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
Graphical Abstract
Keywords
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Article info
Publication history
Published online: November 20, 2022
Accepted:
November 18,
2022
Received in revised form:
October 7,
2022
Received:
July 7,
2022
Identification
Copyright
© 2022 Elsevier B.V. All rights reserved.
