Abstract

Forensic investigations require the rapid and accurate identification of human DNA and sex. Although conventional PCR-based methods are reliable, they are limited by their speed, equipment requirements, and poor suitability for on-site analysis, warranting alternative approaches. Isothermal amplification─particularly, loop-mediated isothermal amplification (LAMP)─is a promising technique because of its speed, sensitivity, and minimal equipment requirements; however, its forensic application is constrained by primer design, extraction requirements, and reagent stability. In this study, we have developed a multiplex LAMP assay that targets the Cyt b and SRY genes for simultaneous human DNA detection and sex identification and further standardized the overall LAMP-based rapid detection workflow. The optimized assay showed a sensitivity of 25 pg, and reagents were formulated into lyophilized beads, which improved portability and long-term on-chip storage (≥9 months). The streamlined workflow integrated sample lysis and amplification and was completed in ≤35 min in a microfluidic device. This enabled rapid and user-friendly point-of-care testing (POCT). Robustness was confirmed by the reliable detection of trace male DNA in mixed samples, and 100% accuracy was confirmed via validation with diverse clinical body fluid specimens. Testing was performed with touch DNA from both simulated and real samples, and the accurate detection rate was 62.5%. Overall, the multiplex LAMP assay provides a sensitive, accurate, and portable solution for the rapid identification of human DNA and sex. The proposed device shows strong potential for forensic POCT application.