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. 2023 Aug 22;16(1):180.
doi: 10.1186/s13104-023-06464-2.

Duplex loop-mediated isothermal amplification assay for simultaneous detection of human and human male DNA

Seiji Kubo  1   2 , Hideki Niimi  3 , Isao Kitajima  4
Affiliations

Duplex loop-mediated isothermal amplification assay for simultaneous detection of human and human male DNA

Seiji Kubo et al. BMC Res Notes. .

Abstract

Objective: Screening of human and human male DNA is necessary for forensic DNA analyses. Although quantitative real-time PCR (qPCR) is commonly used for detecting and quantifying these DNA targets, its use as a screening tool is time-consuming and labor-intensive. To streamline and simplify the screening process, we aimed to develop a duplex loop-mediated isothermal amplification (LAMP) assay capable of simultaneously detecting human and human male DNA in a single tube. We assessed the duplex LAMP assay for forensic application.

Results: For our duplex LAMP assay, we have utilized two fluorescent probes with HEX and FAM fluorophores to specifically detect human and human male DNA, respectively. The HEX (human target) signal was detected from both the male and female DNA samples, and the FAM (male target) signal was detected from only the male DNA sample. This assay has a sensitivity of 10-1 pg of DNA for both targets. Additionally, we successfully detected the two targets in the DNA samples extracted from forensically relevant body fluids, including blood, saliva, semen, and vaginal secretions.

Keywords: Duplex assay; Human DNA; Human male DNA; Loop-mediated isothermal amplification.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Schematic representation of our duplex LAMP assay
Fig. 2
Fig. 2
Confirmation of the duplex LAMP assay. Representative amplification plots of the fluorescence analysis. Male DNA, 2 ng; Female DNA, 20 ng; NTC, no-template control
Fig. 3
Fig. 3
Performance of the duplex LAMP assay. a Sensitivity. Duplex LAMP assay was performed for tenfold serial dilutions (100 ng–1 pg) of the male and female DNA samples. b Mixed DNA samples. Duplex LAMP assay was performed for mixed DNA samples with male:female ratios of 1:1 to 1:10,000 (male DNA = 10 pg). The samples with ratios of 1:0 and 0:1 were the male DNA (10 pg) and female DNA (100 ng), respectively. a, b Data are expressed as mean ± SD (n = 3). Data at a threshold time of 60 min represent samples that were not detected
See this image and copyright information in PMC

References

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