Fig. 3: Combination of RT-RPA and CrisprAIE detects norovirus RNA up to attomolar concentration.

a Schematic of using CrisprAIE assay combined RT-RPA system for norovirus detection, comprising sample RNA extraction, RT-RPA amplification, target activated Cas12a/crRNA trans-cleavage, and fluorescence measurement. b Validation of Q-dsDNA/AIEgen-Q reporters integrated with RT-RPA and Crispr system. Here, Q-dsDNA/AIEgen-Q reporters (group 1) were mixed with other key components needed for RT-RPA and Crispr system (group 2: mixed with RT-RPA reagent; group 3: mixed with Cas12a/crRNA; group 4: mixed with 10 pM targets; group 5: mixed with RT-RPA reagent and Cas12a/crRNA; group 6: mixed with 10 pM targets and RT-RPA reagent; group 7: mixed with 10 pM targets and Cas12a/crRNA; group 8: mixed with 10 pM targets, RT-RPA reagent and Cas12a/crRNA). Relative fluorescence intensity obtain from the analysis of ORF2 gene via RT-RPA integrated Crispr assay based on Q-dsDNA/AIEgens-Q reporters (c) and Q-ssDNA-FAM reporters (d). e Relative fluorescence intensity obtained from RT-RPA integrated CrisprAIE for detecting 15 clinical samples. Samples No. 1–9 represent the positive samples (highlighted in red), while samples No. 10–15 correspond to the negative samples (highlighted in green) as tested by RT-qPCR. f Comparison of test results of RT-qPCR and our developed CrisprAIE assay for 15 clinical samples. Negative samples are highlighted in green, and positive samples are highlighted in red. CrisprAIE assay heatmap represents normalized mean fluorescence values. g ROC curve analysis between the results of CrisprAIE and RT-qPCR, indicating the detection accuracy in clinical applications. Data are mean ± s.d.; n = 3 repeated tests. Two-tailed P values were calculated using unpaired t-tests: *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001; NS, not significant, compared with the group 1 in (b) or nontemplate control (NTC).