Rapid and direct detection of male DNA by recombinase polymerase amplification assay

Screening of male DNA is important in forensic investigations, especially sexual assault cases. Quantitative real-time polymerase chain reaction (qPCR) is widely used for the detection of male DNA. However, the use of this technique as a screening tool is time-consuming and labor-intensive. In this study, we established a recombinase polymerase amplification (RPA) assay targeting the multicopy loci on the Y-chromosome for the rapid detection of male DNA (referred to as Y-RPA). The Y-RPA assay was able to detect male DNA in less than 20 min with a sensitivity of 0.025-0.005 ng/µL. Additionally, the Y-RPA assay was highly tolerant to inhibitors; male DNA was detectable in the presence of up to 1000 ng/µL humic acid, 250 µM indigo carmine, and 500 µM hematin. Then, considering its tolerance to inhibitors, we examined the feasibility of the direct Y-RPA assay. The alkaline lysis protocol (addition of sodium hydroxide and heating at 95 °C for 5 min) was employed for preparing the DNA template. The Y-RPA assay successfully detected male DNA using crude DNA extracted from blood, saliva, and semen samples. This approach enabled the screening of male DNA within approximately 30 min (5 min for lysis and 20 min for Y-RPA). These findings suggest that the Y-RPA assay is a promising screening tool for the rapid, simple, and efficient detection of male DNA.