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Detection of TEM beta-lactamase genes by non-isotopic spot hybridisation

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Abstract

A 998 bp fragment of plasmid pBR322, comprising part of the TEM-1β-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 differentβ-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 strep tavidine/poly alkaline 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 × 106 cells gave optimum positive discrimination. When 90 recent ampicillin-resistant strains ofEscherichia coli isolated from patients with urinary tract infections were screened using the system, 72% gave a positive hybridisation signal.

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Authors and Affiliations

  1. Department of Microbiology & PHLS Laboratory, University Hospital, NG7 2UH, Nottingham, UK

    G. I. Carter, K. J. Towner & R. C. B. Slack

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  1. G. I. Carter
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  2. K. J. Towner
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  3. R. C. B. Slack
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Carter, G.I., Towner, K.J. & Slack, R.C.B. Detection of TEM beta-lactamase genes by non-isotopic spot hybridisation. Eur. J, Clin. Microbiol. 6, 406–409 (1987). https://doi.org/10.1007/BF02013095

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