Specific microbial biomarkers for distinguishing saliva from vaginal secretions: Integration of 16S rRNA sequencing and real-time quantitative PCR

Body fluids are commonly found biological traces at crime scenes. Differentiating body fluid types—especially saliva (SA) from vaginal secretions (VA)—remains a forensic challenge. Currently, microbes play an increasingly vital role in forensic science. In this study, we analyzed bacterial communities from various samples, including SA, VA, semen (SE) and skin (SK), using Illumina HiSeq sequencing of 16S rRNA gene amplicons. We validated the ability to distinguish SA samples from VA samples using real-time quantitative PCR (qPCR) for specific biomarkers. We obtained 8,211,062 tags and identified 4427 operational taxonomic units (OTUs). Bacteria at the phylum level were similar across the four body fluid types, but their abundances varied. At the genus level, different bacteria dominated. Principal coordinates analysis (PCoA) and cluster analysis revealed significant differences among sample types. Additionally, linear discriminant analysis effect size (LEfSe) identified specific bacterial variations across the four body fluids. qPCR validation of the sequencing-derived markers confirmed that eight specific biomarkers reliably distinguish SA from VA. This work enhances the fundamental understanding of microorganisms in different body fluids and aids in distinguishing SA and VA.