Abstract
We have developed a generalizable “smart molecular diagnostic” capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our 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 logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology 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 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.
Highlights
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
In our own previous work, we developed oligonucleotide strand displacement (OSD) probes [12] that were triggered by strand exchange reactions with transiently single-stranded stem-loop sequences [13]
Endpoint OSD fluorescence in all loop-mediatedisothermal isothermal amplification (LAMP) assays seeded with Asian or African Zika virus (ZIKV) genomes was significantly elevated above background noise in assays seeded with dengue virus (DENV) or chikungunya virus (CHIKV) genomic RNA (Figure 3). These results demonstrate that all four degenerate LAMP-OSD assays were capable of lineage-independent ZIKV
Summary
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 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. Viruses 2018, 10, 714 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. Isothermal nucleic acid amplification tests (iNAATs) have been developed that rival
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