Recombinase polymerase amplification for human male determination from semen
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Abstract
Recombinase polymerase amplification (RPA) is a simple, rapid, highly effective, specific, and sensitive technique used for amplification of specific DNA regions at a constant, low temperature (between 37 °C and 42 °C) without using a thermal cycler. The RPA products can be detected using agarose gel electrophoresis. This research developed primers specific to the SRY (sex-determining region Y) gene in RPA assay. RPA primers were designed using parameters according to the instruction manual (TwistDx™) and Primer-BLAST software based on the human SRY gene (accession number: JQ811934.1) obtained from the GenBank database. The designed primers were further tested using RPA at 39 °C for 20 min. The results revealed that the set of developed RPA primers could detect all male DNA from human semen samples. Sensitivity analysis showed that the detection limit was determined to be 0.01 ng for template DNA. Therefore, the RPA primers designed in this research are an alternative valuable tool for sex determination as a part of biological evidence in forensic casework.
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