Real-time polymerase chain reaction assay for vaginal CpG marker, Vag1 region, and trial for precise vaginal fluid identification using a supplemental CpG marker.

The identification of vaginal secretions in forensic samples is often required to reconstruct sexual assault events; however, few conventional confirmatory test exists for vaginal fluid identification. The Vag1 region (cg14991487) is a marker previously reported as vaginal-specific; therefore, we developed a fluorescent-probe-based real-time polymerase chain reaction (PCR) assay suitable for routine forensic analysis that simultaneously detects the methylation status of two adjacent CpG sites within the Vag1 region. The assay yielded a positive methylation signal for vaginal DNA, while showing no cross-reactivity with blood, semen, or saliva DNA; however, a subset of anorectal samples also produced a positive signal. Sensitivity testing showed reliable detection with as little as 0.5 ng of vaginal DNA, although increasing amounts of non-vaginal DNA attenuated the signal in mixed samples. In vaginal-semen mixtures, the semen component masked the signal, but differential extraction of the epithelial fraction restored detection. To address anorectal cross-reactivity, we screened the genome-wide methylation data and identified cg14541992 as an additional marker. The marker is almost completely demethylated in vaginal DNA and highly methylated in anorectal DNA. A duplex assay targeting Vag1 and cg14541992 was established, which could exclude anorectal samples from Vag1-positive samples, although false exclusions of vaginal samples can occur in mixtures (e.g., mixtures with anorectal, semen, or blood DNA). We believe that the rapidity and simplicity of the developed real-time PCR assay are practical advantages for forensic vaginal fluid identification.