G.vag1008 is the only probe with higher sensitivity (97.5%) than our probe, being able to detect one more G. vaginalis strain. This higher sensitivity is due to the Poziotinib solubility dmso presence of a degenerate oligonucleotide in the sequence of the probe (see Table 2),

allowing G.vag1008 to act as two different sequence probes. However, G.vag1008 has 24 oligonucleotides (i.e. 9 nucleotides more than our probe) and it is a DNA probe, which penetrates the cell wall less efficiently [52] and implies need for the use of long hybridization see more periods. GardV probe detected species from several bacterial genera present in vaginal samples, such as Alloscardovia, Parascardovia and Scardovia spp. [53]. G.vag1008 probe hybridized with Aeriscardovia spp. that may also be found in vaginal samples [53] and therefore this represents an important pitfall for the G. vaginalis detection with such probes. It is important to notice that our Gard162 probe is the first PNA probe specifically designed for G. vaginalis detection. Other PNA probes for the detection of learn more lactobacilli [31, 46] revealed several disadvantages when compared to Lac663 probe, as we shown before [26]. Multiplex FISH detection Although numerous authors attempted to correlate differences between healthy and BV vaginal samples [54–57], no consensus was achieved, except that biofilm formation

of G. vaginalis and a decrease in lactobacilli number could be considered as the initial stages in the pathogenesis of BV [10, 58]. Swidsinski and colleagues already conducted an international follow-up study in which vaginal samples from several BV patients were analyzed by DNA-based FISH and a dense as well as active bacterial biofilm on vaginal mucosa was Phosphoprotein phosphatase detected, primarily consisting of G. vaginalis[47]. Therefore, multiplex FISH to analyze G. vaginalis biofilm establishment and subsequently lactobacilli replacement appeared to be a useful molecular methodology for BV diagnosis in vaginal samples. Although several

authors already developed specific probes for G. vaginalis and Lactobacillus spp. detection for FISH, our multiplex method presented new improvements on the method (see Table 2). Due to the difficulty to obtain fresh vaginal samples diagnosed with BV, we devised an in vitro experiment mimicking the shift from healthy vaginal flora to BV HeLa cells were incubated with different concentrations of G. vaginalis and Lactobacillus strains (L. crispatus and L. iners), ranging from normal to BV vaginal microflora contents (1×103 to 1×109 CFU/ml; see Table 4). The HeLa cell line is an established tool in experimental research with lactobacilli. It has not only been used to study attachment of several Lactobacillus species, but also of other pathogens [41–43]. The Lactobacillus strains used here were selected because high concentrations of L. crispatus (in conjugation with low loads or absence of G.