Multiplex quantitative polymerase chain reaction assay for the identification and quantitation of major vaginal lactobacilli

Lactobacilli play a key role in promoting vaginal health. Depletion of these bacteria is associated with bacterial vaginosis (BV), the most common vaginal disorder. Here we describe the development and laboratory validation of a novel single-tube multiplex TaqMan quantitative polymerase chain reaction (qPCR) assay for the identification and quantitative assessment of the four major vaginal Lactobacillus species: L. crispatus, L. jensenii, L. gasseri, and L. iners. The assay utility was evaluated by the analysis of lactobacilli in non-cultured clinical vaginal swab specimens collected from BV patients and healthy individuals. As confirmed by the assay, L. crispatus, L. jensenii, and to a lesser extent L. gasseri, are common in the vagina of healthy women, whereas L. iners dominance is associated with BV. The major assay limitation was preferential detection of dominant Lactobacillus species in samples with mixed lactobacilli resulting in lower sensitivity for minor species. The multiplex qPCR assay described here is an advance in the detection and quantitation of the major vaginal lactobacilli, potentially facilitating the molecular diagnosis of BV and post-therapy restoration of the vaginal microflora.