Rapid sperm lysis and novel screening approach for human male DNA via colorimetric loop-mediated isothermal amplification.

ABSTRACT

Correct identification of probative samples is the first crucial step in the analysis of sexual assault kits (SAKs). We report a nucleic acid-based approach, as an alternative to the widely utilized p30 assay, to screening male DNA from SAKs collected from female victims by combining a rapid lysis protocol with an isothermal amplification method. The enzymatic lysis protocol efficiently digests biological material to release nuclear DNA in 10 min in a single closed tube, including resilient cell types such as sperm cells. The amplification and detection of human male specific DNA is achieved through loop-mediated isothermal amplification (LAMP) accompanied with hydroxynaphthol blue, a colorimetric indicator, producing a visually-distinctive color change in the presence of male DNA. The Y-screen approach demonstrated high specificity to human male DNA, can reliably detect target DNA as low as 50 pg, and correctly identified all probative samples from 14 single-blind mock sexual assault samples. In contrast with the widely used p30 assay which requires at least 2 h incubation time and manual application to a lateral flow pad, this Y-screen assay can be completed in half the time, and can be performed in a 96-well format without the need for a fluorescence detector, making facile high-throughput sample screening possible.