Multiplex bacteria detection using one-pot CRISPR/Cas13a-based droplet microfluidics

High-throughput detection of bacteria at low levels is critical in public health, food safety, and first response. Herein, for the first time, we present a platform based on droplet microfluidics coupling with the recombinase aided amplification (RAA)-assisted one-pot clustered regularly interspaced short palindromic repeats together with CRISPR-associated proteins 13a (CRISPR/Cas13a) assay, and droplet encoding strategy for accurate and sensitive determination of nucleic acids from various foodborne pathogens. The workflow takes full advantage of CRISPR/Cas13a signal amplification and droplet confinement effects, which enhances the detection sensitivity and enables end-point quantitation. Meanwhile, by varying the color of droplets, the number of bacteria detected at the same time is greatly improved. It possesses the capability to simultaneously detect seven different types of foodborne pathogens. Notably, the system is also applied to real food samples with satisfactory results. Overall, in view of superiorities in high sensitivity, outstanding selectivity, and large-scale multiplexing, the one-pot CRISPR/Cas13a-based droplet microfluidic system could be expanded and universalized for identifying other bacteria.