Rapid detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification
Rapid detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification
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通过逆转录重组酶聚合酶扩增快速检测血液和精液 mRNA 标记
DOI：
10.1016/j.fsigen.2022.102665
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Kitajima Isao
中科院分区：
文献类型：
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作者：
Kubo Seiji;Niimi Hideki;Kitajima Isao
体液鉴定对犯罪现场重建至关重要。近年来，信使RNA （mRNA）谱分析已成为体液鉴定的有效方法。一般情况下，通过实时定量逆转录聚合酶链反应（RT-qPCR）或终点RT-PCR检测mRNA；然而，这些传统的方法是耗时的，需要大量的样品处理。为此，我们建立了一种快速简便的逆转录重组酶扩增（RT-RPA）检测血液和精液mRNA标记物的方法。首先，我们筛选了血液和精液中的mRNA标记物，并根据扩增特异性分别选择了血红蛋白β (HBB)和鱼精蛋白1 （PRM1）。在优化的条件下，我们的RT-RPA检测在42℃恒温下20分钟内检测到dhbbandprm1mrna。hbband的检测限为0.01 ng/µL白细胞RNA， prm1的检测限为0.2 ng/µL精液RNA。此外，我们的RT-RPA检测在混合样品中检测hbbandprm1mrna具有很高的特异性和准确性。此外，由于RPA已被报道具有抑制剂耐受性，我们评估了直接RT-RPA检测hbbmrna的可行性。这种直接方法减少了模板制备所需的处理步骤和时间，并在样品制备后45分钟内成功检测到hbbmrna。这些发现表明，RT-RPA是一种有用的基于mrna的血液和精液鉴定方法。
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 detectedHBBandPRM1mRNAs within 20 min at a constant temperature of 42 °C. The detection limits for the assay were 0.01 ng/µL leukocyte RNA forHBBand 0.2 ng/µL semen RNA forPRM1. In addition, our RT-RPA assay exhibited high specificity and accuracy forHBBandPRM1mRNA detection from mixed samples. Furthermore, as RPA has been reported to possess inhibitor tolerance, we evaluated the feasibility of direct RT-RPA forHBBmRNA detection. This direct approach reduced the number of processing steps and time required for template preparation and enabled the successful detection ofHBBmRNA within 45 min from sample preparation. These findings suggest that RT-RPA is a useful method for mRNA-based blood and semen identification.