High-throughput sperm screening using one-step RT-qPCR: Improvement and re-evaluation

Anal Biochem. 2025 Mar:698:115727. doi: 10.1016/j.ab.2024.115727. Epub 2024 Nov 27.

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.

Keywords: PRM2; RT-qPCR; Semen; Sperm.

MeSH terms

  • High-Throughput Screening Assays / methods
  • Humans
  • Male
  • Protamines*
  • RNA, Messenger / analysis
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Spermatozoa* / chemistry
  • Spermatozoa* / metabolism

Substances

  • Protamines
  • RNA, Messenger
  • protamine 2