Multiplex logic processing isothermal diagnostic assays for an evolving virus

Sanchita Bhadra; Miguel A. Saldaña; Hannah Grace Han; Grant L. Hughes; Andrew D. Ellington

While iNAATs have potential as POC diagnostics, they lack the versatility and intelligence to deal with genetically variable and rapidly evolving targets. To fill this need we have created a smart molecular diagnostic composed of a parallel set of degenerate primers that can readily amplify variant targets and an integrated multiplex OR-gated strand exchange logic processor that 'computes' and returns the presence of target relative to even closely related non-target sequences. The fact that multiple, different amplification reactions can be carried out in parallel, and yet the user need only attend to the final, fluorescent signal highlights the power of the diagnostic, and presages the possibility of even more complex integration of molecular signals. For example, strand exchange nucleic acid computation has allowed (i) analysis of repetitive DNA targets of greater than 500 nucleotides in length with single nucleotide specificity, using 'modular probes' composed of multiple short oligonucleotides that undergo competitive hybridization; 42 (ii) sensing and imaging of nucleic acids, enzymes, small molecules, proteins, and tumor cells via hairpin oligonucleotide probes and the hybridization chain reaction (HCR); 43 and (iii) coupling nucleic acid detection to translational reporters via amplicon-mediated strand displacement reactions that couple to reporter protein production in a cell-free translation system.