Multiplex logic processing isothermal diagnostic assays for an evolving virus

Abstract

We have developed a generalizable 'smart molecular diagnostic' capable of accurate point-ofcare (POC) detection of variable nucleic acid targets. Our one-pot isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR gate signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our platform by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with 20 virions / µl being directly detected in human saliva within 90 minutes, and crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity.The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.While point-of-care (POC) diagnostic assays can be performed at or near the site of sample acquisition, they have for the most part been considered to be relatively simplistic tests that provide relatively little information to a clinician or public health worker. Familiar examples include electrochemical sensors for glucose, 1 or rapid immunoassays for metabolites such as human chorionic gonadotropin (the canonical pregnancy test), 2 and pathogens, either directly (influenza viruses) or via immune responses (antibodies against HIV-1/2). 3 The range of conditions and pathogens that could be tested for could likely be greatly expanded by developing POC diagnostics for nucleic acids 3 that would have greater sensitivity and accuracy. However, the current gold standard for molecular diagnostics, the quantitative polymerase chain reaction (qPCR), requires significant technical expertise and expensive and cumbersome equipment. Even portable instruments, such the Cepheid GeneXpert Omni, cost several thousand dollars and rely on expensive qPCR cartridge consumables for individual tests.