PCR detection and quantification of vaginal lactobacilli remains problematic because of the high level of genetic heterogeneity and taxonomic complexity within the genus Lactobacillus. The aim of the present study was to identify conserved sequences among the genomes of major species of vaginal lactobacilli that could be used for the development of a PCR-based method for quantitative determination of vaginal microbiota-specific lactobacilli. Comparative analysis of the genomes of several species of vaginal lactobacilli allowed us to identify conserved regions in the rplK gene, which encodes ribosomal protein L11, and to design group-specific PCR primers and a probe for selected species from the L acidophilus complex, including major vaginal lactobacilli Lactobacillus crispatus, L gasseri, L iners and L jensenii as well as other species that are less common in vaginal microbiota. The applicability of the new assay in routine diagnostic testing was evaluated using a set of clinical samples. The assay was able to detect and quantify vagina-associated lactobacilli within a wide range of initial DNA template concentrations, indicating promising potential for clinical applications. (C) 2016 Elsevier Ltd. All rights reserved.