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A one-pot toolbox based on Cas12a/crRNA enables rapid foodborne pathogen detection at attomolar level.

Bacteria contamination accounts for more than half of food poisoning cases. Conventional methods such as colony-counting and general polymerase chain reaction are time-consuming, instrument-dependent and sometimes not accurate. Herein, we developed a novel One-pot TOolbox with Precision and Ultra Sensitivity (OCTOPUS) platform for foodborne pathogen detection based on the mechanism that Cas12a non-target binding unleashes its collateral DNase activity. We demonstrated its application on two widespread foodborne bacteria, namely E. coli O157:H7 and Streptococcus aureus, using specific crRNA targeting rfbE and nuc gene, respectively. For better sensitivity, recombinase polymerase amplification (RPA) was integrated without product purification. This one-pot detection, that is RPA reagent, crRNA and ssDNA-FQ reporter are all in one tube with the subsequent addition of Cas12a enzyme, was able to detect genomic DNA at attomolar level. It omits extra cap-opening process to avoid practical inconvenience and possible cross-sample contamination. Moreover, we demonstrated this platform for real food matrix. Simple water boiling method for genome extraction together with one-pot assay achieved limit of detection value of 1 CFU/mL in less than 50 minutes. This OCTOPUS technique integrates bacterial genome extraction, preamplification based on RPA and Cas12a/crRNA cleavage assay.