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Proximity Ligation Real Time PCR for the Detection of Spermatozoa

NCJ Number
Date Published
October 2015
20 pages
This report summarizes the findings and methodology for a project that used proximity ligation real-time PCR (PLiRT-PCR) as a microscope-free method for confirming the presence of semen by detecting sperm-specific proteins.

The intent of this project was to develop a method for sperm identification that would eliminate the time-consuming microscopic detection process, thus assisting in reducing the prevalent backlog in the processing of sexual assault cases. The main steps involved in the PLiRT-PCR assay are binding, ligation, and RT-PCR. Each of these steps is described. The kit used for the PLiRT-PCR is the TaqMan Protein Assay from Life Technologies (Foster City, CA). The project results show that the PLiRT-PCR method has the potential for high throughput confirmatory testing for the presence of sperm and other body fluids contingently to the antibody quality. In order to address some identified challenges in this procedure, the report recommends an ideal approach that develops well-characterized monoclonal antibodies that target separate specific antigens of the body fluid-specific protein and optimize the assay with a combination of these. This guarantees the consistency of the antibodies, because the immortal cell lines are producing them. An assay with these antibodies can then be validated for forensic casework, since it will be consistent in time. The approach of testing multiple targets evaluated in the "combo-assay" is also promising. By testing multiple targets on the same sample and by generating a PLiRT-PCR based "serological profile," it is possible to confirm the body fluid type. This process was also determined to be amenable to automation and sample batching. 4 figures, 1 table, and 16 references

Date Published: October 1, 2015