Uracil-DNA glycosylase (UDG) plays essential roles in base excision repair (BER) pathway by eliminating uracil from DNA to sustain the genome integrity. Sensitive detection of UDG activity is of great significance in the study of many fundamental biochemical processes and clinical applications. We develop a label-free method for UDG activity detection using stem-loop primer-mediated exponential amplification (SPEA). In the presence of active UDG, the uracil base in helper hairpin probe (HP) can be excised to generate an abasic site (AP site), which can be cleaved by endonuclease IV (Endo IV) with a blocked primer released. This primer then triggers the strand displacement reaction to produce a dumb-bell structure DNA, which can initiate a loop-mediated isothermal amplification (LAMP) reaction. This reaction generates a large number of long double-strand DNA replicates, which can be stained by SYBR Green (SG) I to deliver enhanced fluorescence for quantitative detection of UDG activity. A linear range from 0.001 U/mL to 1 U/mL and a detection limit down to 0.00068 U/mL are achieved. This strategy has also been demonstrated for UDG assay in complex cell lysates, implying its great potential for UDG based clinical diagnostics and therapeutics.

中文翻译：

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基于茎环引物介导的指数扩增 (SPEA) 的尿嘧啶-DNA 糖基化酶活性检测的无标记和高灵敏度策略

尿嘧啶-DNA 糖基化酶 (UDG) 通过从 DNA 中消除尿嘧啶以维持基因组完整性，在碱基切除修复 (BER) 途径中发挥重要作用。UDG活性的灵敏检测在许多基础生化过程的研究和临床应用中具有重要意义。我们开发了一种使用茎环引物介导的指数扩增 (SPEA) 检测 UDG 活性的无标记方法。在存在活性 UDG 的情况下，辅助发夹探针 (HP) 中的尿嘧啶碱基可被切除以产生脱碱基位点 (AP 位点)，该位点可被内切核酸酶 IV (Endo IV) 切割并释放封闭的引物。然后，该引物触发链置换反应以产生哑铃结构 DNA，从而启动环介导的等温扩增 (LAMP) 反应。该反应产生大量长双链 DNA 复制品，可被 SYBR Green (SG) I 染色以提供增强的荧光，用于 UDG 活性的定量检测。实现了 0.001 U/mL 至 1 U/mL 的线性范围和低至 0.00068 U/mL 的检测限。该策略也已在复杂细胞裂解物中的 UDG 测定中得到证实，这意味着其在基于 UDG 的临床诊断和治疗方面具有巨大潜力。