An evaluation of genetic markers for forensic identification of human body fluids

Abstract

Body fluids are commonly recovered from crime scenes by forensic investigators and their identification are necessary part of forensic casework study. Current body fluid identification techniques rely on enzymatic tests, which have limited sensitivity and specificity, they require large amount of template, use separate assays for various body fluids, and are prone to contamination. Various genes are expressed in different body fluids that could be used as genetic markers for body fluid identification, and are used in forensic investigations.
The aim of this study was to use mRNA markers to identify human body fluids, which included blood, semen, saliva, vaginal secretion and menstrual blood. Initially, ten reference genes (UCE, TEF, GAPDH, 18S rRNA, ACTB, B2M, B-Actin, OAZ1, RPS 29 and S15) were studied to establish an appropriate reference gene in body fluid identification. These are constitutive genes used for normalisation of gene expression data and control of variations in experiments. qRT-PCR efficiency, sensitivity and limit of detection (LOD) were investigated using SYBR Green and Taqman probes. The results of the SYBR Green efficiency test experiment displayed five markers, UCE, TEF, ACTB, B2M, and RPS29 with 90-110% efficiency with a slope -3.33 ± 10%.
Subsequently, Taqman probes were designed for the five markers and then used for the Taqman probe experiment. Reference gene stability test was carried out on body fluid samples stored up to 6 months at room temperature using the designed Taqman probe assays. The results established ACTB and UCE as best candidate reference gene markers in this study as both were most stable in samples stored for 6 months. Furthermore, to identify each of the five body fluids, thirty-two (32) body fluid specific mRNA markers were evaluated, optimised and validated. The experiment was initially carried out with non-fluorescent makers to determine the specificity of the markers. These were analysed using agarose gel electrophoresis. Further optimization was then carried out using fluorescently labelled markers. This was done in five separate multiplexes for each body fluid; –semen-plex, saliva-plex, vaginal secretion-plex, menstrual blood-plex and blood-plex. An attempt was made to combine all the five-separate multiplex into a single multiplex. All body fluids were identified unambiguously with no cross-reactions of non-target body fluids using the combined multiplex assays.
Following further evaluation and validation tests, a total of 14 markers were selected and a capillary electrophoresis (CE) based, multiplex assay was developed to identify blood, saliva, semen and vaginal secretion samples simultaneously. The markers in the developed multiplex assay included ALAS2 and PF4 (blood), STATH and HTN3 (saliva), PRM1, TGM4, MSMB, NKX3-1 (semen), ACTB and UCE (reference genes), CRYP2B7P1, SFTA2, MUC4 and L. crispatus (vaginal secretion). Extensive validation, which include sensitivity, specificity, reduced volume reactions, degradation, reproducibility, mixtures, cycle number and mastermix, was carried out in accordance with the guidelines detailed in Scientific Working Group in DNA Analysis (SWGDAM). The 14-marker CE-based assay displayed high specificity and sensitivity. Each body fluid was detected down to 1:3000 dilution of mRNA except vaginal secretion that was detected down to 1:1500 dilutions of sensitivity. Specificity experiments showed no cross reactions of the assay with non-target body fluids. Reproducibility study displayed similar results reported from an independent laboratory. All body fluids exposed to environmental insult were identified up to at least day 30 of 51, with blood being identified up to day 51. In the mixture study, all body fluids were identified unambiguously using the developed multiplex assay.
In conclusion, the results of this study have led to the development of a new and novel capillary electrophoresis-based mRNA marker assay for forensic body fluid identification, demonstrating its compatibility with forensic laboratory workflows. The use of this assay to profile forensic casework samples for body fluid identification would be a future application of this work.