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High-throughput sperm screening using one-step RT-qPCR: Improvement and re-evaluation
Seiji Kubo1, Keito Amai2, Fumitaka Nakano2
1Department of Clinical Laboratory and Molecular Pathology, Faculty of Medicine, Academic Assembly, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan; Forensic Science Laboratory, Ishikawa Prefectural Police Headquarters, 1-1 Kuratsuki, Kanazawa, 920-8553, Japan.
Abstract:
Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative real-time PCR (RT-qPCR) can enhance the screening by detecting sperm-specific mRNA markers, such as protamine 2 (PRM2). This study aimed to develop a one-step RT-qPCR assay targeting PRM2 mRNA. Our assay was capable of detecting as low as 0.01 μL of semen with high specificity and demonstrated successful detection of PRM2 mRNA in simulated-case samples. Owing to the simple workflow involved, our assay requires <30 min for RNA extraction and <60 min for RT-qPCR. Our assay enables high-throughput sperm screening and offers a promising strategy for enhancing the workflow of sexual assault cases.
