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Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay

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International Journal of Legal Medicine Aims and scope Submit manuscript

Abstract

Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

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Author information

Author notes

  1. Masashi Kitamura and Seiji Kubo contributed equally to this work

Authors and Affiliations

  1. Forensic Science Laboratory, Ishikawa Prefectural Police H.Q, 1-1 Kuratsuki, Kanazawa, Ishikawa, 920-8553, Japan

    Masashi Kitamura, Seiji Kubo & Jin Tanaka

  2. Laboratory of Molecular Pharmacognosy, Division of Pharmaceutical Sciences, Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, 920-1192, Japan

    Masashi Kitamura

  3. Kaneka Corporation, 1-8 Miyamae-cho, Takasago-cho, Takasago, Hyogo, 676-8688, Japan

    Tatsushi Adachi

Authors
  1. Masashi Kitamura
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  2. Seiji Kubo
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  3. Jin Tanaka
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  4. Tatsushi Adachi
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Corresponding author

Correspondence to Masashi Kitamura.

Ethics declarations

This study was approved by the Human Genome/Gene Analysis Research Ethics Committee of the Japanese Association of Forensic Science and Technology. Informed consent was obtained from all individual participants included.

Competing interests

The authors declare that they have no conflict of interest.

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Kitamura, M., Kubo, S., Tanaka, J. et al. Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay. Int J Legal Med 132, 975–981 (2018). https://doi.org/10.1007/s00414-017-1661-z

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  • DOI: https://doi.org/10.1007/s00414-017-1661-z

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