Multiplex logic processing isothermal diagnostic assays for an evolving virus

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

Our results demonstrated that the 4-Plex-LAMP-4GO assay could specifically identify both Asian and African ZIKV without non-specific signaling. However, a fluorimeter was necessary for assay readout due to the relatively high background noise of the 4GO probe. Therefore, we sought to further engineer the logic processing probe in order to achieve a signal-to-noise ratio that could be visually discriminated and thereby allowed easy 'yes/no' assay readout in austere conditions without complex instruments. We surmised that the fluorophore was insufficiently quenched in the five-stranded 4GO probe due to its high degeneracy level. To reduce probe . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 degeneracy, we engineered a bipartite four-input signal processor composed of two OR gated strand exchange modules (2GO) ( Figure 1C ). Each 2GO probe was comprised of 3 degenerate oligonucleotides (S I -S III ) that were partially complementary to each other. The 5'-ends of both S I strands were labeled with the same type of fluorophore while the 3'-ends of S II strands were labeled with the corresponding quencher molecules. Simultaneous hybridization of S I and S II to the unlabeled S III strand would juxtapose the fluorophore and quencher resulting in loss of signal. Short single-stranded toeholds at the 3'-and 5'-ends of S I and S II , respectively could independently initiate strand exchange with their cognate target sequences resulting in separation of the fluorophore from the quencher and a concomitant rise in signal. For instance, the CAN3.2GO probe fluorescence would increase if it underwent strand exchange with either CA or N3 amplicons. Similarly, the N1N5.2GO probe would signal if either or both NS1 and NS5 amplicons were present.