Abstract
Rapid and precise detection of respiratory pathogens is crucial for clinical diagnosis and treatment of respiratory infections. In this study, the multiplex and visual detection of respiratory pathogens is facilitated by specifically designed engineered CRISPR RNA (en-crRNA) to activate the trans-cleavage activity of Cas12a, along with a homemade portable device. The en-crRNA comprised an original crRNA and a DNA reporter molecule that is labelled with both a fluorophore and a quencher. Moreover, the DNA is partially complementary to the variable region of the original crRNA. The proof of concept was demonstrated by simultaneously identifying distinct respiratory pathogens with a detection limit of 10(2) copies per μL. The visual discrimination was subsequently achieved using a homemade portable device that was seamlessly integrated with a smartphone. The specificity of the strategy was validated by comparing with qPCR assays for clinical sample detection, demonstrating exceptional accuracy with areas under the ROC curves of 0.98 for all targets. The research provides a promising avenue for the development of rapid, specific, and on-site detection techniques aimed at multiplex identification of respiratory pathogens.