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.

By rearranging hydrogen bond donor and acceptor groups within a standard Watson–Crick geometry, DNA can add eight independently replicable nucleotides forming four additional not found in standard Terran DNA. For many applications, the orthogonal pairing of standard and nonstandard pairs offers a key advantage. However, other applications require standard and nonstandard nucleotides to communicate with each other. This is especially true when seeking to recruit high-throughput instruments (e.g., Illumina), designed to sequence standard 4-nucleotide DNA, to sequence DNA that includes added nucleotides. For this purpose, PCR workflows are needed to replace nonstandard nucleotides in (for example) a 6-letter DNA sequence by defined mixtures of standard nucleotides built from 4 nucleotides. High-throughput sequencing can then report the sequences of those mixtures to bioinformatic alignment tools, which infer the original 6-nucleotide sequence by analysis of the mixtures. Unfortunately, the intrinsic orthogonality of standard and nonstandard nucleotides often demand polymerases that violate pairing biophysics to do this replacement, leading to inefficiencies in this “transliteration” process. Thus, laboratory in vitro evolution (LIVE) using “anthropogenic evolvable genetic information systems” (AEGIS), an important “consumer” of new sequencing tools, has been slow to be democratized; robust sequencing is needed to identify the AegisBodies and AegisZymes that AEGIS-LIVE delivers. This work introduces a new way to connect synthetic and standard molecular biology: biversal nucleotides. In an example presented here, a pyrimidine analogue (pyridine-2-one, y) pairs with Watson–Crick geometry to both a nonstandard base (2-amino-8-imidazo-[1,2a]-1,3,5-triazin-[8H]-4-one, P, the Watson–Crick partner of 6-amino-5-nitro-[1H]-pyridin-2-one, Z) and a base that completes the Watson–Crick hydrogen bond pattern (2-amino-2'-deoxyadenosine, amA). PCR amplification of GACTZP DNA with dyTP delivers products where Z:P pairs are cleanly transliterated to A:T pairs. In parallel, PCR of the same GACTZP sample at higher pH delivers products where Z:P pairs are cleanly transliterated to C:G pairs. By allowing robust sequencing of 6-letter GACTZP DNA, this workflow will help democratize AEGIS-LIVE. Further, other implementations of the biversal concept can enable communication across and between standard DNA and synthetic DNA more generally.

The ability of nucleic acids to catalyze reactions (as well as store and transmit information) is important for both basic and applied science, the first in the context of molecular evolution and the origin of life and the second for biomedical applications. However, the catalytic power of standard nucleic acids (NAs) assembled from just four nucleotide building blocks is limited when compared with that of proteins. Here, we assess the evolutionary potential of libraries of nucleic acids with six nucleotide building blocks as reservoirs for catalysis. We compare the outcomes of in vitro selection experiments toward RNA-cleavage activity of two nucleic acid libraries: one built from the standard four independently replicable nucleotides and the other from six, with the two added nucleotides coming from an artificially expanded genetic information system (AEGIS). Results from comparative experiments suggest that DNA libraries with increased chemical diversity, higher information density, and larger searchable sequence spaces are one order of magnitude richer reservoirs of molecules that catalyze the cleavage of a phosphodiester bond in RNA than DNA libraries built from a standard four-nucleotide alphabet. Evolved AEGISzymes with nitro-carrying nucleobase Z appear to exploit a general acid–base catalytic mechanism to cleave that bond, analogous to the mechanism of the ribonuclease A family of protein enzymes and heavily modified DNAzymes. The AEGISzyme described here represents a new type of catalysts evolved from libraries built from expanded genetic alphabets.

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a) require no sample transport, (b) require minimal sample manipulation, (c) can be performed by unlicensed individuals, (d) return results on the spot in much less than one hour, and (e) cost no more than a few dollars. The sensitivity need not be as high as normally required by the FDA for screening asymptomatic carriers (as few as 10 virions per sample), as these viral loads are almost certainly not high enough for an individual to present a risk for forward infection. This allows tests specifically useful for this pandemic to trade-off unneeded sensitivity for necessary speed, simplicity, and frugality. In some studies, it was shown that viral load that creates forward-infection risk may exceed 10⁵ virions per milliliter, easily within the sensitivity of an RNA amplification architecture, but unattainable by antibody-based architectures that simply target viral antigens. Here, we describe such a test based on a displaceable probe loop amplification architecture.