Evaluation & Improvement of an mRNA Method for Forensic Body Fluid Identification

Abstract

In forensic casework it can be beneficial to determine the origin of a biological stain found on items of interest or at a crime scene. This information can help reconstruct the course of events and help identify the donor of certain biological material and link this to the crime. Conventional body fluid identification methods are based on enzymatic, chemical or immunological approaches and can be defined as confirmatory or presumptive tests. These often have limited specificity and sensitivity and do not exist for all body fluids, whilst mRNA profiling can be used as a definitive confirmation with greater specificity. This project aimed to evaluate and improve the current method at ESR used for identifying commonly encountered body fluids in forensic work, known as CellTyper 2. There are currently cross reactivity and specificity issues with several of the markers used in CellTyper 2 when there is an abundance of one or more body fluids in a sample. Vaginal material currently only has one body fluid marker and introducing a second marker would help improve result interpretation. A thorough literature search was conducted to identify the candidate markers. Sensitivity testing demonstrated the SMR3A and ESR1 primer sets successfully amplified their target within saliva and vaginal material samples respectively. Additional testing demonstrated SRM3A was specific to saliva and no signals were observed in non-target body fluids. Cross reactions were observed for ESR1 in semen samples. Validation studies are necessary, however SMR3A looks to be a promising marker. Due to the non-specificity of ESR1, this means that additional reporting guidelines will be required and it may need to be used in conjunction with CY2B7P, the other vaginal material marker. mRNA and DNA testing was conducted on finger samples to assess the background levels of DNA and body fluids used in the CellTyper 2 assay found on fingers to help with result reporting, particularly in cases of digital penetration. mRNA profiling of the finger samples demonstrated there were no background levels of body fluids on the fingers. Identifiler™ Plus and Yfiler™ Plus were used to carry out autosomal and male specific Y-STR DNA profiling for the finger swabs and vaginal material to determine if DNA was present from anyone other than the donor. Results demonstrated full single source female profiles for the vaginal material samples and low level partial DNA profiles were observed for the finger samples.