Multicolor Amplification-Free RNA Detection with Cas13a and Cas13b.

The COVID-19 pandemic and recurring outbreaks of infectious diseases underscore the urgent demand for multiplex diagnostics capable of rapid and accurate pathogen identification. Although multiplex nucleic acid amplification tests (NAATs) are widely used for diagnosing diverse infectious diseases, their inherent amplification bias and long turnaround times highlight the demand for faster and reliable alternatives. Here, we present multicolor SATORI (mSATORI), an amplification-free single-molecule genetic test that leverages the complementary activities of CRISPR-Cas13a and Cas13b to achieve simultaneous detection of dual RNA targets. mSATORI identified Influenza A and SARS-CoV-2 RNAs within ∼10 min, with analytical limits of detection (LoD) of 86 aM and 52 aM, respectively. Validation using clinical specimens demonstrated robust diagnostic performance, achieving femtomolar limits of detection (550 aM for Influenza A and 640 aM for SARS-CoV-2), along with sensitivities exceeding 80% and specificities of 100%. Collectively, these results establish mSATORI as a platform for next-generation molecular diagnostics, with broad implications for clinical implementation, outbreak preparedness, and global infectious disease surveillance.