Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 64, Issue 1, Pages 1-8Publisher
ELSEVIER
DOI: 10.1016/j.mimet.2005.04.023
Keywords
Vibrio vulnificus; EMA; RT-PCR; viable cells; dead cells
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Ethidium bromide monoazide (EMA) was utilized to selectively allow the real-time PCR (RT-PCR) amplification of a targeted DNA sequence in viable but not dead cells of Vibrio vulnificus. The optimized light exposure time to achieve cross-linking of DNA by the EMA in dead cells and to photolyse the free EMA in solution was at least 15 min. The use of 3.0 mu g/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable cells of V. vulnificus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 2.5 mu g/ml. Amplification of DNA from dead cells in a mixture with viable cells was successfully inhibited by 2.5 mu g/ml of EMA,. whereas the DNA from viable cells present was successfully amplified by RT-PCR. (c) 2005 Elsevier B.V. All rights reserved.
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