Female sample screening using colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting non-coding RNA XIST

Forensic sample screening is important for establishing an effective DNA typing workflow. The detection of sex-specific markers in forensic samples highlights the necessity for further analysis. Y-chromosome DNA can confirm male contributions, but female contributions are difficult to confirm using DNA-based methods. To address this, we developed a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the long non-coding RNA X-inactive specific transcript (XIST) to screen female samples. Operating at 65 °C for 30 min, the assay yielded results discernible from the color change of the pH indicator dye. The assay showed a detection limit of approximately 0.5 µL of blood. The assay also detected XIST RNA in mixed body fluids and mock samples, indicating its potential applicability to casework samples. Taken together, our assay provides a rapid and simple strategy for screening female samples.