Developmental evaluation of a combinatorial qPCR multiplex for forensic body fluid identification

Forensic body fluid identification (BFID) is an essential step in the biological evidence workflow that can provide critical context clues for crime reconstruction, particularly in sexual assault cases. The ability to link a DNA profile to a cell type or body fluid is important when considering the nature of forensic evidence, where mixtures of body fluids from multiple individuals are commonly observed. Current serological techniques involve enzymatic or immunology-based assays that lack sensitivity/specificity and are subjective to forensic analysts; therefore, molecular methods, such as microRNA (miRNA) analysis or microbial classification, have been extensively researched in the forensic community. Recognition of advantages and disadvantages of each method have led to the proposition that combining molecular markers would increase discrimination efficiency of multiple body fluids from a single assay. While microbial taxonomic classification for BFID is successful in body fluids with high abundances of bacteria, such as vaginal secretions and saliva, miRNA analysis shows promise in body fluids with less bacterial content, such as venous blood, menstrual secretions, and urine. Since miRNAs co-extract with DNA, a separate RNA extraction is not necessary, eliminating a major hurdle to casework implementation. This study aims to synergize on the benefits of both miRNAs and microbial DNA to characterize X body fluids from a single DNA extract. The project will be conducted over 12 months to develop qPCR assay that distinguishes between six forensically relevant body fluids using a combination of miRNAs and microbial DNA targets previously demonstrated as body fluid specific. Microbial species will be used to identify vaginal secretions, feces, and saliva due to their high bacterial content, while semen, blood, and menstrual secretions will be discriminated through miRNA analysis. If possible, autosomal and Y markers will be incorporated to demonstrate human and male DNA in the samples, respectively. The designed assays will be optimized in a qPCR multiplex, allowing for simultaneous identification of all six body fluids at the quantification step of the current DNA workflow. Forensic utility will be addressed through developmental validation studies that demonstrate body fluid specificity in a large population, assay sensitivity and the ability to discriminate body fluids in samples of mixed origin. In addition to the annual and final technical reports, expected scholarly products include a doctoral dissertation, at least one publication in a peer-reviewed journal, and two presentations at scientific meetings. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF