Evaluation of non-radioactive trivalent DNA probe (LT, ST1a, ST1b) for detecting enterotoxigenic Escherichia coli.
OBJECTIVE
To evaluate a digoxigenin-labelled trivalent DNA probe (LT, ST1a, ST1b) for detecting enterotoxigenic Escherichia coli (ETEC), by comparison with a cell culture assay for detecting LT, individual DNA probes for LT, ST1a and ST1b, and an enzyme immunoassay for detecting ST1.


METHODS
A 1268 base pair DNA fragment, containing parts of the genes for E coli heat labile enterotoxin (LT) and heat stable enterotoxins (ST1a and ST1b), was random prime labelled with digoxigenin-dUTP. The labelled DNA was used as a probe in colony hybridisation reactions to examine 180 E coli strains of which 92 had previously been shown by a cell culture assay to produce LT. Six LT negative ST1 positive E coli, 34 Verotoxin producing E coli (VTEC), and 84 organisms from other genera were also examined. All organisms other than VTEC were isolated from travellers returning from abroad with diarrhoea. All E coli strains were retested by cell culture for LT, and were tested by enzyme immunoassay (EIA) for ST1, and by the trivalent and individual DNA probes.


RESULTS
All 81 isolates, that on retesting by cell culture were positive for LT, also hybridised with the trivalent and LT probes; 27 of these were also enzyme immunoassay (EIA) positive for ST1 of which 24 hybridised with the ST1b probe and three with the ST1a probe. Of 99 isolates, that on retesting by cell culture were negative for LT, all were negative by LT probe and only three were EIA positive for ST1; these three were positive by both trivalent and ST1b probes. Four isolates were positive by the trivalent probe but negative by cell culture and EIA; all four were positive by ST1b probe. Compared with the cell culture assay for LT, the probe had a sensitivity and specificity both of 100%; compared with the EIA for ST1, the probe had a sensitivity of 100% and specificity of 88%.


CONCLUSIONS
The trivalent DNA probe is a sensitive, specific, and reliable method for detecting ETEC that should be considered for use by diagnostic microbiology laboratories.
Public Health Laboratory, Northern General Hospital, Sheffield.
P A
PA
Chapman
C M
CM
Daly
D001427:Bacterial Toxins / Q000235:genetics; D002478:Cells, Cultured; D015342:DNA Probes; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000302:isolation & purification* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006801:Humans; D007124:Immunoenzyme Techniques; D012680:Sensitivity and Specificity
D001427:Bacterial Toxins; D015342:DNA Probes; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C034151:heat stable toxin (E coli); C027538:heat-labile enterotoxin, E coli