A multiplex bacterial assay for identifying vaginal fluid in various individual states and mixture stains

Accurate identification of vaginal fluid is imperative for forensic investigations, particularly in resolving sexual assault cases. Body fluid-specific microbiota are considered potential indicators for identifying body fluids. In this study, we selected vaginal core bacteria based on species level and developed a multiplex PCR system for identifying vaginal fluid in various individual states and mixed stains. This system was validated using a set of 350 samples (245 vaginal samples and 105 other body fluid samples). The system demonstrated high sensitivity, stability against inhibitors, and strong species specificity, showing that saliva and skin swabs were not misidentified as vaginal fluid. Menstrual blood and vaginal fluid exhibited extremely similar identification results. Further validation of individual factors showed that the system was applicable to vaginal samples from female individuals across diverse geographical regions, with varying health statuses and sexual practices. Moreover, the system effectively identified vaginal fluid components in all simulated mixed samples and successfully analyzed aged vaginal samples from actual sexual assault cases. In conclusion, this multiplex PCR system provides a promising tool for the forensic identification of vaginal fluid.