Multiplex logic processing isothermal diagnostic assays for an evolving virus

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

To verify that all the four assays could identify both Asian and African lineage ZIKV genomes we individually challenged singleplex NS1, NS3, NS5, and CA LAMP-OSD assays with genomic RNA from 9 Asian ZIKV, and 2 African ZIKV strains (Supplementary Table T4 ). Reaction specificity was assessed by seeding duplicate assays with genomic RNA of related dengue virus (DENV) serotypes 1-4 and chikungunya virus (CHIKV) that often co-circulate with ZIKV. 32 Endpoint OSD fluorescence in all LAMP assays seeded with Asian or African ZIKV genomes was significantly elevated above background noise in assays seeded with DENV or CHIKV genomic RNA (Figure 3) . These results demonstrate that all four degenerate LAMP-OSD assays were capable of lineage-independent ZIKV detection without cross-reaction with or interference from non-target nucleic acids.