Highly sensitive detection of Salmonella based on dual-functional HCR-mediated multivalent aptamer and amplification-free CRISPR/Cas12a system

Salmonella infection severely threatens human health and causes substantial medical and financial concerns. Sensitive and specific detection of Salmonella in food samples is crucial but remains challenging. While some traditional assays for S. typhimurium are reliable, they suffer from various limitations, such as being time-consuming (culture-based methods), involving intricate nucleic molecular extraction (polymerization chain reaction, PCR), and exhibiting inadequate sensitivity (enzyme-linked immunosorbent assay, ELISA). In this case, it is essential to establish a rapid, simple-operation, and sensitive method for monitoring S. typhimurium to preserve food quality and prevent contamination.Herein, an amplification-free detection method for Salmonella was developed by coupling the aptamer magnetic separation with dual-functional HCR (hybridization chain reaction)-scaffold multivalent aptamer and the activity of CRISPR/Cas12a. In the detection system, the dual-functional HCR-scaffold multivalent aptamer with high binding affinity and specificity was fabricated in advance by assembling numerous Salmonella specific aptamers on the long HCR products. In addition to the enhanced affinity, the HCR-multiApt also contains a massive amount of repeated CRISPR-targetable DNA units in its HCR scaffold, which could trigger the trans-cleavage activity of Cas12a. In the presence of target bacteria, the HCR-scaffold multivalent aptamer could attach on the surface of bacteria effectively and amplified the signal of bacteria into CRISPR/Cas12a based fluorescent readout. The proposed detection system allowed for ultrasensitive detection of Salmonella in a linear range from 100 to 107 cfu mL-1 with a LOD (limit of detection) of 2 cfu mL-1.The novel dual-functional HCR-multiApt presents a simple and powerful strategy for improving the aptamer binding affinity toward Salmonella. Simultaneously, integrating this dual-functional HCR-multiApt with the CRISPR/Cas12a system significantly enhances the sensitivity by cascade signal amplification in a nucleic acids amplification-free way. Finally, leveraging the versatility of the aptamer, this highly sensitive method can be further extended for application in the detection of other bacteria, food safety monitoring, or clinical diagnostics.