Protocol for the Use of PCR-Denaturing Gradient Gel Electrophoresis and Quantitative PCR to Determine Vaginal Microflora Constitution and Pathogens in

In healthy women, the vaginal ecosystem is dominated by Lactobacillus spp., but a diverse array of other bacteria can be present in lower amounts. The activity of lactobacilli is essential to protect women from genital infections and to maintain the natural healthy balance of the vaginal microbiota. Bacterial vaginosis (BV) is a complex, polymicrobial disorder characterized by an overgrowth of strict or facultative anaerobic bacteria and a reduction of lactobacilli. Culture-independent techniques based on the analysis of rRNA gene sequences provide powerful tools to reveal the phylogenetic diversity of the vaginal microorganisms in healthy women and patients affected by BV. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis conducted with universal primers for eubacteria allows detecting the most abundant bacterial species of an ecosystem. Sequencing of the DNA fragments and comparison with sequences present in publicly available databases allow identifying the corresponding bacterial species. Quantitative PCR is a powerful technique for the quantitative analysis of a selected genus or species.