Comparison of different PCR tests for detecting Shiga toxin-producing Escherichia coli O157 and development of an ELISA-PCR assay for specific identification of the bacteria.
In an attempt to develop a standard for ELISA-PCR detection of Shiga toxin producing Escherichia coli (STEC) O157, six published PCR tests were tested in a comparative study on a panel of 277 bacterial strains isolated from foods, animals and humans. These tests were based on the detection of the genes rfbE [J. Clin. Microbiol. 36 (1998) 1801] and rfbB [Appl. Environ. Microbiol. 65 (1999) 2954], the 3' end of the eae gene [Epidemiol. Infect. 112 (1994) 449], the region immediately flanking the 5' end of the eae gene [Int. J. Food. Microbiol. 32 (1996) 103], the flicH7 gene [J. Clin. Microbiol. 35 (1997) 656], or a part of the recently described 2634-bp Small Inserted Locus (SILO(157) locus) of STEC O157 [J. Appl. Microbiol. 93 (2002) 250]. Unlike the other PCR assays, those amplifying the rfb sequences were unable to distinguish toxigenic from nontoxigenic O157. These assays were relatively specific to STEC O157, giving essentially a cross reaction with clonally related E. coli O55 and to a lesser extent with E. coli O145, O125, O126. They also detected the Shiga toxin (stx)-negative derivatives of STEC O157. Based on these results, an ELISA-PCR assay consisting of the solution hybridization of amplicons with two probes that ensured the specificity of the amplification was developed. The ELISA-PCR assay, which used an internal control (IC) of inhibition, was able to detect 1 to 10 copies of STEC O157 in the PCR tube. Adaptation of PCR into ELISA-PCR assay format facilitates specific and sensitive detection of PCR amplification products and constitutes a method of choice for screening STEC O157.
Fach
Patrick
P
Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches sur l'Hygiène et la Qualité des Aliments, Unité: Atelier de Biotechnologie, 1-5 rue de Belfort, 94700 Maisons-Alfort, France. p.fach@afssa.fr
Perelle
Sylvie
S
Grout
Joël
J
Dilasser
Françoise
F
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics; D000818:Animals; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D002522:Chlorocebus aethiops; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004927:Escherichia coli Infections / Q000175:diagnosis*; D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005408:Flagellin; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022621:Shiga Toxin / Q000032:analysis / Q000378:metabolism*; D000637:Transaminases / Q000737:chemistry / Q000235:genetics; D014709:Vero Cells
D018829:Adhesins, Bacterial; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005408:Flagellin; C073819:eaeA protein, E coli; D022621:Shiga Toxin; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase