PAM-free CRISPR/Cas12a biosensor for PNAs-assisted isothermal amplification detection of S. typhimurium

CRISPR/Cas12a (Cpf1) have been generally used for detecting pathogenic microorganisms. However, rules for a protospacer adjacent motif (PAM) on target double-stranded DNA (dsDNA) hindered their application. To overcome this shortcoming, we developed a novel isothermal amplification scheme, peptide nucleic acids (PNAs)-assisted self-folding isothermal amplification for a PAM-free CRISPR/Cas12a biosensor (PSCas) to detect salmonella typhimurium (S. typhimurium). The PSCas biosensor uses PNAs to precisely invade the target gene dsDNA to form single-stranded DNA (ssDNA), which in turn binds to a self-folding primer (SP). When the self-folding strand of SP is complementary to the target DNA, the hairpin structure is formed, which exposes the start-up site of the new SP continuous hybridization. Consequently, this amplification used only one primer to produce a large number of ssDNA amplifiers, and exhibited a remarkable diagnostic sensitivity of 3 CFU•mL^-1 S. typhimurium. This platform provided a novel approach for developing innovative PAM-free CRISPR/Cas biosensors for PNAs-assisted isothermal amplification.