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Forensic Science, Medicine and Pathology

, Volume 14, Issue 4, pp 469–477 | Cite as

The detection and identification of saliva in forensic samples by RT-LAMP

  • Li-Chin Tsai
  • Chih-Wen Su
  • James Chun-I Lee
  • Yu-Sheng Lu
  • Hsuan-Chen Chen
  • Yu-Chih Lin
  • Adrian Linacre
  • Hsing-Mei Hsieh
Original Article

Abstract

We report on a novel method for saliva identification by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). In our previous report, real-time RT-LAMP was used for blood identification by using HBB detection as a model but in this advanced study, this method was refined for the identification of the more challenging body fluid of saliva. Expression of the18S rRNA gene was used as the internal control and the Statherin (STATH) gene as the saliva-specific marker. A turbidimeter was used for real-time detection of the RT-LAMP products, and confirmation was obtained that the real products were generated using: agarose gel electrophoresis, calcein fluorescence detection and/or enzymatic digestion. The specificity of the test was performed using 42 samples including 7 different body fluids, and the expression of STATH was only observed in all the saliva samples (6) with a threshold time of 39.4 ± 2.9 min. Sensitivity testing showed that RT-LAMP products for STATH were stably detected when the RNA template was not less than 6.25 ng. When the primer concentrations for STATH were two times that of 18S rRNA, saliva could be identified in the body fluid mixtures even at a ratio (saliva:semen) of 1:3 (without loop primer)/1:5 (with loop primer). A multiplex RT-LAMP was established to simultaneously amplify the 18S rRNA and STATH genes, and applied to the identification of saliva on ten non-probative cigarette butts. A positive result for saliva was obtained from all ten butts, even for those that returned a negative or ambiguous result using the amylase test. A direct RT-LAMP test is also reported where the RNA extraction step was omitted to speed the collection of data and all tests using either the simplex or multiplex RT-LAMP resulted in a positive response if saliva was present. Our data provide a simple and effective means to detect the presence of saliva.

Keywords

Saliva identification RT-LAMP Statherin (STATH) Multiplex RT-LAMP Non-probative evidence samples 

Notes

Acknowledgements

This study was supported by the Ministry of Science and Technology in Taiwan (grant number NSC 101-2320-B-015-001 and MOST 104-2320-B-015 -001 -MY2).

Funding

This study was funded by the Ministry of Science and Technology in Taiwan (grant numbers NSC 101–2320-B-015-001 and MOST 104–2320-B-015-001 -MY2).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The human body fluids used in this study were collected from the volunteers using procedures approved by Antai Medical Care Cooperation Antai- Tian-Sheng Memorial Hospital Institutional Review Board in Taiwan (TSMH IRB No./ Protocol No.: 14–085-B1).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

