
==== Front
medRxiv
MEDRXIV
medRxiv
Cold Spring Harbor Laboratory

10.1101/2024.09.09.24313328
preprint
1
Article
Harnessing non-standard nucleic acids for highly sensitive icosaplex (20-plex) detection of microbial threats
Kawabe Hinako
Manfio Luran
Pena Sebastian Magana
Zhou Nicolette A.
Bradley Kevin M.
Chen Cen
McLendon Chris
Benner Steven A.
Levy Karen
Yang Zunyi
Marchand Jorge A.
Fuhrmeister Erica R.
10 9 2024
2024.09.09.24313328https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://medrxiv.org/lookup/doi/10.1101/2024.09.09.24313328
nihpp-2024.09.09.24313328.pdf
Abstract

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. We benchmarked this assay using a low-cost, portable sequencing platform (Oxford Nanopore) on wastewater, soil, and human stool samples. Using parallelized multi-target TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay, there was 74% agreement on positive calls and 97% agreement on negative calls. Additionally, we show how sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish sub-species. 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.
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pmc
