Respiratory infection caused by pathogens is among the most prevalent health issues affecting people worldwide. Accurate and rapid screening of respiratory pathogens is crucial for selecting appropriate treatments to control epidemics. However, it is often challenged by two aspects: first, the low concentration of pathogens in the early stages of infection; second, the difficulty of analyzing multiple pathogens. Herein, we report a mass spectrometry strategy combining the CRISPR/Cas12a system with DNA nanomachine for respiratory pathogens detection. Thanks to the high sensitivity of the CRISPR/Cas12a-enhanced DNA nanomachine and the multiple analysis of elemental mass spectrometry, the proposed method was successfully applied for clinical sample analysis with a low detection limit of 28 amol, 30 amol, and 38 amol for SARS-CoV-2, influenza A virus subtype H1N1, and Mycoplasma pneumoniae, respectively.

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