A proof of principal study on the use of direct PCR of semen and spermatozoa and development of a differential isolation protocol for use in cases of alleged sexual assault

Abstract

Sexual assault samples are some of the most common samples encountered in forensic analysis. These samples can require a significant time investment due to differential extraction processes. We report on the first record of successful direct amplification of semen for STR analysis. Neat seminal fluid, dilutions ranging from 1:5 to 1:160 and GEDNAP samples were successfully amplified using a direct method. A mild differential isolation technique to enrich spermatozoa was developed and successfully implemented to separate and directly amplify a mixture of semen and female epithelial cells. Aliquots of samples subjected to the differential isolation protocol were stained with Haemotoxylin and Eosin for sperm scoring. Samples stained after PCR showed a complete lack of intact spermatozoa demonstrating that the cells are lysed during the PCR process. This paper demonstrates the potential to incorporate direct PCR in cases of sexual assault to more rapidly obtain results and achieve a higher sensitivity.

The effects of dithiothreitol (DTT) on fluorescent qPCR dyes

“…Because this method bypasses quantification, many laboratories have only adopted it for processing of reference samples, which are exempt from the FBI QA Standard 9.4 requiring human DNA quantification [10]. However, direct amplification has recently been explored in the literature for rapid screening of some forensic evidence samples from higher volume crimes, such as those seen in sexual assault cases [5,11]. Several prominent studies have even developed and tested microdevices for processing sexual assault samples [12][13][14][15][16].…”

Review of direct PCR and Rapid DNA approaches to streamline sexual assault kit testing

“…Prior to DNA processing, serology for SAK samples includes presumptive screening for various body fluids often followed by confirmatory testing [9,10]. "National Best Practices", as recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the NIJ, suggests that labs could take a "direct to DNA" or Yscreening approach for SAK samples, whereby all sexual assault samples proceed straight to DNA extraction with serology being performed later, only as necessary [11][12][13].…”

“…Sexual assault kits may contain various items such as swabs collected from the victim’s body, clothing, and fingernail scrapings, where small amounts of perpetrator DNA may be left behind [9]. The most probative items in SAKs are usually intimate swabs from the genitals of the victim, where semen or other body fluids from the perpetrator can be used for identification and to corroborate that a sexual interaction took place.…”

A review of direct polymerase chain reaction of DNA and RNA for forensic purposes

“…The influence of this may be somewhat minimized by using kits which are resistant to PCR inhibitors, in addition to the use of internal PCR controls to differentiate between a lack of profiling results caused by inhibitors, as opposed to insufficient template DNA (Ensenberger, Hill, McLaren, Sprecher, & Storts, 2014;Gray et al, 2014). A differential extraction is required for sexual assault samples and a direct PCR method could include separation of spermatozoa and epithelial cells and lysing of spermatozoa, which was demonstrated to be successful by Tobe et al (2016). The removal of the quantification step in most direct PCR protocols may also complicate analysis, as the PCR reactions generally operate within a particular range of amount of input template.…”