CST-Based Molecular Profiling of Vaginal Microbiota Validated by qRT-PCR and 16S Amplicon Sequencing

The vaginal microbiome is commonly classified into five Community State Types (CSTs) based on the dominance and relative abundance of Lactobacillus species, which play a central role in maintaining vaginal homeostasis through the production of lactic acid and hydrogen peroxide. Disruption or depletion of Lactobacillus spp. Leads to elevated vaginal pH, thereby promoting the overgrowth of anaerobic bacteria associated with bacterial vaginosis (BV). Although next-generation sequencing (NGS)-based microbiome profiling has been widely used to characterize vaginal microbial communities, its routine clinical application remains limited by high cost, long turnaround time, and reduced suitability for rapid, species-specific quantification. In this study, we developed and validated species-specific quantitative real-time polymerase chain reaction (qRT-PCR) primer sets targeting four health-associated Lactobacillus species (Lactobacillus crispatus, Lactobacillus. gasseri, Lactobacillus. iners, and Lactobacillus. jensenii) and four BV-associated bacterial taxa (Atopobium vaginae, Clostridiales genomosp. BVAB1, Prevotella bivia, and Megasphaera type 1). Quantitative results obtained by qRT-PCR were systematically compared with relative abundance profiles derived from 16S rRNA gene V3-V4 sequencing, revealing a strong concordance between the two platforms in identifying key vaginal microbial signatures. Collectively, these findings demonstrate that qRT-PCR represents a rapid, cost-effective, and sensitive approach for targeted vaginal microbiome assessment and supports its potential utility as a molecular diagnostic tool for the precise detection of vaginal microbiota imbalance.