Multiplex logic processing isothermal diagnostic assays for an evolving virus

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

More importantly, the robustness of our smart molecular diagnostic allows it to perform with little or no error in a variety of inclement environments, including with crude preparations of macerated mosquitoes. LAMP-based smart molecular assays of ZIKV-infected and un-infected mosquitoes could be performed with results that were on par with those obtained by qPCR analyses. As few as 190 copies of ZIKV RNA could be readily identified using 4-Plex-LAMP-2GO assays without any false positive reactions. Similarly, both single and multiplex LAMPbased assays could identify 2 x 10 4 /ml ZIKV virions spiked in human saliva within 90 min. These assays were equally adept at identifying both Asian and African lineages of ZIKV without cross-reaction with related or co-circulating viruses. The assay thus finds itself in a relatively rarified set of molecular diagnostics that are truly capable of performing in demanding field conditions. For instance, RPA has demonstrated near 100% sensitivity and specificity in field tests for detection of many viruses, bacteria, and parasites. 44, 45, 46 Similarly, the isothermal nicking enzyme amplification reaction (NEAR) automated on the Clinical Laboratory Improvement Amendments (CLIA)-waived portable device Alere i (Alere, Scarborough, MA) is being used for POC diagnosis of infectious agents such as influenza viruses. 47 Overall, though, our envisioned smart molecular diagnostics platform has much greater versatility for field use going forward. For instance, most field applications of RPA involve purification of nucleic acid analytes from clinical specimens. In contrast, our one-pot reactions can directly analyze crude biospecimens without necessitating complicated sample preparation and nucleic acid purification. Field applications of RPA and NEAR typically employ commercial instruments costing a few thousand dollars for assay readout 46 whereas our assay requires a simple instrument-free visual 'yes/no' score for presence or absence of signal at endpoint.