2. Academic Validation
  3. An isothermal amplification-based field on-site rapid test for direct detection of male DNA via the Y-specific TSPY4 gene

An isothermal amplification-based field on-site rapid test for direct detection of male DNA via the Y-specific TSPY4 gene

  • Forensic Sci Int. 2026 Jun:383:112858. doi: 10.1016/j.forsciint.2026.112858.
Lei You 1,  Zhiwei Li 2,  Jumei Zhang 1,  Mingyang Han 1,  Luyao Zhong 1,  Xianming Shi 1,  Jun Lv 2,  Junhong Zhao 2,  Chen Li 1,  Lanlan Zheng 3,  Yonghong Zhang 4
Affiliations

Affiliations

  • 1 Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, School of Basic Medicine, Hubei University of Medicine, Shiyan 442000, China; Shiyan Key Laboratory of Medicinal Plants and Evolutionary Genetics, Hubei Key Laboratory of Wudang local Chinese Medicine Research, Hubei University of Medicine, Shiyan 442000, China.
  • 2 Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, School of Basic Medicine, Hubei University of Medicine, Shiyan 442000, China.
  • 3 Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, School of Basic Medicine, Hubei University of Medicine, Shiyan 442000, China; Shiyan Key Laboratory of Medicinal Plants and Evolutionary Genetics, Hubei Key Laboratory of Wudang local Chinese Medicine Research, Hubei University of Medicine, Shiyan 442000, China. Electronic address: [email protected].
  • 4 Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, School of Basic Medicine, Hubei University of Medicine, Shiyan 442000, China; Shiyan Key Laboratory of Medicinal Plants and Evolutionary Genetics, Hubei Key Laboratory of Wudang local Chinese Medicine Research, Hubei University of Medicine, Shiyan 442000, China. Electronic address: [email protected].
PMID: 41702134 DOI: 10.1016/j.forsciint.2026.112858
Abstract

Recombinase polymerase amplification (RPA) is a promising method for rapid, portable, and isothermal DNA detection in forensic applications. In this study, we developed and optimized an RPA assay targeting the Y-chromosome-specific TSPY4 gene, referred to as FORT-TSPY (Field On-site Rapid Test), for the rapid and direct detection of male DNA. An optimal primer pair was identified through multiple rounds of screening using a primer-walking strategy. The optimal primer concentration for efficient amplification was determined using male DNA samples, revealing that primer concentration is not simply a matter of higher or lower being better, but rather that an optimal concentration exists. The assay demonstrated excellent sensitivity, detecting as little as 1.2 × 101 copies (∼10-11 ng) of plasmid DNA within 30 min, without the need for complex laboratory equipment. Furthermore, the method exhibited high specificity for male DNA, with no cross-reactivity observed with female or non-human DNA. Randomized testing under simulated forensic conditions indicated the assay's potential for on-site male DNA detection. Collectively, these results demonstrate that RPA-based FORT‑TSPY provides a rapid, equipment‑minimal alternative or adjunct to laboratory PCR workflows and illustrates the broader adaptability of the FORT Field On‑site Rapid Test platform for point‑of‑evidence nucleic acid detection.

Keywords

Field on-site rapid Test; On-site detection; Primer walking strategy; Rapid male DNA detection; Recombinase polymerase amplification.

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