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Improved reverse transcription-recombinase polymerase amplification assay for blood mRNA screening: comparison with one-step RT-qPCR assay

  • Seiji Kubo
    Correspondence
    Corresponding author at: Department of Clinical Laboratory and Molecular Pathology, Faculty of Medicine, Academic Assembly, University of Toyama, 2630 Sugitani, Toyama 930–0194, Japan.
    Affiliations
    Department of Clinical Laboratory and Molecular Pathology, Faculty of Medicine, Academic Assembly, University of Toyama, 2630 Sugitani, Toyama 930–0194, Japan

    Forensic Science Laboratory, Ishikawa Prefectural Police Headquarters, 1–1 Kuratsuki, Kanazawa 920–8553, Japan
    Search for articles by this author
  • Hideki Niimi
    Correspondence
    Corresponding author.
    Affiliations
    Department of Clinical Laboratory and Molecular Pathology, Faculty of Medicine, Academic Assembly, University of Toyama, 2630 Sugitani, Toyama 930–0194, Japan
    Search for articles by this author
  • Isao Kitajima
    Affiliations
    Administrative office, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan
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Published:November 20, 2022DOI:https://doi.org/10.1016/j.fsigen.2022.102808

      Highlights

      • An improved RT-RPA assay was developed to screen blood mRNA (HBB).
      • The RT-RPA assay could detect HBB in 10−4 ng of leukocyte RNA and 10−3 µL of blood.
      • The RT-RPA assay could directly detect HBB in blood samples heated in TCEP/EDTA.
      • The RT-RPA assay is a promising tool for blood mRNA screening.

      Abstract

      mRNA profiling is effective for body fluid identification because of its sensitivity, specificity, and multiplexing capability. Body fluid mRNA markers can typically be detected using RT-qPCR, RT-PCR followed by capillary electrophoresis, or targeted RNA sequencing. However, due to the multiple handling steps involved, the analysis of many forensic samples using these methods requires time and effort. Here, we describe a rapid and simple method for detecting the blood mRNA marker hemoglobin β (HBB), intended for use in screening before definitive blood identification. We employed a reverse transcription-recombinase polymerase amplification (RT-RPA) assay that can detect target mRNA within 20 min in a single tube. For comparison, we used a one-step RT-qPCR assay. We optimized the RT-RPA assay and found that it could detect HBB from 10−3–10−4 ng of leukocyte RNA and approximately 10−3 µL of blood. The sensitivity was 10-fold lower than that of the one-step RT-qPCR assay but higher than that of the comprehensive analysis methods for definitive blood identification. Thus, the rapidity and sensitivity of the RT-RPA assay support its use as a screening tool. We also found that the RT-RPA assay was highly tolerant to common inhibitors such as humic acid, hematin, tannic acid, and melanin. Considering the inhibitor tolerability, we integrated a simple lysis method (addition of TCEP/EDTA and heating at 95 °C for 5 min) without the RNA purification process into the RT-RPA assay. This direct assay successfully detected HBB in crude blood samples. Our findings suggest that the RT-RPA assay for HBB is a promising strategy for mRNA-based blood screening.

      Graphical Abstract

      Keywords

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