Development of a Quantitative Real-time PCR Assay for Identification of Streptococcus thermophilus Present in Artisanal Raw Cow’s Milk Cheese

Abstract

The aim of this paper was to develop a real-time PCR method targeting a gene sequence encoding a bacteriocin ABC transporter ATP-binding protein for the detection of Streptococcus thermophilus in cheese produced from raw cow milk. A real-time quantitative PCR assay was designated to identify and count S. thermophilus cells in ripened cheese. The developed real-time PCR primers and probe were highly specific for S. thermophilus CNRZ1066, CNRZ8232, LMD-9, LMG 18311, CNRZ 002 and CNRZ 03 but not for Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Leuconostoc spp., Enterococcus spp. that are phylogenetically very similar to S. thermophilus, and this is the reason why they are difficult to differentiate using culture-based methods. The real-time PCR allowed for quantification with a detection of 101–103 cfu/g of product. qPCR-standard curves were linear over seven log units down to 101 copies per reaction; PCR efficiencies ranged from 1.00 to 1.03. The newly developed qPCR method could be applied to specific detection and precise enumeration of S. thermophilus in raw milk and cheese.