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Confirmatory Detection of Sperm and Semen Via Proximity Ligation real-Time PCR

NCJ Number
255193
Date Published
December 2015
Length
3 pages
Author(s)
Sarah Riman; Chin H. Shek; Valerie Clermont-Beaudain; Michelle Peck; Jaclyn Benjamin; Caitlin Ayub; Lori Prugh; Daniele Podini
Agencies
NIJ-Sponsored
Annotation
This article reports on the development of a confirmatory method that uses the Proximity Ligation Real Time PCR (PLiRT-PCR) for the identification of semen and sperm from sexual assault evidence.
Abstract
Semen and sperm identification play a critical role in forensic investigation for understanding the circumstances surrounding a crime and determining whether a sexual act occurred. Yet current methods for sperm detection vary widely in speed, sensitivity, and specificity. Also, the only undisputable confirmatory test for the presence of semen is the microscopic observation of spermatozoa. This process can be time-consuming and labor intensive, since each sample must be processed and examined individually. In samples with low levels or no spermatozoa, analysts may spend hours searching a slide. Also, failure to identify sperms by microscopic examination is not conclusive for their absence. The current project developed a confirmatory method that employs Proximity Ligation Real Time PCR (PLiRT-PCR) for the identification of semen and sperm from sexual assault evidence. PLiRT-PCR is designed to detect and quantitate the expression of protein markers through an antibody-protein binding reaction followed by qPCR. Two protein targets have been chosen as candidates for the assay: cysteine-rich secretor protein 2 (CRISP-2) and prostate-specific antigen (PSA). CRISP-2 is specifically expressed in the male reproductive tract and localized inside the acrosome of spermatozoa. The acrosomal location protects CRISP-2 from environmental damage until lysis, enabling successful detection even on aged forensic samples. Also, its location on the surface of the acrosome makes it likely for epitopes to be accessible to antibody probes. PSA was selected as a second candidate due to its high concentration in semen and its useful role when processing samples from azoospermic or vasectomized perpetrators. This study discusses the identification, specificity, and the limit of detection of Crisp2 and PSA markers in samples containing body fluid mixtures. Data demonstrate the potential for PLiRT-PCR as a confirmatory test for semen. The approach enables high throughput semen and sperm detection to be processed in parallel on a 96 well plate, enabling analysts to make the most informed decisions on the best analytical path forward in each case, preserve valuable evidence, overcome the drawbacks associated with the microscopic observation of spermatozoa, and shorten analysis time. (publisher abstract modified)

Date Created: July 20, 2021