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 create a multiplexed, internally redundant assay that simultaneously amplifies and detects CA, NS1, NS3, and NS5 sequences, all 21 LAMP primers and 4 hemi-duplex OSD reporters were combined in a single reaction (4-Plex-LAMP-OSD). To verify that each individual reverse transcription LAMP-OSD reaction was functional in this multiplex environment, replicate multiplex assays were supplemented with all 21 primers but only a single type of OSD reporter at a time. In the presence of all four ZIKV synthetic RNA templates, multiplexed 4-Plex-LAMP-OSD demonstrated exponential fluorescence accumulation. Despite co-mingling of multiple degenerate primers and OSD probes, no spurious signals were observed in the absence of specific templates ( Figure 4A) . As a result, brightly fluorescent ZIKV-positive assays containing only a few hundred copies of ZIKV templates could be readily distinguished from dark ZIKVnegative reactions by simple visual examination ( Figure 4B ). Each individual LAMP assay was functional in the multiplex assay, as indicated by accumulation of fluorescence in 4-Plex-LAMP-OSD reactions probed with individual OSD reporters (Figures 4C, D, E, F) . The multiplexed 4-Plex-LAMP-OSD assay could also readily identify the more complex ZIKV genomic RNA from both Asian and African lineages without cross-reaction with DENV and CHIKV genomic nucleic acids ( Figure 4G ).