Contamination-free V-shaped ultrafast reaction cascade transferase signal amplification driven CRISPR/Cas12a magnetic relaxation switching biosensor for bacteria detection

Foodborne pathogenic bacteria seriously endanger human health and must be rapidly identified for control. Magnetic relaxation switching biosensors (MRS) are ideal for rapid bacteria detection due to their high signal-to-noise ratio and immunity to sample matrix signal interference. However, conventional MRS still has some challenges in terms of sensitivity, specificity, and stability due to insufficient cross-linking or non-specific binding of magnetic nanoparticles (MNPs) to the target. To address these challenges, we firstly proposed a novel contamination-free uracil-DNA glycosylase (UDG) assisted V-shaped PCR driven CRISPR/Cas12a-MRS (UPC-MRS) biosensor, which combines contamination-free ultrafast nucleic acid amplification and powerful CRISPR/Cas12a system. It has an extremely specific quadruple signal guarantee realized by the merits of UDG anti-contamination, PCR primer specificity matching, the CRISPR/Cas12a system's precise recognition abilities, and magnetic probe signal unaffected by the sample matrix. As a cascade combined with original terminal deoxynucleotidyl transferase (Tdt)-mediated signal amplification technology, this platform can achieve Salmonella detection at concentrations as low as 53 CFU/mL, which is more sensitive than most existing MRS sensors, and it displays accuracy and applicability in real sample detection. This novel UPC-MRS biosensors avoid the common aerosol pollution problem of previous CRISPR/Cas12a systems which after combining with nucleic acid amplification, hence not only offers an alternative toolbox for Salmonella and other pathogen detection with satisfactory specificity and sensitivity, but also has potential for future applications across diverse fields.