International Journal of Legal Medicine

, Volume 132, Issue 4, pp 975–981 | Cite as

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

  • Masashi Kitamura
  • Seiji Kubo
  • Jin Tanaka
  • Tatsushi Adachi
Original Article


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.


Loop-mediated isothermal amplification Forensic science Sex identification On-site detection 


Compliance with ethical standards

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.

Supplementary material

414_2017_1661_MOESM1_ESM.pdf (203 kb)
ESM 1 (PDF 203 kb)


  1. 1.
    Old J, Schweers BA, Boonlayangoor PW, Fischer B, Miller KW, Reich K (2012) Developmental validation of RSID-Semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen. J Forensic Sci 57(2):489–499CrossRefPubMedGoogle Scholar
  2. 2.
    Murakami H, Yamamoto Y, Yoshitome K, Ono T, Okamoto O, Shigeta Y, Doi Y, Miyaishi S, Ishizu H (2000) Forensic study of sex determination using PCR on teeth samples. Acta Med Okayama 54(1):21–32PubMedGoogle Scholar
  3. 3.
    Nicklas JA, Buel E (2006) Simultaneous determination of total human and male DNA using a duplex real-time PCR assay. J Forensic Sci 51(5):1005–1015CrossRefPubMedGoogle Scholar
  4. 4.
    Kamodyová N, Durdiaková J, Celec P, Sedláčková T, Repiská G, Sviežená B, Minárik G (2013) Prevalence and persistence of male DNA identified in mixed saliva samples after intense kissing. Forensic Sci Int Genet 7(1):124–128CrossRefPubMedGoogle Scholar
  5. 5.
    Nakanishi H, Shojo H, Ohmori T, Hara M, Takada A, Adachi N, Saito K (2015) A novel method for sex determination by detecting the number of X chromosomes. Int J Legal Med 129(1):23–29CrossRefPubMedGoogle Scholar
  6. 6.
    Holt A, Wootton SC, Mulero JJ, Brzoska PM, Langit E, Green RL (2016) Developmental validation of the Quantifiler(®) HP and Trio Kits for human DNA quantification in forensic samples. Forensic Sci Int Genet 21:145–157CrossRefPubMedGoogle Scholar
  7. 7.
    Ewing MM, Thompson JM, McLaren RS, Purpero VM, Thomas KJ, Dobrowski PA, DeGroot GA, Romsos EL, Storts DR (2016) Human DNA quantification and sample quality assessment: developmental validation of the PowerQuant(®) system. Forensic Sci Int Genet 23:166–177CrossRefPubMedGoogle Scholar
  8. 8.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16(3):223–229CrossRefPubMedGoogle Scholar
  10. 10.
    Kurosaki Y, Magassouba N, Bah HA, Soropogui B, Doré A, Kourouma F, Cherif MS, Keita S, Yasuda J (2016) Deployment of a reverse transcription loop-mediated isothermal amplification test for Ebola virus surveillance in remote areas in Guinea. J Infect Dis 214(suppl 3):S229–S233CrossRefPubMedGoogle Scholar
  11. 11.
    Ravan H, Amandadi M, Sanadgol N (2016) A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7. Microb Pathog 91:161–165CrossRefPubMedGoogle Scholar
  12. 12.
    Sasaki Y, Fujimoto T, Aragane M, Yasuda I, Nagumo S (2009) Rapid and sensitive detection of Lophophora williamsii by loop-mediated isothermal amplification. Biol Pharm Bull 32(5):887–891CrossRefPubMedGoogle Scholar
  13. 13.
    Kitamura M, Aragane M, Nakamura K, Watanabe K, Sasaki Y (2016) Development of loop-mediated isothermal amplification (LAMP) assay for rapid detection of Cannabis sativa. Biol Pharm Bull 39(7):1144–1149CrossRefPubMedGoogle Scholar
  14. 14.
    Kitamura M, Aragane M, Nakamura K, Watanabe K, Sasaki Y (2017) Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay. J Nat Med 71(1):86–95CrossRefPubMedGoogle Scholar
  15. 15.
    Nogami H, Tsutsumi H, Komuro T, Mukoyama R (2008) Rapid and simple sex determination method from dental pulp by loop-mediated isothermal amplification. Forensic Sci Int Genet 2(4):349–353CrossRefPubMedGoogle Scholar
  16. 16.
    Watthanapanpituck K, Kiatpathomchai W, Chu E, Panvisavas N (2014) Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe. Int J Legal Med 128(6):923–931CrossRefPubMedGoogle Scholar
  17. 17.
    Duan Y, Zhang X, Ge C, Wang Y, Cao J, Jia X, Wang J, Zhou M (2014) Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum. Sci Rep 4:7094CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Miyamoto S, Sano S, Takahashi K, Jikihara T (2015) Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes. Anal Biochem 473:28–33CrossRefPubMedGoogle Scholar
  19. 19.
    Nakahara H, Fujii K, Mizuno N, Yoshida K, Kasai K (2007) Evaluations of DNA quantification methods for forensic biological samples. Jpn J Forensic Sci Tech 12:13–26CrossRefGoogle Scholar
  20. 20.
    Kouprina N, Ebersole T, Koriabine M, Pak E, Rogozin IB, Katoh M, Oshimura M, Ogi K, Peredelchuk M, Solomon G et al (2003) Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes. Nucleic Acids Res 31(3):922–934CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Aboud MJ, Gassmann M, McCord B (2015) Ultrafast STR separations on short-channel microfluidic systems for forensic screening and genotyping. J Forensic Sci 60(5):1164–1170CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Masashi Kitamura
    • 1
    • 2
  • Seiji Kubo
    • 1
  • Jin Tanaka
    • 1
  • Tatsushi Adachi
    • 3
  1. 1.Forensic Science Laboratory, Ishikawa Prefectural Police H.QKanazawaJapan
  2. 2.Laboratory of Molecular Pharmacognosy, Division of Pharmaceutical Sciences, Graduate School of Medical SciencesKanazawa UniversityKanazawaJapan
  3. 3.Kaneka CorporationTakasagoJapan

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