Abstract
Identifying body fluids from forensic samples can provide valuable evidence for criminal investigations. Messenger RNA (mRNA)-based body fluid identification was recently developed, and highly sensitive parallel identification using reverse transcription polymerase chain reaction (RT-PCR) has been described. In this study, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a simple, rapid assay for identifying three common forensic body fluids, namely blood, semen, and saliva, and evaluated its specificity and sensitivity. Hemoglobin beta (HBB), transglutaminase 4 (TGM4), and statherin (STATH) were selected as marker genes for blood, semen, and saliva, respectively. RT-LAMP could be performed in a single step including both reverse transcription and DNA amplification under an isothermal condition within 60 min, and detection could be conveniently performed via visual fluorescence. Marker-specific amplification was performed in each assay, and no cross-reaction was observed among five representative forensically relevant body fluids. The detection limits of the assays were 0.3 nL, 30 nL, and 0.3 μL for blood, semen, and saliva, respectively, and their sensitivities were comparable with those of RT-PCR. Furthermore, RT-LAMP assays were applicable to forensic casework samples. It is considered that RT-LAMP is useful for body fluid identification.
Similar content being viewed by others
References
Gaensslen RE. Sourcebook in forensic serology, immunology, and biochemistry. Washington DC: National Institute of Justice; 1983.
Kishi K, Takizawa H, Yamamoto S. Forensic serology: illustrated technical manual. Kanehara: Tokyo; 1990.
Virkler K, Lednev IK. Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int. 2009;188:1–17.
Gefrides L, Welch K. Forensic biology: serology and DNA. In: Mozayani A, Noziglia C, editors. The forensic laboratory handbook procedure and practice. New York: Humana Press; 2011. p. 15–50.
Li R. Forensic biology. 2nd ed. Boca Raton: CRC Press; 2015.
Juusola J, Ballantyne J. Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Sci Int. 2003;135:85–96.
Juusola J, Ballantyne J. Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int. 2005;152:1–12.
Nussbaumer C, Gharehbaghi-Schnell E, Korschineck I. Messenger RNA profiling: a novel method for body fluid identification by real-time PCR. Forensic Sci Int. 2006;157:181–6.
Juusola J, Ballantyne J. mRNA profiling for body fluid identification by multiplex quantitative RT-PCR. J Forensic Sci. 2007;52:1252–62.
Sakurada K, Ikegaya H, Fukushima H, Akutsu T, Watanabe K, Yoshino M. Evaluation of mRNA-based approach for identification of saliva and semen. Legal Med (Tokyo). 2009;11:125–8.
Sakurada K, Akutsu T, Watanabe K, Fujinami Y, Yoshino M. Expression of statherin mRNA and protein in nasal and vaginal secretions. Legal Med (Tokyo). 2011;13:309–13.
Richard M, Harper K, Craig R, Onorato A, Robertson J, 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.
Sakurada K, Akutsu T, Watanabe K, Miyasaka S, Kasai K. Identification of body fluid stains using real-time RT-PCR: discrimination between salivary, nasal, and vaginal secretions. Jpn J Forensic Sci Technol. 2013;18:1–11.
Xu Y, Xie J, Cao Y, Zhou H, Ping Y, Chen L, et al. Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification. PLoS One. 2014;9:e100123.
Matsumura S, Matsusue A, Waters B, Kashiwagi M, Hara K, Kubo S. Application of mRNA expression analysis to human blood identification in degenerated samples that were false-negative by immunochromatography. J Forensic Sci. 2016;61:903–12.
Hanson E, Lubenow H, Ballantyne J. Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Anal Biochem. 2009;387:303–14.
Zubakov D, Boersma AWM, Choi Y, van Kuijk PF, Wiemer EAC, Kayser M. MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. Int J Legal Med. 2010;124:217–26.
Lee HY, Park MJ, Choi A, An JH, Yang WI, Shin KJ. Potential forensic application of DNA methylation profiling to body fluid identification. Int J Legal Med. 2012;126:55–62.
Wasserstorm A, Frumkin D, Davidson A, Shpitzen M, Herman Y, Gafny R. Demonstration of DSI-semen-A novel DNA methylation-based forensic semen identification assay. Forensic Sci Int Genet. 2013;7:136–42.
