Human semen stain analysis in casework sample by HRM-qPCR

Determining the biological origin of body fluid evidence from crime scenes is extremely important for criminal investigation, especially in sexual assault cases. In Brazil, sexual crimes still present low-resolution rates, where approximately 8% of cases are judged. The determination of the presence of semen in samples from crime scenes as a test prior to DNA analysis is a mandatory requirement in forensic analysis and can help to better understand the dynamics of the event. This report aims to present the methodological strategy used in a criminal case of a home invasion where a t-shirt containing visible stains similar to human semen was found at the site. Convencional tests to detect the presence of PSA and sperm cells were performed on the fabric cutouts which showed negative results. We then processed the fabric samples for genetic analysis after two-years-storage, where were performed automated method for the genetic material isolation (QIAcube, Qiagen). The Real-time PCR analysis were carried out using the SOLIScript 1-step SolisGreen (SolisBiodyne) kit, with specific primers to the TGM4 (Transglutaminase 4) gene, in the Rotor Gene Q-5Plex HRM equipment (Qiagen). The results obtained for the melt curve indicated the presence of human semen in the analyzed samples. After the HRM-qPCR assay we also analyzed the a-STR and Y-STR markers. All the results were useful for the criminal process, which led to the identification of the author of the crime.