2025 AIChE Annual Meeting

(134f) Harnessing Non-Standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance

Authors

Luran Manfio, Foundation for Applied Molecular Evolution
Sebastian Magana Pena, Foundation for Applied Molecular Evolution
Nicolette A. Zhou, University of Washington
Kevin M. Bradley, Foundation for Applied Molecular Evolution
Cen Chen, Foundation for Applied Molecular Evolution
Chris McLendon, Firebird Biomolecular Sciences LLC
Steven A. Benner, The Westheimer Institute for Science and Technology
Karen Levy, University of Washington
Zunyi Yang, Foundation for Applied Molecular Evolution
Jorge A. Marchand, University of California, Berkeley
Erica R. Fuhrmeister, University of Washington
The emergence and evolution of pathogens continuously threaten global public health. To understand and track disease burden through environmental surveillance, there is an urgent need for a molecular detection assay that is sensitive, specific, and highly multiplexable. Many existing assays rely on the polymerase chain reaction (PCR), including quantitative and digital PCR (q/dPCR). However, these assays are constrained in multiplexability due to the limited number of available fluorophores, along with reduced sensitivity and specificity from off-target effects such as primer dimerization and non-specific amplification. Here, we present an icosaplex (20-plex) PCR assay capable of simultaneously detecting 18 enteropathogen and two antimicrobial resistance genes. The primers in this assay incorporate two sets of non-standard nucleotides: the self-avoiding molecular recognition systems (SAMRS) reduce primer dimerization, and the artificially expanded genetic information systems (AEGIS) reduce non-specific amplification. PCR amplification was subsequently followed by next-generation sequencing using both nanopore and Illumina platforms. The SAMRS-AEGIS 20-plex assay was evaluated on 10 wastewater, 10 soil, and 10 fecal samples. Compared to a standard DNA 20-plex, the SAMRS-AEGIS 20-plex had between 2-8 times more target alignment and 2-4 times fewer primer alignment. We also compared this assay to the TaqMan array card (TAC), a qPCR-based assay, and found 90% agreement on positive calls and 89% agreement on negative calls. Of the discrepancies, 47 out of 62 were targets the SAMRS-AEGIS 20-plex detected, but the TAC did not. Moreover, the sequencing data enabled subspecies and allelic variant differentiation, highlighting the advantages of sequencing over a fluorophore-based readout. This work demonstrates the benefits of integrating non-standard bases in a highly multiplexable detection assay, offering a versatile tool to support environmental monitoring and diagnostics.