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

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.