Quantitative polymerase chain reaction used for the rapid detection ofCarnobacteriumspecies from French soft cheeses

Abstract

An identification method by PCR, specific to the Carnobacterium genus, was optimised by testing it on 28 bacterial strains. Primers from the literature were tested to differentiate Carnobacterium strains present among various bacterial species. The DNA of Carnobacterium species (C. alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C. mobile, C. viridans), specifically amplified by the Cb1–Cb2R primer couple at a hybridization temperature of 69 °C, gave only one band of 340 bp. The validation of this technique was carried out on a French soft cheese made with pasteurised milk inoculated with C. maltaromaticum LMA 28. Using classical PCR, detection was not possible for decimal dilutions of the cheese above 1 g L⁻¹. With Sybr Green I real time PCR, the specificity of the reaction was confirmed by the T_m value. The standard curve constructed using the main threshold cycle and various concentrations of C. maltaromaticum LMA 28 (ranging from 10⁰ to 10⁸ cfu mL⁻¹) showed good linearity and a sensitivity limit of 10⁴ cfu g⁻¹ of cheese. This technique was applied on commercially available cheeses made from raw cow's milk. The Sybr Green I real time PCR method constitutes an effective and easy-to-perform method to quantify Carnobacterium sp. in cheese.