Multiplex logic processing isothermal diagnostic assays for an evolving virus

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

ZIKV genomic sequences were analyzed for variation using the National Center for Biotechnology Information (NCBI) Virus Variation Resource. 51 ZIKV sequences were also compared to related and co-circulating viruses, such as DENV and CHIKV, using MUSCLE. 52, 53 Four relatively conserved genomic regions in ZIKV capsid, NS1, NS3, and NS5 genes were chosen for primer design. The Primer Explorer v5 LAMP primer design software (Eiken Chemical Co., Japan) was used for generating LAMP primer sets composed of the outer primers F3 and B3 and the inner primers FIP and BIP. Primer design was constrained to include at least a 40 base pair (bp) gap between the F1 and F2 as well as between the B1 and B2 priming sites. Loop primers and stem primers were manually designed. Primer specificity for ZIKV isolates and a corresponding lack of significant cross-reactivity to other nucleic acids of human or pathogenic origin was further assessed using NCBI BLAST. 54, 55 Polymorphic loci in the primers were substituted with degenerate bases although, some polymorphisms near the 5'end of F3 or B3 or near the middle of FIP or BIP were ignored to reduce the degeneracy burden and to ensure efficient amplification. 15 Our previous work had shown that such mismatches minimally disrupted LAMP primer efficiency. 15 The nucleic acid circuit design software NUPACK 56 was used to design four OSD probes, each specific to one of the four ZIKV amplicons, according to our previously published design rules. 12 Unique, fluorophore-labeled OSD strands were designed to bind between B1 and B2 sequences of NS5 and NS3 amplicons and between F1 and F2 sequences of NS1 and capsid amplicons. The quencher-labeled OSD strands were designed to be partially complementary to the fluorophore-labeled strand. Single-stranded toeholds at the 3'-end or 5'-end of fluorophorelabeled strands were designed to be 10 or 13 nucleotides long. All 3'-OH ends were blocked with inverted deoxythymidine (dT) to prevent extension by DNA polymerase. Polymorphic loci in all four OSD probes were substituted with appropriate degenerate bases. These degenerate OSD probe strands were then used as a basis for designing the two-input 2GO and four-input . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/424440 doi: bioRxiv preprint 4GO probes. NUPACK was used to visualize and optimize probe architecture with variables such as buffer composition, temperature, and oligonucleotide concentrations. Assays analyzed using 2GO probes were supplemented with 50 mM trehalose and 100 nM of the fluorophore-labeled probe strand pre-annealed with the complementary gate and quencherlabeled oligonucleotides as described in sub-section "Assembly of strand exchange probes". 4-Plex assays read using the complete bipartite signal transducer contained 100 nM each of both CAN3.2GO and N1N5.2GO probes. Assays read using 4GO probes were also supplemented . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/424440 doi: bioRxiv preprint with 50 mM trehalose along with 80 nM of the fluorophore-labeled strand annealed with the remaining four strands of the 4GO probe as described in the sub-section "Assembly of strand exchange probes".