Quantitative Determination of Vibrio parahaemolyticus by Polymerase Chain Reaction

Abstract

A method for quantitative detection of Vibrio parahaemolyticus, based on the polymerase chain reaction (PCR), was developed. Several lysis methods were compared and a recently developed lysis solution proved effective. The PCR amplification products were visualized after agarose gel electrophoresis with the nucleic acid stains GelStar and ethidium bromide. The relative fluorescent intensity of the DNA bands was analyzed using the NIH Image 1.61 software program. The GelStar stain was found to be more sensitive than ethidium bromide. The limit of detection by staining DNA bands with GelStar was 16 CFU per PCR and 48 with ethidium bromide. A log-linear relationship between the number of CFU per PCR reaction and the fluorescent intensity of the DNA bands was obtained and calibration curves were generated.

Keywords:

• Vibrio parahaemolyticus
• Quantitative PCR
• Lysis
• Sodium azide
• Triton x-100

Acknowledgments

This study was supported by a Special Seafood Safety grant (award #2002-34423-11960) from the U.S.D.A.