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 test 4GO probe function, multiplex reverse transcription LAMP reactions containing all 21 degenerate primers and the degenerate 4GO probe (4-Plex-LAMP-4GO) were spiked with a single type or a combination of all four of the ZIKV synthetic RNA templates NS1, NS3, NS5, and CA. As a control to assess signal specificity, duplicate reactions were assembled using a non-specific RNA template and its cognate LAMP primers that would lead to generation of nonspecific LAMP amplicons. Following LAMP amplification, 4GO probe fluorescence was found to be elevated in all reactions containing even a few hundred copies of at least one of the ZIKV templates (Figures 5A-E) . This ZIKV-specific signal remained consistently above noise generated in the absence of specific amplicons. These results demonstrate that the 4GO probe was able to function specifically in multiplex LAMP to identify and signal the presence of a few hundred copies of one or all four ZIKV amplicons without interference from non-specific templates or amplicons.