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 byproducts. Here, we developed a portable lateral flow colorimetric biosensor, termed CALL (dCas9-assisted LAMPbased 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.

Automated summary

Foodborne pathogens pose a serious threat to human health worldwide. In this study, a portable lateral flow colorimetric biosensor called CALL was developed using dCas9 as an intermediate bridge to distinguish target amplicons and byproducts. The biosensor showed high specificity and sensitivity in detecting Salmonella, enabling on-site screening of low concentrations in a short time.