Development of a PCR method for detecting proteolytic psychrotrophic bacteria in raw milk

Abstract

The application of polymerase chain reaction (PCR) to detect microorganisms is largely affected by DNA template quality prepared from food samples. In this study, milk filtration prior to DNA purification was evaluated to detect Pseudomonas fluorescens using PCR. The modified filtration method coupled with an amplification of a P. fluorescens-specific 16S rDNA fragment allowed a limit detection of 102 cfu mL−1 of P. fluorescens inoculated into sterilised whole milk. However, in raw milk, this P. fluorescens-specific fragment was detected when the count of Pseudomonas was 107 cfu mL−1, indicating that other species in addition to P. fluorescens could be present. To detect multiple target bacterial species simultaneously, gene specific primers designed to target the proteases genes of Acinetobacter ssp., P. fluorescens, Aeromonas hydrophila and Serratia marcescens were used in multiplex PCR. This approach demonstrated the potential of multiplex PCR to check the quality of raw milk before being processed.