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Development of mRNA-based body fluid identification using reverse transcription loop-mediated isothermal amplification

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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.

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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.

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Authors and Affiliations

  1. Forensic Science Laboratory, Kumamoto Prefectural Police Headquarters, 6-18-1 Suizenji, Chuo-ku Kumamoto-shi, Kumamoto, 862-8610, Japan

    Tetsuya Satoh, Seiya Kouroki, Keita Ogawa, Yorika Tanaka, Kazutoshi Matsumura & Susumu Iwase

Authors
  1. Tetsuya Satoh
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  2. Seiya Kouroki
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  3. Keita Ogawa
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  4. Yorika Tanaka
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  5. Kazutoshi Matsumura
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  6. Susumu Iwase
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Corresponding author

Correspondence to Tetsuya Satoh.

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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.

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The authors declare that they have no conflict of interest.

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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

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  • DOI: https://doi.org/10.1007/s00216-018-1088-5

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