Highlights
- •Y-RPA was developed for rapid and simple detection of male DNA.
- •Y-RPA could detect male DNA within 20 min at 39 °C.
- •Y-RPA directly detected male DNA from crude body fluids heated in NaOH for 5 min.
- •Y-RPA is an efficient tool for screening male DNA from forensic samples.
Abstract
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.
Graphical Abstract
Graphical Abstract
Keywords
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Article info
Publication history
Published online: April 04, 2022
Accepted:
April 1,
2022
Received in revised form:
March 31,
2022
Received:
December 27,
2021
Identification
Copyright
© 2022 Elsevier B.V. All rights reserved.
