Improved reverse transcription-recombinase polymerase amplification assay for blood mRNA screening: comparison with one-step RT-qPCR assay
Improved reverse transcription-recombinase polymerase amplification assay for blood mRNA screening: comparison with one-step RT-qPCR assay
复制标题
用于血液 mRNA 筛查的改进逆转录重组酶聚合酶扩增测定：与一步 RT-qPCR 测定的比较
DOI：
10.1016/j.fsigen.2022.102808
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发表时间：
2023
期刊：
影响因子：
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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
由于其敏感性、特异性和多路复用能力，mRNA谱分析在体液鉴定中是有效的。通常可以使用RT-qPCR、RT-PCR后毛细管电泳或靶向RNA测序来检测体液mRNA标记物。然而，由于涉及多个处理步骤，使用这些方法分析许多法医样本需要时间和精力。在这里，我们描述了一种快速而简单的检测血液mRNA标记物血红蛋白β(HBB)的方法，旨在用于最终血液鉴定之前的筛选。我们采用了逆转录重组酶聚合酶扩增（RT-RPA）方法，可以在单管20分钟内检测到目标mRNA。为了进行比较，我们使用了一步RT-qPCR检测。我们优化了RT-RPA法，发现它可以从10−3 - 10−4ng的白细胞RNA和大约10−3µL的血液中检测到thbb。灵敏度比一步RT-qPCR法低10倍，但高于确定血液鉴定的综合分析方法。因此，RT-RPA检测的快速和敏感性支持其作为筛选工具。我们还发现，RT-RPA检测对常见抑制剂如腐植酸、血红素、单宁酸和黑色素具有高度耐受性。考虑到抑制剂的耐受性，我们将一种简单的裂解方法（加入TCEP/EDTA并在95°C加热5分钟）整合到RT-RPA实验中，而不需要RNA纯化过程。该方法成功地检测了粗血样品中的dhbbin。我们的研究结果表明，hbbb的RT-RPA分析是一种很有前途的基于mrna的血液筛查策略。
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 markerhemoglobin 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 detectHBBfrom 10−3–10−4ng of leukocyte RNA and approximately 10−3µL 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 detectedHBBin crude blood samples. Our findings suggest that the RT-RPA assay forHBBis a promising strategy for mRNA-based blood screening.