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 evaluate assay performance in natural biological matrices, Ae. aegypti mosquitoes were fed a blood meal spiked with ZIKV virions and then individually tested for viral nucleic acids after 14 days. Half of these mosquitoes that were exposed to ZIKV generated bright endpoint fluorescence in both singleplex (NS1 LAMP-OSD and Capsid LAMP-OSD) and multiplex (4plex-LAMP-2GO) degenerate reverse transcription LAMP-based assays (Figure 8, Supplementary Figure 2) . Parallel tests performed in the absence of primers, and hence absence of LAMP amplification and concomitant probe activation, revealed negligible signal inflation due to sample auto-fluorescence, indicating the positive LAMP signal was due to these mosquitoes developing a viral infection. These mosquitoes also tested positive by ZIKV NS2b TaqMan qRT-PCR assay and were found to contain ~6x10 3 to 4x10 5 NS2b RNA copies per . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 mosquito (Figure 8 ). Since only a 1/33 fraction of each mosquito was used for a LAMP test, these results suggest that the LAMP-based assays were able to generate a readily discernable bright visible fluorescence starting from as few as 190 copies of ZIKV genomic material embedded in crude virus-infected mosquito matrix.