Sensitive and accurate identification of single-nucleotide mutations (SNMs) has become crucial in the field of molecular diagnosis and personalized medicine. Here, we developed a highly sensitive fluorometric assay for detecting multiple SNMs by tandem gene amplification; reverse transcription PCR (RT-PCR) coupled with rolling circle amplification generating G-quadruplex (GQ-RCA). In tandem gene amplification, RT-PCR-amplified DNA from sample RNA was digested with lambda exonuclease, which degraded the strand harboring the phosphorylated 5′-end of amplicon DNA to generate single-stranded DNA (ssDNA). The resulting ssDNA was hybridized with padlock probe DNA, which can discriminate a single base mismatch. Depending on the presence of mismatched bases between the amplicon ssDNA and padlock probe DNA, ligation of both ends of the padlock DNA was evaluated. If ligation occurred, a circular form of padlock DNA was generated, which was eligible for RCA. The RCA process generates long stretches of ssDNA containing tandem repeats of G-quadruplex structures. The amplified ssDNA harboring G-quadruplex was fluorescently visualized and quantified using Thioflavin T fluorophore. Our assay detected mutant RNA containing an SNM as low as 8.3 fg and detected mutant RNA with a concentration as low as 0.53% in the mixture with wild-type RNA. The fluorometric SNM detection method with tandem gene amplification was successfully applied for multiplex detection of SNMs in clinical samples derived from patients with chronic myeloid leukemia. Our method would be useful in clinical diagnosis for early detection and monitoring of multiple SNMs in cancer patients with proper treatment.

敏感和准确地鉴定单核苷酸突变（SNM）在分子诊断和个性化医学领域已变得至关重要。在这里，我们开发了一种高灵敏的荧光测定法，可通过串联基因扩增检测多个SNM。逆转录PCR（RT-PCR）结合滚环扩增产生G-四链体（GQ-RCA）。在串联基因扩增中，用λ核酸外切酶消化来自样品RNA的RT-PCR扩增的DNA，该酶降解包含扩增子DNA磷酸化5'端的链，生成单链DNA（ssDNA）。将所得的ssDNA与锁式探针DNA杂交，可以区分单个碱基错配。根据扩增子ssDNA和挂锁探针DNA之间是否存在错配的碱基，评估了挂锁DNA两端的连接。如果发生连接，则会生成圆形形式的挂锁DNA，可以用于RCA。RCA过程产生长段的ssDNA，其中包含G-四链体结构的串联重复序列。使用硫黄素T荧光团荧光可视化并定量了带有G-四链体的扩增的ssDNA。我们的检测方法检测到含有SNM值低至8.3 fg的突变RNA，并检测到与野生型RNA混合物的浓度低至0.53％的突变RNA。具有串联基因扩增的荧光SNM检测方法已成功地用于慢性髓样白血病患者临床样品中SNM的多重检测。