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References

  1. 1.
    Willott GM. An improved test for the detection of salivary-amylase in stains. J Forensic Sci Soc. 1974;14:341–4.CrossRefGoogle Scholar
  2. 2.
    Liang T, Roy R. Ultraviolet-visible spectrophotometry (UV-VIS) and SALIgAE® qualitative and semi-quantitative tools for the analysis of salivary amylase. J Forensic Res. 2014;5:247.Google Scholar
  3. 3.
    Old JB, Schweers BA, Boonlayangoor PW, Reich KA. Developmental validation of RSID™-saliva: a lateral flow immunochromatographic strip test for the forensic detection of saliva. J Forensic Sci. 2009;54:866–73.CrossRefGoogle Scholar
  4. 4.
    Harbison SA, Fleming RI. Forensic body fluid identification: state of the art. Res Rep Forensic Med Sci. 2016;6:11–23.Google Scholar
  5. 5.
    Nakanishi H, Kido A, Ohmori T, Takada A, Hara M, Adachi N, et al. A novel method for the identification of saliva by detecting oral streptococci using PCR. Forensic Sci Int. 2009;183:20–3.CrossRefGoogle Scholar
  6. 6.
    Choi A, Shin KJ, Yang WI, Lee HY. Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA. Int J Legal Med. 2014;128:33–41.CrossRefGoogle Scholar
  7. 7.
    Juusola J, Ballantyne J. Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int. 2005;152:1–12.CrossRefGoogle Scholar
  8. 8.
    Sakurada K, Ikegaya H, Fukushima H, Akutsu T, Watanabe K, Yoshino M. Evaluation of mRNA-based approach for identification of saliva and semen. Leg Med (Tokyo). 2009;11:125–8.CrossRefGoogle Scholar
  9. 9.
    Zubakov D, Hanekamp E, Kokshoorn M, van Ijcken W, Kayser M. Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples. Int J Legal Med. 2008;122:135–42.CrossRefGoogle Scholar
  10. 10.
    Silva SS, Lopes C, Teixeira AL, Carneiro de Sousa MJ, Medeiros R. Forensic miRNA: potential biomarker for body fluids? Forensic Sci Int Genet. 2015;14:1–10.CrossRefGoogle Scholar
  11. 11.
    Mayes C, Seashols-Williams S, Hughes-Stamm S. A capillary electrophoresis method for identifying forensically relevant body fluids using miRNAs. Leg Med (Tokyo). 2018;30:1–4.CrossRefGoogle Scholar
  12. 12.
    Haas C, Klesser B, Maake C, Bär W, Kratzer A. mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Sci Int Genet. 2009;3:80–8.CrossRefGoogle Scholar
  13. 13.
    Schwartz SS, Hay DI, Schluckebier SK. Inhibition of calcium phosphate precipitation by human salivary statherin: structure-activity relationships. Calcif Tissue Int. 1992;50:511–7.CrossRefGoogle Scholar
  14. 14.
    Goobes R, Goobes G, Campbell CT, Stayton PS. Thermodynamics of statherin adsorption onto hydroxyapatite. Biochemistry. 2006;45:5576–86.CrossRefGoogle Scholar
  15. 15.
    Richard ML, Harper KA, Craig RL, Onorato AJ, Robertson JM, Donfack J. Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis. Forensic Sci Int Genet. 2012;6:452–60.CrossRefGoogle Scholar
  16. 16.
    Lindenbergh A, de Pagter M, Ramdayal G, Visser M, Zubakov D, Kayser M, et al. A multiplex (m) RNA-profiling system for the forensic identification of body fluids and contact traces. Forensic Sci Int Genet. 2012;6:565–77.CrossRefGoogle Scholar
  17. 17.
    Juusola J, Ballantyne J. mRNA profiling for body fluid identification by multiplex quantitative RT-PCR. J Forensic Sci. 2007;52:1252–62.PubMedGoogle Scholar
  18. 18.
    Young ST, Moore JR, Bishop CP. A rapid, confirmatory test for body fluid identification. J Forensic Sci. 2018;63:511–6.CrossRefGoogle Scholar
  19. 19.
    Hanson E, Ingold S, Haas C, Ballantyne J. Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing. Forensic Sci Int Genet. 2018;34:206–21.CrossRefGoogle Scholar
  20. 20.
    Su CW, Li CY, Lee JC, Ji DD, Li SY, Daniel B, et al. A novel application of real-time RT-LAMP for body fluid identification: using HBB detection as the model. Forensic Sci Med Pathol. 2015;11:208–15.CrossRefGoogle Scholar
  21. 21.
    Nakanishi H, Ohmori T, Hara M, Takada A, Shojo H, Adachi N, et al. A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification. J Forensic Sci. 2011;56:S158–61.CrossRefGoogle Scholar
  22. 22.
    Nogami H, Tsutsumi H, Komuro T, Mukoyama R. Rapid and simple sex determination method from dental pulp by loop-mediated isothermal amplification. Forensic Sci Int Genet. 2008;2:349–53.CrossRefGoogle Scholar
  23. 23.
    Watthanapanpituck K, Kiatpathomchai W, Chu E, Panvisavas N. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe. Int J Legal Med. 2014;128:923–31.CrossRefGoogle Scholar
  24. 24.
    Kitamura M, Kubo S, Tanaka J, Adachi T. Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay. Int J Legal Med. 2018;132:975–81.CrossRefGoogle Scholar
  25. 25.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:e63.CrossRefGoogle Scholar
  26. 26.
    Nie K, Qi SX, Zhang Y, Luo L, Xie Y, Yang MJ, et al. Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction for the detection of human enterovirus 71 subgenotype C4 in nasopharyngeal swab specimens. PLoS One. 2012;7:e52486.CrossRefGoogle Scholar
  27. 27.
    Takehara S, Yanagishita M, Podyma-Inoue KA, Kawaguchi Y. Degradation of MUC7 and MUC5B in human saliva. PLoS One. 2013;8:e69059.CrossRefGoogle Scholar
  28. 28.
    Seyama K, Nukiwa T, Takahashi K, Takahashi H, Kira S. Amylase mRNA transcripts in normal tissues and neoplasms: the implication of different expressions of amylase isogenes. J Cancer Res Clin Oncol. 1994;120:213–20.CrossRefGoogle Scholar
  29. 29.
    Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002;16:223–9.CrossRefGoogle Scholar
  30. 30.
    Hayashida M, Ota T, Ishii M, Iwao-Koizumi K, Murata S, Kinoshita K. Direct detection of single nucleotide polymorphism (SNP) by the TaqMan PCR assay using dried saliva on water-soluble paper and hair-roots, without DNA extraction. Anal Sci. 2014;30:427–9.CrossRefGoogle Scholar
  31. 31.
    Ambers A, Wiley R, Novroski N, Budowle B. Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ® swabs. Forensic Sci Int Genet. 2018;32:80–7.CrossRefGoogle Scholar
  32. 32.
    Lee JW, Jung JY, Lim SK. Simple and rapid identification of saliva by detection of oral streptococci using direct polymerase chain reaction combined with an immunochromatographic strip. Forensic Sci Int Genet. 2018;33:155–60.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Forensic ScienceCentral Police UniversityTaoyuanRepublic of China
  2. 2.Forensic Biology DivisionCriminal Investigation Bureau, National Police AdministrationTaipeiRepublic of China
  3. 3.Department of Forensic Medicine, College of MedicineNational Taiwan UniversityTaipeiRepublic of China
  4. 4.Taichung City Government Police DepartmentTaichungRepublic of China
  5. 5.College of Science & EngineeringFlinders UniversityAdelaideAustralia

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