A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory.
A multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 10(2)-10(3) DEC CFU/PCR in the presence of 10(6) non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 10(6) CFU/mL stool sample.
Persson
S
S
Department of Bacteriology, Mycology and Parasitology, Unit of Gastrointestinal Infection, Statens Serum Institut, Copenhagen, Denmark. SPN@ssi.dk
Olsen
K E P
KE
Scheutz
F
F
Krogfelt
K A
KA
Gerner-Smidt
P
P
D004403:Dysentery / Q000175:diagnosis / Q000235:genetics* / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics*; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics; D022641:Shiga Toxin 2; D022603:Shiga Toxins / Q000235:genetics
D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D022603:Shiga Toxins; C430449:verocytotoxin 2