Development of a Codetection System Based on STRs and Biofluid‐Specific CpG Markers

The detection of body fluids in biological stains at crime scenes and their association with donors is increasingly important for the validity of evidence in case investigations and prosecutions. While autosomal short tandem repeats (STR) and DNA methylation markers can effectively analyze personal identification and body fluid origin, there is currently no integrated system for simultaneous STR typing (subsource level) and biofluid identification (source level). This study has developed a codetection system based on DNA methylation markers (cytosine-phosphate-guanine [CpG]) and an STR amplification system for simultaneous personal identification and biofluid origin determination using capillary electrophoresis. This system integrates a 7-plex amplification system, comprising 5 biofluid-specific CpG markers (cg05261336 for semen, cg04011671 for peripheral blood, cg09107912 for saliva, cg15988970 for vaginal secretion, and cg18069290 for menstrual blood) and 2 control markers, with a 20-plex STR amplification system. The combined system successfully distinguished five types of body fluids from single sources (peripheral blood, semen, saliva, menstrual blood, and vaginal secretion) and generated DNA methylation profiles in mixed samples of two to four body fluids in varying proportions. The cumulative discrimination power for personal identification was calculated to be 1-3.5120 × 10^-25. Additionally, semen and vaginal secretion with 0.1 ng DNA input or kept at room temperature for 5 months could still be efficiently identified. This study provides a promising method for simultaneously identifying body fluids and their donors in forensic science.