Practical and economical method for using biotinylated DNA probes with bacterial colony blots to identify diarrhea-causing Escherichia coli.
A simple and economical method was developed for using biotinylated DNA probes to hybridize with bacterial colonies belonging to the various categories of diarrhea-causing Escherichia coli. Simplification and cost containment were achieved by using Whatman no. 541 filter papers instead of nitrocellulose, by minimizing the concentration of proteinase K (an expensive but necessary reagent used to pretreat the colony blots prior to hybridization with biotin-labeled DNA probes) and by reusing hybridization solution containing labeled probe DNA. After exposing the colony blots to lysing solution and steam, followed by lysozyme (1.5 mg/ml), sucrose (25%), and proteinase K (10 micrograms/ml) treatments, biotinylated probes were used to detect enterotoxigenic, enteropathogenic, enterohemorrhagic, diffuse adherence, and enteroinvasive categories of diarrhea-causing E. coli with a high level of sensitivity and specificity. Three independent observers who were experienced in reading DNA blots recorded remarkably similar results, while less satisfactory results were obtained when the blots were read by an inexperienced observer. This technique will be useful in laboratories in which radioactive isotopes are unavailable or impractical and in which budgets are restricted.
Gicquelais
K G
KG
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore 21201.
Baldini
M M
MM
Martinez
J
J
Maggi
L
L
Martin
W C
WC
Prado
V
V
Kaper
J B
JB
Levine
M M
MM
D001710:Biotin; D015169:Colony Count, Microbial; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics; D003967:Diarrhea / Q000382:microbiology*; D019286:Endopeptidase K; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000382:microbiology*; D005069:Evaluation Studies as Topic; D006801:Humans; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization; D012697:Serine Endopeptidases
D015342:DNA Probes; D004269:DNA, Bacterial; D001710:Biotin; D012697:Serine Endopeptidases; D019286:Endopeptidase K