(182ah) An Icosaplex (20-plex) Environmental Surveillance Assay Using Non-Standard Nucleic Acids

Infectious disease and antimicrobial resistance present significant challenges to global public health. Environmental surveillance is critical in monitoring and understanding disease burden. As these microbial threats become more diverse, there is an urgent need for a sensitive, cost-effective, and highly multiplexed surveillance assay. While many current methods rely on quantitative and digital polymerase chain reaction (q/dPCR), multi-target detection is constrained by a limited range of available fluorescent dyes and high cost. Moreover, increasing the number of primers also leads to off-target effects such as primer dimerization and non-specific amplification.

In this work, we present a highly sensitive PCR-based assay capable of detecting 20 targets simultaneously by integrating two sets of non-standard bases in the primers. Amplification is followed by next-generation sequencing for target identification. We validated this assay in wastewater, soil, and fecal matrices, demonstrating that primers containing non-standard bases outperform those containing standard bases in minimizing off-target effects. We also compared this assay to the TaqMan Array Card (TAC), an existing qPCR assay, and found high agreement between the two methods. Additionally, we utilized the sequencing data to gain further insight into allelic variants and target subspecies. This assay is cost-effective, sensitive, and robust, leveraging non-standard bases for sensitive and specific target detection. While this work focused on 20 targets, future assays will aim to detect additional targets with increased multiplexability (e.g. 50-plex) to support efforts in environmental monitoring, wastewater-based epidemiology, and diagnostics.