- •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.
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.
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- Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification.Forensic Sci. Int. 2003; 135: 85-96https://doi.org/10.1016/S0379-0738(03)00197-X
- Multiplex mRNA profiling for the identification of body fluids.Forensic Sci. Int. 2005; 152: 1-12https://doi.org/10.1016/j.forsciint.2005.02.020
- Messenger RNA profiling: a novel method for body fluid identification by real-time PCR.Forensic Sci. Int. 2006; 157: 181-186https://doi.org/10.1016/j.forsciint.2005.10.009
- mRNA profiling for body fluid identification by multiplex quantitative RT-PCR.J. Forensic Sci. 2007; 52: 1252-1262https://doi.org/10.1111/j.1556-4029.2007.00550.x
- mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR.Forensic Sci. Int. Genet. 2009; 3: 80-88https://doi.org/10.1016/j.fsigen.2008.11.003
- mRNA profiling for the identification of sperm and seminal plasma.Forensic Sci. Int. Genet. Suppl. Ser. 2009; 2: 534-535https://doi.org/10.1016/j.fsigss.2009.08.109
- The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.Forensic Sci. Int. Genet. 2010; 4: 244-256https://doi.org/10.1016/j.fsigen.2009.10.006
- Selection of highly specific and sensitive mRNA biomarkers for the identification of blood.Forensic Sci. Int. Genet. 2011; 5: 449-458https://doi.org/10.1016/j.fsigen.2010.09.006
- Successful mRNA profiling of 23 years old blood stains.Forensic Sci. Int. Genet. 2012; 6: 274-276https://doi.org/10.1016/j.fsigen.2011.04.007
- A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.Forensic Sci. Int. Genet. 2012; 6: 565-577https://doi.org/10.1016/j.fsigen.2012.01.009
- A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results.Forensic Sci. Int. Genet. 2014; 10: 40-48https://doi.org/10.1016/j.fsigen.2014.01.006
- A 17-month time course study of human RNA and DNA degradation in body fluids under dry and humid environmental conditions.Int. J. Leg. Med. 2016; 130: 1431-1438https://doi.org/10.1007/s00414-016-1373-9
- Identification of aged bloodstains through mRNA profiling: experiments results on selected markers of 30- and 50-year-old samples.Forensic Sci. Int. 2017; 272: e1-e6https://doi.org/10.1016/j.forsciint.2017.01.006
- Predicting the origin of stains from next generation sequencing mRNA data.Forensic Sci. Int. Genet. 2018; 34: 37-48https://doi.org/10.1016/j.fsigen.2018.01.001
- Body fluid identification using a targeted mRNA massively parallel sequencing approach – results of a EUROFORGEN/EDNAP collaborative exercise.Forensic Sci. Int. Genet. 2018; 34: 105-115https://doi.org/10.1016/j.fsigen.2018.01.002
- Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing.Forensic Sci. Int. Genet. 2018; 34: 206-221https://doi.org/10.1016/j.fsigen.2018.02.020
- Body fluid identification and assignment to donors using a targeted mRNA massively parallel sequencing approach – results of a second EUROFORGEN / EDNAP collaborative exercise.Forensic Sci. Int. Genet. 2020; 45102208https://doi.org/10.1016/j.fsigen.2019.102208
- Recombinase polymerase amplification for diagnostic applications.Clin. Chem. 2016; 62: 947-958https://doi.org/10.1373/clinchem.2015.245829
- Recombinase polymerase amplification: basics, applications and recent advances.Trends Anal. Chem. 2018; 98: 19-35https://doi.org/10.1016/j.trac.2017.10.015
- DNA detection using recombination proteins.PLoS Biol. 2006; 4: 1115-1121https://doi.org/10.1371/journal.pbio.0040204
TwistDx,TwistAmp® DNA Amplification Kits Combined Instruction Manual. https://www.twistdx.co.uk/wp-content/uploads/2021/04/ta01cmanual-combined-manual_revo_v1-3b.pdf.
TwistDx, TwistAmp® DNA Amplification Kits Assay Design Manual. https://www.twistdx.co.uk/wp-content/uploads/2021/04/twistamp-assay-design-manual-v2-5.pdf.
- Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples.J. Mol. Diagn. 2014; 16: 127-135https://doi.org/10.1016/j.jmoldx.2013.08.003
- A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria.Lab Chip. 2016; 16: 2309-2316https://doi.org/10.1039/c6lc00329j
- Development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses.Sci. Rep. 2017; 740244https://doi.org/10.1038/srep40244
- Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae.Mol. Cell. Probes. 2017; 35: 44-56https://doi.org/10.1016/j.mcp.2017.06.004
- Rapid and direct detection of male DNA by recombinase polymerase amplification assay.Forensic Sci. Int. Genet. 2022; 59102704https://doi.org/10.1016/j.fsigen.2022.102704
- Rapid detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification.Forensic Sci. Int. Genet. 2022; 58102665https://doi.org/10.1016/j.fsigen.2022.102665
- Field-deployable viral diagnostics using CRISPR-Cas13.Science. 2018; 360: 444-448https://doi.org/10.1126/science.aas8836
- SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification.Proc. Natl. Acad. Sci. USA. 2020; 117: 24450-24458https://doi.org/10.1073/pnas.2011221117
- Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria.Proc. Natl. Acad. Sci. USA. 2020; 117: 25722-25731https://doi.org/10.1073/pnas.2010196117
- An enhanced isothermal amplification assay for viral detection.Nat. Commun. 2020; 11https://doi.org/10.1038/s41467-020-19258-y
- A study of PCR inhibition mechanisms using real time PCR.J. Forensic Sci. 2010; 55: 25-33https://doi.org/10.1111/j.1556-4029.2009.01245.x
- Integrating reverse transcription recombinase polymerase amplification with CRISPR technology for the one-tube assay of RNA.Anal. Chem. 2021; 93: 12808-12816https://doi.org/10.1021/acs.analchem.1c03456
- Enhanced isothermal amplification for ultrafast sensing of SARS-CoV-2 in mcrodroplets.Anal. Chem. 2022; 94: 4135-4140https://doi.org/10.1021/acs.analchem.2c00008
- Identification of nasal blood by real-time RT-PCR.Leg. Med. 2012; 14: 201-204https://doi.org/10.1016/j.legalmed.2012.01.014
- A novel application of real-time RT-LAMP for body fluid identification: using HBB detection as the model.Forensic Sci. Med. Pathol. 2015; 11: 208-215https://doi.org/10.1007/s12024-015-9668-6
- Development of mRNA-based body fluid identification using reverse transcription loop-mediated isothermal amplification.Anal. Bioanal. Chem. 2018; 410: 4371-4378https://doi.org/10.1007/s00216-018-1088-5
- The detection and identification of saliva in forensic samples by RT-LAMP.Forensic Sci. Med. Pathol. 2018; 14: 469-477https://doi.org/10.1007/s12024-018-0008-5
- A novel loop-mediated isothermal amplification method for identification of four body fluids with smartphone detection.Forensic Sci. Int. Genet. 2020; 45102195https://doi.org/10.1016/j.fsigen.2019.102195
- Optimization of novel loop-mediated isothermal amplification with colorimetric image analysis for forensic body fluid identification.J. Forensic Sci. 2021; 66: 1033-1041https://doi.org/10.1111/1556-4029.14682
- Developmental validation of the Quantifiler® HP and Trio Kits for human DNA quantification in forensic samples.Forensic Sci. Int. Genet. 2016; 21: 145-157https://doi.org/10.1016/j.fsigen.2015.12.007
- Human DNA quantification and sample quality assessment: developmental validation of the PowerQuant® system.Forensic Sci. Int. Genet. 2016; 23: 166-177https://doi.org/10.1016/j.fsigen.2016.04.007
- Isothermal DNA amplification strategies for duplex microorganism detection.Food Chem. 2015; 174: 509-515https://doi.org/10.1016/j.foodchem.2014.11.080
- Isothermal solid-phase amplification system for detection of Yersinia pestis.Anal. Bioanal. Chem. 2016; 408: 671-676https://doi.org/10.1007/s00216-015-9177-1
- Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection.Microchim. Acta. 2017; 184: 1453-1462https://doi.org/10.1007/s00604-017-2144-0
- Nucleic acid detection with CRISPR-Cas13a/C2c2.Science. 2017; 356: 438-442https://doi.org/10.1126/science.aam9321
Published online: November 20, 2022
Accepted: November 18, 2022
Received in revised form: October 7, 2022
Received: July 7, 2022
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