Rapid detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification

Body fluid identification is crucial for crime scene reconstruction. Recently, messenger RNA (mRNA) profiling has been an effective approach for body fluid identification. In general, mRNA is detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) or end-point RT-PCR; however, these conventional methods are time-consuming and require extensive sample processing. Therefore, we developed a rapid and simple method for the detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification (RT-RPA). First, we screened mRNA markers for blood and semen and selected hemoglobin beta (HBB) and protamine 1 (PRM1), respectively, based on amplification specificity. Under optimized conditions, our RT-RPA assay detected HBB and PRM1 mRNAs within 20 min at a constant temperature of 42 °C. The detection limits for the assay were 0.01 ng/µL leukocyte RNA for HBB and 0.2 ng/µL semen RNA for PRM1. In addition, our RT-RPA assay exhibited high specificity and accuracy for HBB and PRM1 mRNA detection from mixed samples. Furthermore, as RPA has been reported to possess inhibitor tolerance, we evaluated the feasibility of direct RT-RPA for HBB mRNA detection. This direct approach reduced the number of processing steps and time required for template preparation and enabled the successful detection of HBB mRNA within 45 min from sample preparation. These findings suggest that RT-RPA is a useful method for mRNA-based blood and semen identification.