An integrated extraction-free LAMP platform with colorimetric, real-time, and immunochromatographic strip detection for rapid male-specific DNA screening in forensic triage.

Rapid identification of male-derived biological evidence enhances forensic casework efficiency, particularly in sexual assault investigations that require prioritization of male DNA for downstream short tandem repeat (STR) analysis. Here, we present an extraction-free direct loop-mediated isothermal amplification (LAMP) workflow targeting the Y-chromosomal sex-determining region Y (SRY) gene for male-specific DNA detection, with the autosomal human leukocyte antigen DRA (HLA-DRA) gene incorporated as a human-specific internal control. Three detection formats were systematically evaluated within a single extraction-free framework: colorimetric LAMP enabled visual SRY detection within 30 min at 65 °C; multiplex real-time LAMP simultaneously detected both SRY and HLA-DRA within 15 min, achieving limits of detection of 100 pg per reaction for male DNA and 10 pg per reaction for total human DNA; and an immunochromatographic strip (ICS)-based format provided instrument-free visual discrimination between male, female, and non-human samples through simultaneous dual-target readout on a single strip. To assess field applicability, five direct lysis reagents were evaluated for compatibility with each detection modality. Nuclease-free water preserved the pH conditions required for colorimetric detection, enabling reliable male DNA identification from whole blood diluted up to 1:25 and saliva diluted up to 1:5. NaOH-based lysis improved DNA release and amplification kinetics in real-time LAMP, enabling detection from blood diluted up to 1:125 and saliva diluted up to 1:625. A UDG/dUTP anti-contamination system was optimized for the ICS format to suppress carry-over-derived false-positive signals while maintaining robust visual band intensity-an essential requirement for reliable point-of-care interpretation. The optimized workflow was validated across multiple forensically relevant matrices including whole blood, saliva, swab-collected samples, and cigarette butt mock evidence, consistently discriminating male- from female-derived biological material. These results demonstrate that direct LAMP, when paired with appropriate lysis chemistry and detection modality, provides a rapid, sensitive, and operationally practical strategy for on-site male DNA screening and forensic evidence triage.