Visual Detection of Bacterial Pathogens via PNA-Based Padlock Probe Assembly and Isothermal Amplification of DNAzymes

We have developed a self-reporting isothermal system for visual bacterial pathogen detection with single base resolution. The new DNA diagnostic is based on combination of peptide nucleic acid (PNA) technology, rolling circle amplification (RCA) and DNAzymes. PNAs are used as exceedingly selective chemical tools that bind genomic DNA at a predetermined sequence under nondenaturing conditions. After assembly of the PNA-DNA construct a padlock probe is circularized on the free strand. The probe incorporates a G-quadruplex structure flanked by nicking enzyme recognition sites. The assembled circle serves as a template for a novel hybrid RCA strategy that allows for exponential amplification and production of short single-stranded DNA pieces. These DNA fragments fold into G-quadruplex structures and when complexed with hemin become functional DNAzymes. The catalytic activity of each DNAzyme unit leads to colorimetric detection and provides the second amplification step. The combination of PNA, RCA, and DNAzymes allows for sequence-specific and highly sensitive detection of bacteria with a colorimetric output observed with the naked eye. Herein, we apply this method for the discrimination of Escherichia coli, Salmonella typhimurium, and Clostridium difficile genomes.