Rapid and direct detection of male DNA by recombinase polymerase amplification assay
Rapid and direct detection of male DNA by recombinase polymerase amplification assay
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DOI：
10.1016/j.fsigen.2022.102704
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发表时间：
2022-04-11
影响因子：
3.1
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
男性DNA筛查在法医调查中非常重要，特别是性侵犯案件。实时定量聚合酶链反应（qPCR）广泛用于男性DNA的检测。然而，使用这种技术作为筛选工具是费时费力的。在这项研究中，我们建立了一个重组酶聚合酶扩增（RPA）检测靶向的多拷贝位点的Y染色体上的男性DNA的快速检测（简称为Y-RPA）。Y-RPA检测能够在不到20分钟的时间内检测出男性DNA，灵敏度为0.025-0.005 ng/µL。此外，Y-RPA检测对抑制剂具有高度耐受性;在高达1000 ng/µL腐殖酸、250 µM靛蓝胭脂红和500 µM血红素的存在下可检测到男性DNA。然后，考虑到其对抑制剂的耐受性，我们研究了直接Y-RPA测定的可行性。采用碱裂解方案（添加氢氧化钠并在95 °C下加热5分钟）来制备DNA模板。Y-RPA检测使用从血液、唾液和精液样本中提取的粗DNA成功检测出男性DNA。该方法能够在约30分钟内筛选雄性DNA（5分钟用于裂解，20分钟用于Y-RPA）。这些发现表明，Y-RPA检测是一种有前途的筛选工具，快速，简单，有效地检测男性DNA。
Screening of male DNA is important in forensic investigations, especially sexual assault cases. Quantitative real-time polymerase chain reaction (qPCR) is widely used for the detection of male DNA. However, the use of this technique as a screening tool is time-consuming and labor-intensive. In this study, we established a recombinase polymerase amplification (RPA) assay targeting the multicopy loci on the Y-chromosome for the rapid detection of male DNA (referred to as Y-RPA). The Y-RPA assay was able to detect male DNA in less than 20 min with a sensitivity of 0.025–0.005 ng/µL. Additionally, the Y-RPA assay was highly tolerant to inhibitors; male DNA was detectable in the presence of up to 1000 ng/µL humic acid, 250 µM indigo carmine, and 500 µM hematin. Then, considering its tolerance to inhibitors, we examined the feasibility of the direct Y-RPA assay. The alkaline lysis protocol (addition of sodium hydroxide and heating at 95 °C for 5 min) was employed for preparing the DNA template. The Y-RPA assay successfully detected male DNA using crude DNA extracted from blood, saliva, and semen samples. This approach enabled the screening of male DNA within approximately 30 min (5 min for lysis and 20 min for Y-RPA). These findings suggest that the Y-RPA assay is a promising screening tool for the rapid, simple, and efficient detection of male DNA.