Matsubara Y, Ikeda H, Endo H, Narisawa K. Dried blood spot on filter paper as a source of mRNA. Nucleic Acids Res. 1992;20:1998.
Karlsson H, Guthenberg C, von Dobeln U, Kristenssson K. Extraction of RNA from dried blood on filter papers after long-term storage. Clin Chem. 2003;49:979–81.
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.
Zubakov D, Kokshoorn M, Kloosterman A, Kayser M. New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains. Int J Legal Med. 2009;123:71–4.
Alvarez M, Juusola J, Ballantyne J. An mRNA and DNA co-isolation method for forensic casework samples. Anal Biochem. 2004;335:289–98.
Watanabe K, Iwashima Y, Akutsu T, Sekiguchi K, Sakurada K. Evaluation of a co-extraction method for real-time PCR-based body fluid identification and DNA typing. Legal Med (Tokyo). 2014;16:56–9.
Bowden A, Fleming R, Harbison S. A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ™ system. Forensic Sci Int Genet. 2011;5:64–8.
Akutsu T, Kitayama T, Watanabe K, Sakurada K. Comparison of automated and manual purification of total RNA for mRNA-based identification of body fluids. Forensic Sci Int Genet. 2015;14:11–7.
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.
Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. Loop-mediated isothermal amplification reaction using a nondenatured template. Clin Chem. 2001;47:1742–3.
Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun. 2001;289:150–4.
Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002;16:223–9.
Mori Y, Kitao M, Tomita N, Notomi T. Real-time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods. 2004;59:145–57.
Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc. 2008;3:877–82.
Poon LLM, Leung CSW, Tashiro M, Chan KH, Wong BWY, Yuen KY, et al. Rapid detection of the severe acute respiratory syndrome (SARS) coronavirus by a loop-mediated isothermal amplification assay. Clin Chem. 2004;50:1050–2.
Hara-Kudo Y, Yoshino M, Kojima T, Ikedo M. Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol Lett. 2005;253:155–61.
Annaka T, Yoshino M, Momoda T, Nemoto J, Sunada A, Kojima T, et al. Simple detection of Legionella species by LAMP, a new DNA amplification method. J Jpn Soc Clin Microbiol. 2003;13:19–25.
Nakahara H, Mizuno N, Fujii K, Sekiguchi K. Human DNA specific detection from forensic biological samples. Rep Natl Res Inst Police Sci. 2007;58:66–74.
Tanaka J. ABO genotyping by loop-mediated isothermal amplification (LAMP). Jpn J Forensic Sci Technol. 2009;14:1–9.
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(suppl s1):S158–61.
Su C, Li C, Lee JC, Ji D, Li S, 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.
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. 2017; https://doi.org/10.1007/s00414-017-1661-z.
Sun B, Shen F, McCalla SE, Kreutz JE, Karymov MA, Ismagilov RF. Mechanistic evaluation of the pros and cons of digital RT-LAMP for HIV-1 viral load quantification on a microfluidc device and improved efficiency via a two-step digital protocol. Anal Chem. 2013;85:1540–6.
Myers FB, Henrikson RH, Bone J, Lee LP. A handheld point-of-care genomic diagnostic system. PLoS One. 2013;8:e70266.
Damhorst GL, Duarte-Guevara C, Chen W, Ghonge T, Cunningham BT, Bashir R. Smartphone-imaged HIV-1 reverse-transcription loop-mediated isothermal amplification (RT-LAMP) on a chip from whole blood. Engineering. 2015;1:324–35.
Durate C, Salm E, Dorvel B, Reddy B Jr, Bashir R. On-chip parallel detection of foodborne pathogens using loop-mediated isothermal amplification. Biomed Microdevices. 2013;15:821–30.
Acknowledgements
The real-time RT-LAMP measurements were performed at the Kumamoto Prefectural Institute of Public-Health and Environment Science.
Funding
This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP26933005 and JP15H00672.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
All participants in this study provided informed consent, and the study design was approved by the Human Genome/Gene Analysis Research Ethics Committee of the Japanese Association of Forensic Science and Technology. Written informed consent was obtained from each participant.
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Satoh, T., Kouroki, S., Ogawa, K. et al. Development of mRNA-based body fluid identification using reverse transcription loop-mediated isothermal amplification. Anal Bioanal Chem 410, 4371–4378 (2018). https://doi.org/10.1007/s00216-018-1088-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-018-1088-5