mRNA profiling for vaginal fluid and menstrual blood identification

The detection and identification of human biological fluids, including vaginal secretions and menstrual blood, are highly important in forensic biology. Previous studies have proposed a few mRNA and bacterial markers for vaginal fluid detection, but they have not proven to be specific and reliable. The aim of this project was to develop, validate and evaluate a reliable, specific test for vaginal fluid identification that would combine detection of vaginal mRNAs and Lactobacilli. We have developed a hexaplex that detects HBD1 (human beta-defensin 1), MUC4 (mucin 4), menstrual blood marker MMP11 (matrix metalloproteinase 11), housekeeping gene G6PDH (glucose 6-phosphate dehydrogenase) and the 16S-23S rRNA intergenic spacer region of Lactobacillus crispatus and Lactobacillus gasseri/Lactobacillus johnsonii. We analysed the specificity of the markers and variations among women, as well as the sensitivity of the test and its ability to detect vaginal fluid in mixtures with semen and blood. This approach allows for the detection of vaginal fluid in stains that were up to 2 years old, if stored at room temperature and up to 18 years old if stored frozen. Through simultaneous analysis of 5 vaginal markers, the proposed hexaplex ensures high specificity and reliability in the detection of vaginal material.