Identification of the vaginal secretion donor in mixture stains using polymorphic cSNPs on mRNA biomarkers

In recent years, RNA profiling has become an important technique in identifying the origin of human body fluids/tissues. Both perpetrators and victims can be identified from stains involving vaginal secretions (VS), such as sperm/VS mixtures left on condoms, bed sheets, or papers, etc. Body fluid specific RNA typing could link the source of body fluids/tissues and the identity of the donor. In this study, we aimed to trace the donor of VS in mixture stains using body fluid-specific mRNA markers and construct a coding single nucleotide polymorphism (cSNP) typing system for VS. We screened 8 VS-specific mRNA biomarkers (MUC4, SFTA2, CYP2A6, MYOZ1, FUT6, ESR1, SPINK5, and SERPINB13) encompassing 18 cSNPs. The RNA obtained from various body fluid/tissue samples was treated with reverse transcription polymerase chain reaction (RT-PCR) and then followed by a multiplex PCR and SNaPshot mini-sequencing assay. The detection limit of the assay was 0.08 ng RNA. For single-source body fluid, the positive cSNP typing was only shown in VS and void in non-VS body fluids/tissues. For laboratory-generated VS-containing mixtures, the minor VS contributor could be successfully detected at a ratio of 1:10-1:500. We also confirmed the concordance of DNA typing and mRNA typing for the cSNPs in this system. In summary, we established an 18-cSNP typing system for VS with high sensitivity and specificity, which could identify both the donor and the tissue origin simultaneously. This was shown to be a powerful tool for identifying the VS donor in those VS-containing mixture stains.