Proximity Ligation Real time PCR for the Detection of Spermatozoa

In forensic biology laboratories it is common practice to process sexual assault evidence for the identification of seminal fluid. Seminal fluid is presumptively identified using an alternate light source as an enhancement tool followed by testing for Seminal Acid Phosphatase. If positive, the next step in some laboratories is to test for Prostate Specific Antigen (PSA or p30) with commercially available immunochromatography kits. PSA has been identified at very low levels in other body fluids, thus a positive PSA result may not be considered confirmatory for seminal fluid by all practitioners. The only undisputable confirmatory test for the presence of semen is the microscopic observation of spermatozoa. Microscopic observation of spermatozoa can be extremely time-consuming. Particularly in samples with low levels or no spermatozoa, analysts may spend hours searching a slide. The physical demand of working on a microscope for hours at a time can cause analyst burnout and increases the possibility of sperm cells going unnoticed in low level samples. Automated sperm searcher systems decrease the time spent on a single sample; still these systems can only process samples one at a time. Finally, the cost of fluorescent microscopes and automated sperm searching technology is a large financial commitment for a laboratory. Sexual assault samples are a large contribution to the backlog that many laboratories face. As technology improves for DNA identification, it is likely that the common time since intercourse restriction (3 days) will be expanded for the collection of sexual assault evidence. This will only further increase the number of samples that will need to be tested for seminal fluid. The time, cost, and limited automation capabilities of microscopic observation limit the reduction in backlog. Forensic laboratories would greatly benefit from the implementation of a faster, cost effective, and amenable to automation method for the identification of spermatozoa. Proximity ligation real-time PCR (PLiRT-PCR) has the potential to be the technology that meets these requirements. PLiRT-PCR is a molecular assay that enables detection and quantitation of a target protein. Using antibody probes that are specific for the target, a representative DNA molecule is generated if the antigen is present, which can then be detected by Real Time PCR (RT-PCR). The amount of signal from this surrogate amplicon is indicative of the amount of protein in the sample. It is a powerful and highly sensitive assay that combines the specificity of an immunological reaction with the sensitivity of PCR simply using RT-PCR technology, which is commonly used in crime labs. During a proof of concept study we demonstrated that a PLiRT-PCR assay for PSA was able to detect PSA in extremely low concentrations (1:5,000,000 dilution) of semen. These results encouraged us to continue our research and develop a PLiRT-PCR assay targeting sperm cell specific proteins such as acrosomal membrane proteins SP-10 and CRISP-2. Although more work needs to be done, the preliminary data obtained are very promising. The goal of this proposed project is the development and optimization of a PLiRT-PCR assay targeting sperm specific proteins. Detection of these proteins will serve as a confirmatory test for the presence of spermatozoa and will allow faster and more efficient processing of sexual assault evidence. ca/ncf