Detection of TEM beta-lactamase genes by non-isotopic spot hybridisation.
A 998 bp fragment of plasmid pBR322, comprising part of the TEM-1 beta-lactamase gene, was labelled with biotin-11-dUTP for use as a DNA probe in a rapid non-isotopic spot hybridisation test. Diluted broth cultures of bacteria producing different beta-lactamases were filtered onto nitrocellulose and lysed in situ. Following pre-hybridisation treatment with proteinase K, hybridisation with the labelled probe was demonstrated using a commercially available streptavidine/polyalkaline phosphatase-based detection system. The probe was highly specific, reacting only with strains producing either the TEM-1 or structurally similar TEM-2 enzyme. An inoculum of 3-4 X 10(6) cells gave optimum positive discrimination. When 90 recent ampicillin-resistant strains of Escherichia coli isolated from patients with urinary tract infections were screened using the system, 72% gave a positive hybridisation signal.
Carter
G I
GI
Department of Microbiology, University Hospital, Nottingham, UK.
Towner
K J
KJ
Slack
R C
RC
D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004927:Escherichia coli Infections / Q000382:microbiology; D005796:Genes; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011237:Predictive Value of Tests; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D014552:Urinary Tract Infections / Q000382:microbiology; D001618:beta-Lactamases / Q000235:genetics*
D004269:DNA, Bacterial; D001618:beta-Lactamases