Using dCas9 as an intermediate bridge of loop-mediated isothermal amplification-based lateral flow colorimetric biosensor for point-of-care Salmonella detection

Foodborne pathogens pose a serious threat to human health worldwide. Combining isothermal amplification techniques (e.g., loop-mediated isothermal amplification, LAMP) with lateral flow biosensor (LFB) is ideal for point-of-care testing of foodborne pathogens, but challenges remain in distinguishing target amplicons and by-products. Here, we developed a portable lateral flow colorimetric biosensor, termed CALL (dCas9-assisted LAMP-based LFB), using dCas9 as an intermediate bridge to address these challenges. As a proof-of-concept, we chose Salmonella as the model. Strategically, LAMP amplicons of the target gene contain many contiguous repeats that can be recognized and unlocked by dCas9/single-guide RNA, which then serve as scaffolds to assemble many gold nanoparticles (GNPs) into chainlike structures on the test line. This strategy eliminates false positives caused by by-products and enables all repeats of LAMP amplicon to participate in capturing GNPs, greatly improving specificity and sensitivity. The biosensor integrates rapid extraction, LAMP amplification, dCas9-based assembly and LFB analysis without requiring complex equipment, enabling on-site screening of Salmonella as low as 41 CFU/mL in 40 min. The practicability of the biosensor was further demonstrated by testing real foods. The success of CALL provides a promising direction for developing foodborne pathogen detection tools.