Thus, cultureindependent methods allow to overcome biases associated to the culturing step. The detection of microbial populations from total DNA or RNA extracted directly from food matrices can give a more realistic and reliable “picture” of cheese microbiota. In order to monitor the presence and viability of L. lactis throughout cheese manufacturing and ripening, a highly selective qPCR protocol was optimized. The detection of L. lactis with respect to other LAB species, which normally colonize ripened cheeses, was reached by selective primer design on tuf gene codyfing a GTP binding protein and widespread in eubacteria genomes. Tuf gene has been generally recognized as a housekeeping gene ; moreover, its stability was confirmed by studying its expression throughout L. lactis growth curve. SYBR green fluorescent chemistry was chosen and good results were obtained, in terms of specificity, correlation coefficient and efficiency, by increasing the stringency of the thermal cycle and using primers in unbalanced concentration. In particular, the high annealing temperature, used in qPCR and RT-qPCR protocols, allowed the specific detection of L. lactis and no fluorescent signal was detected when the protocol was applied to the other LAB species. Thus, tuf gene represented a suitable target for the specific detection and quantification of L. lactis as also highlighted by other authors. Moreover, the efficiency of the protocols was improved by the choice of nucleic acid extraction protocols specifically designed for the treatment of fatty matrices, highlighting, once again, how this step heavily influence the performance of the subsequent amplification. The high quality of the extracted RNA and the set amplification conditions allowed to obtain standard curves with a good linearity range covering 6 orders of magnitude, from 102 to 108 CFU/g. Other authors optimized qPCR protocols to detect L. lactis in milk, under simulated conditions of cheese manufacture, in ultrafiltered milk cheese models and in the manufacturing of raw milk soft cheeses. The only study dealing with the monitoring of active population of L. lactis in cheese Y-27632 dihydrochloride ripening by RT-qPCR has been focused on Cheddar cheese, for which undefined starter cultures containing L. lactis subsp. lactis and L. lactis subsp. cremoris are commonly used. In particular, the authors evaluated the impact of milk heat treatments and ripening temperatures on lactococcal starter and NSLAB throughout maturing of Cheddar cheese and the results showed that lactococci remained dominant throughout the ripening process. The results presented in this study, however, do not shed light into the possible contribution of L. lactis in terms of organoleptic characteristics of the final cheese product. Thus, as future prospective, it will be important to investigate the role, in terms of metabolic activities, of this microorganism during cheese ripening. In particular, it will be interesting to understand which L. lactis functions are being carried out in each specific phase of the production, with the final aim of improving technological processes and cheese quality.