Detection in Escherichia coli of the Genes Encoding the Major Virulence Factors, the Genes Defining the O157:H7 Serotype, and Components of the Type 2 Shiga Toxin Family by Multiplex PCR

Abstract

Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include enterohemorrhagic E. coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E. coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control. Those genes detected were stx 1 , stx 2 , stx 2c , stx 2d , stx 2e , stx 2f , EHEC hlyA, and eaeA, as well as rfbE, which encodes the E. coli O157 serotype; fliC, which encodes the E. coli flagellum H7 serotype; and the E. coli 16S rRNA, which was included as an internal control. A total of 129 E. coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated. Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx. Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive. All STEC strains were identified by this procedure. In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method. A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion. The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E. coli while differentiating O157:H7 from non-O157 isolates.

Genetic diversity of Shiga toxin-producing Escherichia coli O157 : H7 recovered from human and food sources

“…Detection of stx 1 and stx 2 genes was performed by PCR using primer sequences and cycling conditions previously described (Botteldoorn et al, 2003) (Table S1). Isolates that showed a positive result for stx 2 were further tested for the presence of stx 2a and stx 2c by their respective PCR (Wang et al, 2002).…”

Laboratory-based surveillance of human verocytotoxigenic Escherichia coli infection in the Republic of Ireland, 2002–2004

“…VT2 is further subdivided into five subtypes VT2, VT2c, VT2d, VT2e and VT2f (Wang et al, 2002). Variants of VT1 have also been described (Paton et al, 1995); however, VT1 is more homogeneous than VT2.…”

Campylobacter jejuni infection and virulence-associated genes in children with moderate to severe diarrhoea admitted to emergency rooms in northeastern Brazil

“…In order to certify the quality of our extraction procedures, we performed an internal control PCR for all DNA extracted directly from faecal aliquots just before the PCR diagnosis procedures. This internal control PCR amplified the rrs (16S rRNA) gene (Wang et al, 2002; Brandal et al, 2007). Positive control DNA was extracted from C. jejuni ATCC 33291 and C. coli INCQS 00263 (kindly provided by Oswaldo Cruz Foundation, Rio de Janeiro, Brazil).…”