developed for the detection of E. coli O157:H7 using several selective differentiating media, immunological assays, and DNA probes (Feng 1993; Huck et al. 1995; Nataro and Kaper 1998; Paton and Paton 1998a). The cultural methods have exploited the biochemical characteristics of the O157:H7 strains, which do not produce b-glucuronidase and usually do not ferment D-sorbitol rapidly. The agar medium most commonly used for isolation of E. coli O157:H7 is the sorbitol-MacConkey (SMAC) agar containing cefixime and tellurite (Nataro and Kaper 1998). It permits the growth of Stx-producing E. coli O157:H7 and inhibits the growth of most of the other E. coli strains, especially nontoxigenic E. coli O157 of H-types other than H7, which could be found in human infections, foods and animals. The disadvantage of these cultural methods is that they do not differentiate between toxigenic and stx-negative Correspondence to: Sylvie Perelle, Agence Franc ¸aise de Se´curite´Sanitaire des Aliments (AFSSA), Laboratoire d’Etudes et de Recherches sur l’Hygie`ne et la Qualite´des Aliments, Unite´: Atelier de Biotechnologie, 1–5 rue de Belfort, 94700 Maisons-Alfort, France (e-mail: s.perelle@afssa.fr). ª 2002 The Society for Applied Microbiology Journal of Applied Microbiology 2002, 93, 250–260