Preamplification-Free Detection of Viable Microorganisms in Fermentation Using Tandem CRISPR Nuclease Probe

Accurate detection of viable bacteria is crucial for evaluating and monitoring the fermentation process. However, the complexity of fermentation samples presents challenges to developing precise and rapid detection tools. Here, we present a Cas13a-Csm6 tandem nuclease probe capable of the one-pot detection of viable microorganisms during fermentation, eliminating the need for nucleic acid preamplification. The RNA-activated CRISPR-Cas13a generates cleavage substrates that serve as activators for the CRISPR/Cas III-A Csm6 system. Leveraging the high specificity and efficient amplification capacity of the CRISPR cascade, this nuclease probe can detect 1% of viable <i>Lactobacillus</i> and <i>Bacillus,</i> facilitating the monitoring of bacterial populations throughout fermentation. This approach completes detection within 30 min and improves sensitivity for bacterial profiling by 16-fold compared with using Cas13 alone. The Cas13a-Csm6 tandem nuclease probe offers a precise and rapid analytical tool for the on-site quality monitoring of fermented foods.