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Commercial detection methods for biotinylated gene probes: Comparison with32P-labeled DNA probes

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Abstract

This study had two objectives: (a) to determine whether biotinylated DNA probes could be substituted for32P-labeled DNA probes to detect the presence of the TEM-1 β-lactamase gene in crude bacterial preparations, and (b) to evaluate two commercial detection systems for biotinylated probes—an alkaline phosphatase kit produced by Bethesda Research Laboratories (BRL) and an acid phosphatase kit produced by Enzo Biochem. Both the kits produced nonspecific reactions with TEM-1-negative organisms. Treatment with chloroformphenol and proteinase K did not remove these nonspecific reactions. When plasmid DNA was purified by electrophoresis and transferred to nitrocellulose filters by the Southern blot method, there was no qualitative difference between the biotinylated and radioactive probes. However, the32P-labeled probes were quantitatively 100 times more sensitive than the biotinylated probes. In addition, the Enzo Biochem kit and the32P-labeled probes could be used with charged nylon membranes, whereas the BRL kit could be used only with nitrocellulose filters.

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Authors and Affiliations

  1. Laboratory Services, Veterans Administration Medical Center, 27705, Durham, North Carolina, USA

    Peter Zwadyk Jr.

  2. Department of Pathology, Duke University, Durham, North Carolina, USA

    Peter Zwadyk Jr.

  3. Antimicrobics and Infection Mechanisms Branch, Hospital Infections Program, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia, USA

    Robert C. Cooksey & Clyde Thornsberry

Authors
  1. Peter Zwadyk Jr.
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  2. Robert C. Cooksey
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  3. Clyde Thornsberry
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Zwadyk, P., Cooksey, R.C. & Thornsberry, C. Commercial detection methods for biotinylated gene probes: Comparison with32P-labeled DNA probes. Current Microbiology 14, 95–100 (1986). https://doi.org/10.1007/BF01568410

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  • DOI: https://doi.org/10.1007/BF01568410

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