The clinical methods to detect RNA viruses and disease-related RNAs suffer from time-consuming processes, high false-positive rates, or limited sensitivity. Here, we propose a strategy for rapid RNA detection through intra-enzyme chain replacement-mediated Cas13a cascade cyclic reaction without target amplification. A hairpin RNA mediator (a cleavage substrate for target-activated Cas13a) and a guiding RNA recognized by the cleavage product through intra-enzyme chain replacement were designed and optimized. Upon the recognition and binding of the target RNA to the Cas13a/CrRNA complex, Cas13a is initially activated to cleave the mediator, and the cleavage products recognize the corresponding Cas13a/CrRNA complex by intra-enzyme chain replacement and initiate the circular cascade of Cas13a cleavage and activation. The accumulated active Cas13a cleaves fluorescent reporter probe for achieving target RNA detection. This “mix & read” RNA detection at room temperature was performed in total 30 min. Using miRNA-21 as the target, the changes in fluorescence intensity were linearly correlated to the concentrations from 10 fM to 50 pM with the detection limit of 75 aM, while no significant changes in fluorescence intensity were detected for non-targets. This method applied to the clinical sputum respiratory syncytial virus-positive samples gave results consistent with those from the clinical fluorescence immunoassay. Thus, intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction for detection of RNA viruses in the “mix & read” mode at room temperature is rapid, simple, convenient, and efficient for RNA detection and can be adapted to point-of-care testing for high throughput screening of RNA virus infections.

检测RNA病毒和疾病相关RNA的临床方法存在过程耗时、假阳性率高或灵敏度有限的问题。在这里，我们提出了一种通过酶内链替换介导的 Cas13a 级联循环反应快速检测 RNA 的策略，无需靶标放大。设计并优化了发夹 RNA 介质（靶点激活的 Cas13a 的裂解底物）和通过酶内链替换被裂解产物识别的引导 RNA。当目标RNA识别并与Cas13a/CrRNA复合物结合后，Cas13a首先被激活以裂解介质，裂解产物通过酶内链置换识别相应的Cas13a/CrRNA复合物，并启动Cas13a裂解的循环级联和激活。积累的活性Cas13a切割荧光报告探针以实现目标RNA检测。这种在室温下“混合并读取”的 RNA 检测总共进行了 30 分钟。以miRNA-21为靶标，从10 fM到50 pM，荧光强度的变化与浓度呈线性相关，检测限为75 aM，而非靶标则没有检测到荧光强度的显着变化。该方法应用于临床呼吸道合胞病毒阳性痰标本，结果与临床荧光免疫分析结果一致。因此，在室温下以“混合&读取”模式检测RNA病毒的酶内链替换促进的Cas13a级联循环反应对于RNA检测来说是快速、简单、方便和高效的，并且可以适应点--of-RNA检测。 RNA 病毒感染高通量筛查的护理测试。