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

Authors

Sydney G. Nelson, Antibiotic Resistance Action Center, Department of Environmental and Occupational Health, Milken Institute School of Public Health, George Washington University, Washington, DC, 20052, USA.
Megan Spyers-Duran, Antibiotic Resistance Action Center, Department of Environmental and Occupational Health, Milken Institute School of Public Health, George Washington University, Washington, DC, 20052, USA.
Maliha Aziz, Antibiotic Resistance Action Center, Department of Environmental and Occupational Health, Milken Institute School of Public Health, George Washington University, Washington, DC, 20052, USA.
Danielle Marcone, Antibiotic Resistance Action Center, Department of Environmental and Occupational Health, Milken Institute School of Public Health, George Washington University, Washington, DC, 20052, USA.
Deneen Nault, George Washington University Hospital, Washington, DC, 20037, USA.
Gizachew Demessie, George Washington University Hospital, Washington, DC, 20037, USA.
Rupert Kaul, Department of Immunology, University of Toronto, Toronto, ON, Canada.
Ronald M. Galiwango, Rakai Health Sciences Program, Kalisizo, Uganda.
Rebecca Yee, George Washington University School of Medicine and Health Sciences, Washington, DC, 20052, USA.
Cindy M. Liu, Antibiotic Resistance Action Center, Department of Environmental and Occupational Health, Milken Institute School of Public Health, George Washington University, Washington, DC, 20052, USA. cindyliu@gwu.edu.

Document Type

Journal Article

Publication Date

10-29-2025

Journal

Scientific reports

Volume

15

Issue

1

DOI

10.1038/s41598-025-21406-7

Keywords

Dialister micraerophilus; Peptostreptococcus anaerobius; Prevotella bivia; Bacteria associated with HIV seroconversion; Bacterial vaginosis; Genital anaerobes; Immune cells; Inflammation

Abstract

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 × 10 to 1.0 × 10 copies/µL for P. bivia and D. micraerophilus assays, and 2.5 × 10 to 1.0 × 10 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.

APA Citation

Nelson, Sydney G.; Spyers-Duran, Megan; Aziz, Maliha; Marcone, Danielle; Nault, Deneen; Demessie, Gizachew; Kaul, Rupert; Galiwango, Ronald M.; Yee, Rebecca; and Liu, Cindy M., "Real-time PCR assay design and validation for Prevotella bivia, Peptostreptococcus anaerobius, and Dialister micraerophilus, bacteria associated with increased HIV susceptibility" (2025). GW Authored Works. Paper 8213.
https://hsrc.himmelfarb.gwu.edu/gwhpubs/8213

Department

Environmental and Occupational Health