Multiplex logic processing isothermal diagnostic assays for an evolving virus

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

4GO probes were also able to specifically identify all 9 Asian and both African ZIKV strains tested without cross reaction with related viruses (Figure 5F ). Endpoint fluorescence signals in all degenerate 4-Plex-LAMP-4GO assays seeded with Asian or African lineage ZIKV genomic RNA were consistently elevated above background noise in assays containing non-specific DENV genomes. In contrast, a previously reported Asian ZIKV NS2b gene-specific TaqMan qRT-PCR assay 33 was able to detect only Asian lineage viral genomes while failing to amplify African lineage genomic RNA ( Figure 5G ). Asian genomic RNA copy numbers estimated from this qRT-PCR assay (Supplementary Figure 1) suggested that 4-Plex-LAMP-4GO assays could readily detect a few hundred copies of viral genomes ( Figure 5H ). These results demonstrate that a single one-pot assay comprised of 21 degenerate LAMP primers and a fivestranded degenerate logic processing strand exchange probe could reliably detect viral variants within a genus and distinguish them from related viruses. Furthermore, by integrating signals from four separate inputs into one output via a single fluorophore-quencher pair, the fivestranded 4GO probe could capture the viral diversity at a fraction of the cost of four individuallylabeled OSD probes.