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Ultrasensitive Detection of RNA with Single-Base Resolution by Coupling Electrochemical Sensing Strategy with Chimeric DNA Probe-Aided Ligase Chain Reaction
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-12-07 , DOI: 10.1021/acs.analchem.0c03563
Guang-Xian Zhong 1 , Chen-Liu Ye 2 , Hong-Xiang Wei 1 , Liang-Yong Yang 3 , Qing-Xia Wei 3 , Zhou-Jie Liu 3 , Leng-Xi Fu 4 , Xin-Hua Lin 5 , Jin-Yuan Chen 4
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-12-07 , DOI: 10.1021/acs.analchem.0c03563
Guang-Xian Zhong 1 , Chen-Liu Ye 2 , Hong-Xiang Wei 1 , Liang-Yong Yang 3 , Qing-Xia Wei 3 , Zhou-Jie Liu 3 , Leng-Xi Fu 4 , Xin-Hua Lin 5 , Jin-Yuan Chen 4
Affiliation
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Accurate and sensitive detection of single-base mutations in RNAs is of great value in basic studies of life science and medical diagnostics. However, the current available RNA detection methods are challenged by heterogeneous clinical samples in which trace RNA mutants usually existed in a large pool of normal wild sequences. Thus, there is still great need for developing the highly sensitive and highly specific methods in detecting single-base mutations of RNAs in heterogeneous clinical samples. In the present study, a new chimeric DNA probe-aided ligase chain reaction-based electrochemical method (cmDNA-eLCR) was developed for RNA mutation detection through the BSA-based carrier platform and the horseradish peroxidase-hydrogen peroxide-tetramethylbenzidine (HRP-H2O2-TMB) system. The denaturing polyacrylamide gel electrophoresis and a fluorophore-labeled probe was ingeniously designed to demonstrate the advantage of cmDNA in ligation to normal DNA templated by RNA with the catalysis of T4 RNA ligase 2 as well as its higher selectivity than DNA ligase system. Finally, the proposed cmDNA-eLCR, compared with the traditional eLCR, showed excellent performance in discriminating single base-mismatched sequences, where the signal response for mismatched targets at a high concentration could overlap completely with that for the blank control. Besides, this cmDNA-eLCR assay had a wide linear range crossing six orders of magnitude from 1.0 × 10–15 to1.0 × 10–10 M with a limit of detection as low as 0.6 fM. Furthermore, this assay was applied to detect RNA in real sample with a satisfactory result, thereby demonstrating its great potential in diagnosis of RNA-related diseases.
中文翻译：
嵌合DNA探针辅助的连接酶链反应的耦合电化学传感策略超灵敏地检测具有单碱基分辨率的RNA。
准确，灵敏地检测RNA中单碱基突变在生命科学和医学诊断的基础研究中具有重要价值。然而，当前可用的RNA检测方法受到异质临床样品的挑战，其中痕量RNA突变体通常存在于大量正常野生序列中。因此，仍然非常需要开发用于检测异质临床样品中RNA的单碱基突变的高度灵敏和高度特异性的方法。在本研究中，开发了一种新的基于嵌合DNA探针的连接酶链反应电化学方法（cmDNA-eLCR），用于通过基于BSA的载体平台和辣根过氧化物酶-过氧化氢-四甲基联苯胺（HRP-H）进行RNA突变检测。2 O 2-TMB）系统。变性聚丙烯酰胺凝胶电泳和荧光团标记的探针经过精心设计，以证明cmDNA在与RNA模板化的正常DNA连接中具有T4 RNA连接酶2的催化优势，并且具有比DNA连接酶系统更高的选择性。最后，与传统的eLCR相比，建议的cmDNA-eLCR在区分单个碱基不匹配的序列中表现出出色的性能，其中高浓度错配靶标的信号响应可能与空白对照的信号响应完全重叠。此外，此cmDNA-eLCR分析的线性范围很宽，从1.0×10 –15到1.0 ×10 –10六个数量级M，检出限低至0.6 fM。此外，该测定法被用于检测真实样品中的RNA，结果令人满意，从而证明了其在诊断RNA相关疾病中的巨大潜力。
更新日期：2021-01-19
中文翻译：
嵌合DNA探针辅助的连接酶链反应的耦合电化学传感策略超灵敏地检测具有单碱基分辨率的RNA。
准确，灵敏地检测RNA中单碱基突变在生命科学和医学诊断的基础研究中具有重要价值。然而，当前可用的RNA检测方法受到异质临床样品的挑战，其中痕量RNA突变体通常存在于大量正常野生序列中。因此，仍然非常需要开发用于检测异质临床样品中RNA的单碱基突变的高度灵敏和高度特异性的方法。在本研究中，开发了一种新的基于嵌合DNA探针的连接酶链反应电化学方法（cmDNA-eLCR），用于通过基于BSA的载体平台和辣根过氧化物酶-过氧化氢-四甲基联苯胺（HRP-H）进行RNA突变检测。2 O 2-TMB）系统。变性聚丙烯酰胺凝胶电泳和荧光团标记的探针经过精心设计，以证明cmDNA在与RNA模板化的正常DNA连接中具有T4 RNA连接酶2的催化优势，并且具有比DNA连接酶系统更高的选择性。最后，与传统的eLCR相比，建议的cmDNA-eLCR在区分单个碱基不匹配的序列中表现出出色的性能，其中高浓度错配靶标的信号响应可能与空白对照的信号响应完全重叠。此外，此cmDNA-eLCR分析的线性范围很宽，从1.0×10 –15到1.0 ×10 –10六个数量级M，检出限低至0.6 fM。此外，该测定法被用于检测真实样品中的RNA，结果令人满意，从而证明了其在诊断RNA相关疾病中的巨大潜力。
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