![]() |
![]() |
||||||||||||
|
02534 QUANTITATIVE AND QUALITATIVE DETERMINATION OF THE VAGINAL MICROBIAL FLORA BY REAL TIME PCR Meacci Francesca*, In each ecological niche of the human body, including the vagina, the microbial commensal flora is composed of about 100 to 600 different bacterial species. While a minority of these organism grows well in vitro, the vast majority is not cultivable by standard laboratory techniques. Since it is of great importance to asses the variation of the microbial flora connected with the use of microbicides, as well as in specific pathological conditions, we developed an efficient new method based on quantitative real-time PCR. Aim of this work is the development and validation of a novel molecular diagnostic approach for characterisation vaginal flora, both for cultivable and uncultivable bacterial species. Real time PCR is used as a culture-independent tool for characterisation of the shifts in the composition of microbial population of the vagina. After construction of a database, several PCR primer sets, able to amplify defined groups of bacteria, were designed. Three different sets of primer were designed to identify/quantify the predominant species of the human vagina flora which correlated to normality (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii). For the recognition/quantification of the major phylogenetic groups of bacteria a series of 10 primer sets were designed. For few pathogens specific primer sets were designed (Chlamydia trachomatis, Trichomonas vaginalis, Candida albicans). The PCR assays have been validated on controls of cloned DNA and specificity and sensitivity were checked. The quantitative real time PCR assay developed was shown to be able to quantify efficiently selected species in spiked samples over a wide range of serial dilutions. Dr. Francesca Meacci |
|||||||||||||