Quantification of Vibrio vulnificus using the polymerase chain reaction

An efficient procedure was developed for the quantitative PCR detection of Vibrio vulnificus in pure culture. The procedure involved boiling cell suspensions in TZ solution (2% Triton X-100 and 2.5 mg mL(-1) NaN3 in 0.1 M Tris-HCl buffer at pH 8.0). Serial dilutions of lysed cell suspensions were then used for PCR. The method of visualizing amplified bands in agarose gels was evaluated by comparing a new nucleic acid dye (GelStar) to ethidium bromide (EB). GelStar stain was found to yield discernible amplified bands with lower levels of target DNA than could be achieved with EB. The minimum detection level with the GelStar stain was 16 CFU per PCR reaction compared to 40 CFU with EB. The relative fluorescence intensity of the DNA bands was analyzed with the NIH Image 1.61 software program. Calibration curves relating fluorescence intensity of amplified bands to lysed cells were obtained and the method was found suitable for the quantification of genomic DNA derived from similar to 10(1)-10(3) CFU per PCR reaction.