Real-time PCR assay design and validation for Prevotella bivia, Peptostreptococcus anaerobius, and Dialister micraerophilus, bacteria associated with increased HIV susceptibility

Many genital anaerobes, including Prevotella bivia, Peptostreptococcus anaerobius, and Dialister micraerophilus, are underexplored despite being Bacteria Associated with HIV Seroconversion, Inflammation, and immune Cells (BASICs). This is partly due to the lack of molecular tools for their detection and quantification. To address this gap, we designed and validated three real-time qPCR assays for rapid and cost-effective analysis. qPCR assays were designed based on the species-specific core genomes of the three target genital anaerobic species. Assay sensitivity, specificity, and quantification characteristics were assessed using a synthetic oligonucleotide and DNA extracted from closely related species (n = 27-42) and human urogenital swabs (n = 111-114). The resultant assays demonstrated 100% sensitivity and specificity for bacterial isolates and high sensitivity (94.6%-97.7%) and specificity (92.8%-95.7%) for human urogenital swabs. The linear dynamic range was 5.0 × 10¹ to 1.0 × 10⁷ copies/µL for P. bivia and D. micraerophilus assays, and 2.5 × 10² to 1.0 × 10⁷ copies/µL for the P. anaerobius assay. Assay efficiency ranged from 99.0% to 112.0%. These assays provide a highly sensitive and specific method for the analysis of bacterial isolates, urogenital samples, and in vitro or ex vivo experiments, which can enhance our understanding of the epidemiology, clinical impact, and biology of key genital anaerobes associated with HIV risk.