One-step MCDA-CRISPR/Cas12b platform for point-of-care testing of Shewanella algae

Shewanella algae (S. algae) is an emerging marine zoonotic pathogen capable of causing various infections with a high mortality rate. Existing detection methods for S. algae tend to be nonspecific, complicated, and time-consuming. Here, we developed a CRISPR-based detection method, named MCTOS (Multiple Cross Displacement Amplification CRISPR/Cas12b-based Testing in One-Step), for S. algae detection. Through pan-genome analysis of 193 S. algae genomes against 103 other Shewanella genomes, we identified a S. algae-specific gene designated SA, as the detection target. MCTOS combines MCDA for target pre-amplification and CRISPR/Cas12b-based cleavage for signal readout in one-pot at a constant temperature of 61 °C for 45 min. Including a 15 min rapid nucleic acid extraction step, the total assay time is 60 min with real-time fluorescence for results visualization, or 65 min with an additional 5 min lateral flow biosensor readout. The limit of detection was 10 copies, and no cross-reactivity was observed against 14 common non-S. algae templates. In evaluations of 50 isolates and 47 samples, MCTOS showed 100% (27/27) specificity and sensitivities of 98.6% (69/70) and 95.7% (67/70) for the real-time fluorescence instrument and lateral flow biosensor, respectively. Compared with recent one-pot CRISPR methods and the loop-mediated isothermal amplification method for S. algae detection, MCTOS simplifies primer design and achieves higher sensitivity. In summary, MCTOS is a simple, rapid, convenient, highly sensitive, and specific method for pathogen detection that can serve as a point-of-care testing tool for clinical diagnosis and epidemiological surveillance, and is especially suited for basic, field, and clinical laboratories.