Developmental validation of the SF 28CS typing system: a robust 6-dye multiplex for forensic human identification

Short tandem repeats (STRs) are central to human identification and kinship analysis owing to their high polymorphism. We report the developmental validation of the SF28CS typing system, a novel six-dye multiplex PCR assay that simultaneously amplifies 28 loci, including 23 autosomal STRs (18 expanded CODIS loci, Penta D, Penta E, and three additional markers), three Y-STRs (DYS391, DYS576, DYS518), one Y-chromosomal insertion/deletion marker, and the Amelogenin locus for sex determination. Validation was conducted in accordance with SWGDAM guidelines using the Applied Biosystems 3500 Genetic Analyzer. The system demonstrated high sensitivity, generating complete profiles with as little as 62.5 pg of DNA, and successfully genotyped challenging forensic samples such as bloodstains, semen stains, and decade-old materials. It showed strict species specificity and stability under common PCR inhibitors, including calcium and EDTA. Mixture studies indicated reliable resolution at contributor ratios up to 1:4. A population study of 500 unrelated individuals further confirmed the high discriminatory power and informativeness of the assay, supporting its application in forensic databases. Compared with existing multiplex kits, the SF28CS exhibited superior robustness for degraded samples and complex mixtures, while its inclusion of Y-chromosomal markers enhances its utility in kinship testing and male identification. These results establish the SF28CS system as a sensitive, reliable, and comprehensive tool for forensic casework and human identification.