Enhancing the sexual assault workflow: Development of a rapid male screening assay incorporating molecular non-microscopic sperm identification

Abstract

Sexual assault samples are among the most difficult sample types encountered by forensic laboratories. Typically, a sexual assault sample poses multiple challenges including small quantity of male DNA and a relatively high quantity of female DNA. Differential extraction procedures are time-consuming and labor-intensive, particularly with microscopic sperm identification. A rapid upfront screening or triage assay prior to use of differential extraction procedures could ensure that male DNA is present prior to the use of full DNA workflows. Previously reported rapid male screening assays do not provide a confirmation of whether sperm are present and therefore still require the use of microscopic sperm identification. This work was focused on an improved rapid male DNA screening assay with an upfront sperm identification using mRNA profiling.The rapid male screening assay consists of a brief lysis using only a small tip portion of a swab, to obtain both RNA (eluted) and DNA (extraction 'waste') followed by a one-step reverse transcription-high resolution melt assay for PRM2, a sperm-specific mRNA. Without additional purification, the DNA 'waste' fraction can be quantitated and used to obtain an upfront Y-STR profile of the sperm donor. The assay takes 30 min for lysis followed by ∼2 h for quantitation and sperm identification.Here, we demonstrate the specificity of the assay to detect male DNA in semen samples with no cross reactivity with other body fluids and no PRM2 detection in vasectomized males. RNA and DNA profiling results were obtained with as little as ∼0.15 μl of semen in vaginal-semen admixtures. In all samples tested, Y-STR profiling results were obtained when male DNA was indicated based on quantitation results.

High‐throughput seminal fluid identification by automated immunoaffinity mass spectrometry

“…• Immunoaffinity-MS provides reliable and sensitive results from SAK samples. approach, with serological screening performed only when needed, if at all [1][2][3]. However, the presence or absence of semen may be key to reconstructing alleged events and corroborating witness accounts in a sexual assault case.…”

“…With an emphasis on processing evidence from sexual assaults, serological testing seeks to confirm the presence of sperm cells or a molecular component of seminal fluid recovered from sexual assault kits (SAKs), whereas genetic testing aims to individualize the source of the stain. Advances in DNA processing of sexual assault evidence have prompted the forensic community to adopt a “direct‐to‐DNA” approach, with serological screening performed only when needed, if at all [1–3]. However, the presence or absence of semen may be key to reconstructing alleged events and corroborating witness accounts in a sexual assault case.…”

Forensic transcriptome analysis using massively parallel sequencing

“…It is also possible that non-microscopical identification of spermatozoa will be possible due to the presence of sperm specific RNAs (e.g. protamines such as PRM1) [166]. It is likely that the transcriptome will be a constituent part of a (multi)omics/data integration process for evidence analysis that will include the not-isolated biological entities of the genome, transcriptome, methylome, proteome and microbiome.…”

Interlaboratory Comparison of SpermX™ and Conventional Differential Extractions Indicated High Male DNA Recovery by the SpermX Method

“…Without significant increases in funding or improved technology to increase case capacity, these trends are likely to continue, and backlogs will continue to increase. The challenges with SAK backlogs and the differential extraction method are well known to the forensic community [8]. Much R&D investment has gone into trying to develop alternative methods to improve and automate the differential extraction process, but other methods that are commercially available either have similar or worse sperm recovery to standard manual differential methods [6,9].…”