Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance

Environmental surveillance
and clinical diagnostics heavily rely
on the polymerase chain reaction (PCR) for target detection. A growing
list of microbial threats warrants new PCR-based detection methods
that are highly sensitive, specific, and multiplexable. Here, we introduce
a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen
and two antimicrobial resistance genes. This multiplexed PCR assay
leverages the self-avoiding molecular recognition system (SAMRS) to
avoid primer dimer formation, the artificially expanded genetic information
system (AEGIS) for amplification specificity, and next-generation
sequencing for amplicon identification. Using parallelized multitarget
TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay
on wastewater, soil, and human stool samples, we found 90% agreement
on positive calls and 89% agreement on negative calls. Additionally,
we show how long-read and short-read sequencing information from the
20-plex can be used to further classify allelic variants of genes
and distinguish subspecies. The strategy presented offers sensitive,
affordable, and robust multiplex detection that can be used to support
efforts in wastewater-based epidemiology, environmental monitoring,
and human/animal diagnostics.