Solid-Phase PCR-Amplified DNAzyme Activity for Real-Time FO-SPR Detection of the MCR-2 Gene

The polymerase chain reaction (PCR) has been the gold standard molecular analysis technique for decades and has seen quite some evolution in terms of reaction components, methodology, and readout mechanisms. Nucleic acid enzymes (NAzymes) have been used to further exploit the applications of PCR, but so far the work was limited to the colorimetric G-quadruplex or fluorescent substrate cleaving NAzymes. In this study, a solid-phase, fiber optic surface plasmon resonance (FO-SPR) technique is presented as an alternative readout for PCR utilizing NAzymes. First, the surface cleavage activity of DNAzyme-extended amplicons (DNAzyme-amps) is established, followed by optimization of the PCR conditions, which are required for compatibility with the FO-SPR system. Next, by integrating the complement of a 10-23 DNAzyme into the primer pair, PCR-amplified DNAzyme-amps were generated, tested, and validated on qPCR for the detection of the antimicrobial resistance gene MCR-2. Once validated, this primer concept was developed as a one-step assay, driven by PCR-amplified DNAzymes, for FO-SPR-based sensitive and specific detection. Using gold nanoparticle labeled RNA-DNA hybrid strands as substrate for the DNAzyme, PCR-amplified DNAzyme-amps generated in the presence of MCR-2 gene were monitored in real-time, which resulted in an experimental limit of detection of 4 × 10⁵ copy numbers or 6.6 fM. In addition, the DNAzyme-based FO-PCR assay was able to discriminate between the MCR-1 and MCR-2 genes, to further prove the specificity of this assay. Henceforth, this DNAzyme-based fiber optic PCR assay provides a universally applicable, real-time system for the detection of virtually any target NA, in a specific and sensitive manner.