Development of a time-effective and highly specific quantitative real-time polymerase chain reaction assay for the identification of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in artisanal raw cow’s milk cheese

The first objective of this work included the development of real-time polymerase chain reaction (RT-PCR) which is also known as quantitative polymerase chain reaction (qPCR) assays to quantify two species of lactic acid bacteria which play a very important role in cheese ripening: Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The second objective was the comparison of qPCR and plate counts of these two species present in raw cow’s milk cheese samples during different stages of ripening. Thirty-three deoxyribonucleic acid (DNA) samples coming from seven different bacterial species, which were phylogenetically related or commonly isolated from raw milk and dairy products, were chosen as positive and negative controls. The qPCR assays showed a high quantification capacity characterised by their linearity (R2 > 0.998), PCR efficiencies which were within the range 78.0-90.0% for L. delbrueckii subsp. bulgaricus, and 93.6-100.5% for S. thermophilus, and quantification limit (103 gene copies/ml for L. delbrueckii subsp. bulgaricus and 10 gene copies/ml for S. thermophilus). The importance of our study is in the monitoring of changes in populations of L. delbrueckii subsp. bulgaricus and S. thermophilus contributing to cheese ripening using the newly designed qPCR assay