The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3′end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.

中文翻译：

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RNase H 依赖性扩增提高滚环扩增结合环介导等温扩增 (RCA-LAMP) 的准确性

结合滚环扩增和环介导等温扩增 (RCA-LAMP) 的混合方法被开发用于量化非常少量的不同类型的 RNA，例如 miRNA。RCA-LAMP 可以通过挂锁探针 (PLP) 环化帮助检测短序列，并表现出强大的 DNA 扩增。然而，决定 RCA-LAMP 检测限的因素之一是非特异性扩增。在这项研究中，我们通过应用 RNase H 依赖 PCR (rhPCR) 技术提高了 RCA-LAMP 的准确性。在该方法中，通过使用rh引物抑制非特异性扩增，该引物通过阻断3'端的修饰以停止DNA聚合酶反应并使用RNase H2酶用RNA替换从末端开始的第6个DNA分子而设计。传统的 RCA-LAMP 在没有靶向反应的情况下从线性 PLP 扩增非特异性扩增子，而带有 rh 引物和 RNase H2 的 RCA-LAMP 抑制非特异性扩增。相反，我们确定了在 RNA 靶向步骤中使用夹板 R 连接酶进行 PLP 环化反应所带来的风险，即使在 RNA 阴性条件下也会发生，这是确定 RCA-LAMP 检测限的另一个因素。因此，本研究有助于提高使用 RCA-LAMP 进行 RNA 定量的准确性。这是决定 RCA-LAMP 检测限的另一个因素。因此，本研究有助于提高使用 RCA-LAMP 进行 RNA 定量的准确性。这是决定 RCA-LAMP 检测限的另一个因素。因此，本研究有助于提高使用 RCA-LAMP 进行 RNA 定量的准确性。