A novel co-amplification system for simultaneous amplification of 23 Y-STR and identification of spermatozoa.

In alleged sexual assault cases, identification of the presence of spermatozoa at the crime scene, or on items of eventual significance, or associated with the body of the victim, is integral to the forensic investigation to support or refute the proposition that sexual act has occurred. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) system has been developed previously to identify spermatozoa based on the presence or absence of DNA methylation. This assay showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa even when there was excessively more DNA isolated from vaginal fluid than DNA from a semen extract (80 ng/0.1 ng) or a mix of the menstrual blood/semen DNA (5 ng/0.1 ng). In this study, we combine spermatozoa detection with co-amplification of 23 Y-STR loci. We perform standard validation steps to present a novel test that saves time and uses the same sample for both DNA typing and spermatozoa detection in the same reaction. The combined assay can identify Y-STR and spermatozoa simultaneously using just 0.1 ng semen DNA, even in the presence of 5 ng of DNA from a female (male/female:1/50). No other body fluid tested, such as saliva, gave a result for the presence of spermatozoa. A total of 9 non-probative forensic samples from 7 sexual assault cases were tested by this co-amplification system. In all cases, the same sperm-positive data were obtained, concordant with our previous study analyzed by only 3-plex MSRE-PCR, and the Y-STR results were also consistent with that analyzed by only PowerPlex® Y23 kit. The co-amplification will be beneficial for the limited samples in many criminal cases.

Spermatozoa identification by the 3-plex MSRE-PCR assay: a collaborative exercise.

Identification of semen and spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. In cases of alleged sexual assault where there is a long time gap between the incident and sample collection, or in cases of low sperm count, current methods have limitations of specificity, in the case of presumptive tests for semen, or the problem of recording spermatozoa by microscopy if they are few in number. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay using a spermatozoa-specific DNA methylated marker to identify spermatozoa has been reported previously by our laboratory. A key advantage over current methods is the increased sensitivity and specificity. A transition from a research tool to operational use requires blind trial testing and inter-laboratory trials. We report on a collaborative exercise where reagents of the 3-plex MSRE-PCR were sent to six participating laboratories. Each laboratory used their own equipment, consumables, and the presumptive reagents conventionally for body fluid (such as acid phosphatase or PSA), DNA extraction, and quantification in practical casework. The reagents and protocol for the 3-plex MSRE-PCR assay and 9 samples were provided by the organizing laboratory. The participating laboratories were requested to fill in the questionnaire after testing. The reported results from all the six participating laboratories were concordant and the expected correct results for the presence of spermatozoa. These outcomes verified the reproducibility and feasibility of the 3-plex MSRE-PCR assay. The results also indicated that the 3-plex MSRE-PCR assay was readily accessible to forensic laboratories for integrating it into current forensic casework processes.

Identification of spermatozoa using a novel 3-plex MSRE-PCR assay for forensic examination of sexual assaults.

Identification of semen and then spermatozoa is essential to verify that sexual activity has occurred in alleged cases of sexual assault. Microscopic examination commonly used for spermatozoa identification is however time-consuming and can often lead to false-negative results for samples with deformed and, or, limited number of spermatozoa. To address this limitation, we report on a novel 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay to specifically identify spermatozoa. This assay is comprised of 3 markers: a digestive control marker (DC), sperm-specific marker (SP), and Y chromosome marker (SRY). A total of 214 samples from 10 body fluids or tissues were analyzed. Specificity testing showed that all the normal semen samples were unambiguously identified as being sperm-positive, and no other body fluid (or tissues) showed a sperm-specific signal in the electropherogram. Testing for sensitivity showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa by this assay. Mixture analyses illustrated the sensitivity of the assay when the vaginal/semen DNA ratio (80/0.1) was under 800 or the menstrual blood/semen DNA ratio (5/0.1) was under 50, the trace amounts (approximately 0.1 ng) of DNA from semen can still be identified by this 3-plex MSRE-PCR assay. This assay was also applied to the identification of 31 non-probative forensic samples from 18 sexual assault cases. The case studies showed that the 3-plex MSRE-PCR assay was an improvement in the sensitivity of spermatozoa detection.