In this study, a fluorescent sensing strategy has been developed for rapid and ultrasensitive detection of BCR-ABL1 mRNA in chronic myeloid leukemia (CML) based on DNAzyme Cleavage-induced Rolling circle amplification (DCR for short). In the presence of BCR-ABL1 mRNA, DNAzyme was activated to split the target sequence into two fragments, producing the 3′ terminus on the forward cleavage fragments. After T4 polynucleotide kinase (PNK) modification reaction, the forward cleavage fragments were extended by rolling circle amplification (RCA). Plenty of long single DNA strands were produced and partially hybridized with the fluorescence-quenching decorator probes, thus inducing the separation of fluorophore and quencher decorator probes and recovery of fluorescence. Highly sensitive detection of BCR-ABL1 was achieved with a limit of detection at 9.4 fM. In addition, the DCR strategy was adopted to successfully in situ image the BCR-ABL1 mRNA in the cytoplasm of human leukemia bone marrow cells. Moreover, results of the BCR-ABL1 mRNA expression in clinical samples achieved by DCR sensing were well consistent with that of reverse transcription PCR (RT-PCR) and fluorescence in situ hybridization (FISH) analysis. Therefore, this developed DCR sensing strategy might provide a potential alternative tool for precise diagnosis of CML.

在这项研究中，开发了一种荧光传感策略，用于基于DNAzyme裂解诱导的滚环扩增（简称DCR）的快速超灵敏检测慢性粒细胞白血病（CML）中的BCR-ABL1 mRNA。在存在BCR-ABL1 mRNA的情况下，DNAzyme被激活以将靶序列分为两个片段，在正向裂解片段上产生3'末端。在T4多核苷酸激酶（PNK）修饰反应后，正向裂解片段通过滚环扩增（RCA）进行延伸。产生了许多长的单DNA链，并与荧光猝灭修饰子探针部分杂交，从而诱导了荧光团和淬灭修饰子探针的分离以及荧光的恢复。BCR-ABL1的高灵敏度检测达到了9.4 fM的检测限。人白血病骨髓细胞胞浆中的BCR-ABL1 mRNA原位成像。此外，通过DCR传感获得的临床样品中BCR-ABL1 mRNA表达的结果与逆转录PCR（RT-PCR）和荧光原位杂交（FISH）分析的结果非常一致。因此，这种发达的DCR传感策略可能为CML的精确诊断提供潜在的替代工具。