In this report, a simple, label-free and highly efficient nucleic acid amplification technique is developed for ultrasensitive detection of single-nucleotide polymorphism (SNP). Briefly, a designed padlock probe is first circularized by a DNA ligase when it perfectly complements to a mutant gene. Then, the mutant gene functions as a primer to initiate branched rolling circle amplification reaction (BRCA), generating a large number of branched DNA strands and a lot of pyrophosphate molecules which is equivalent to the number of nucleotides consumed. With the addition of a terpyridine–Zn(II) complex, pyrophosphate molecules can be sensitively detected owing to the formation of a fluorescent terpyridine–Zn(II)–pyrophosphate complex. The fluorescence intensity is directly associated with the content of the mutant gene in a sample solution. On the other hand, the circulation of the padlock probe is prohibited when it hybridizes with the wild-type gene. In this assay, the accumulative nature of the BRCA process produces a detection limit of 0.1 pM and an excellent selectivity factor of 1000 toward SNP. As little as 0.1% mutant in the wild-type gene can be successfully detected. The simple procedure, high sensitivity, and high selectivity of this assay offer a potentially viable alternative for routine SNP analysis.

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在本报告中，开发了一种简单，无标记且高效的核酸扩增技术，用于单核苷酸多态性（SNP）的超灵敏检测。简而言之，当设计的挂锁探针与突变基因完美互补时，首先要用DNA连接酶将其环化。然后，突变基因充当引物，引发分支滚环扩增反应（BRCA），产生大量分支DNA链和大量焦磷酸盐分子，相当于消耗的核苷酸数量。加入三联吡啶-Zn（II）络合物后，由于形成了荧光三联吡啶-Zn（II）-焦磷酸盐络合物，因此可以灵敏地检测到焦磷酸盐分子。荧光强度与样品溶液中突变基因的含量直接相关。另一方面，当挂锁探针与野生型基因杂交时，其循环被禁止。在此分析中，BRCA过程的累积性质产生了0.1 pM的检测限和对SNP的1000的出色选择性。可以成功检测到野生型基因中仅有0.1％的突变体。该测定法的简单程序，高灵敏度和高选择性为常规SNP分析提供了潜在可行的替代方法。

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