Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR.
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 microg/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 microg/ml. Amplification of DNA from dead cells in a mixture with viable cells was successfully inhibited by 2.5 microg/ml of EMA, whereas the DNA from viable cells present was successfully amplified by RT-PCR.
Wang
Shishan
S
Department of Food Science, Massachusetts Agricultural Experiment Station, University of Massachusetts, Amherst, MA 01003, USA.
Levin
Robert E
RE
D000345:Affinity Labels / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D001431:Bacteriological Techniques; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000737:chemistry / Q000145:classification* / Q000378:metabolism; D008027:Light; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D041261:Vibrio vulnificus / Q000235:genetics* / Q000302:isolation & purification
D000345:Affinity Labels; D001386:Azides; D004269:DNA, Bacterial; C014535:8-azidoethidium