Direct Detection of Conserved Viral Sequences and Other Nucleic Acid Motifs with Solid-State Nanopores

Sethi, Komal; Dailey, Gabrielle P.; Zahid, Osama K.; Taylor, Ethan Will; Růžička, Jan; Hall, Adam R.

doi:10.1021/acsnano.0c10887

Cited by 22 publications

(15 citation statements)

References 52 publications

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“…Conserved sequences of the HIV RNA genome have been detected using biotinylated ssDNA probes and monovalent streptavidin. 10 DNA nanostructures were used to report the presence of a Zika virus gene by folding in its presence, as well as a microRNA biomarker for human lung cancer. 11−13 A loopmediated isothermal amplification scheme was used to generate many amplicon copies that can be counted to develop qualitative and quantitative nucleic acid tests.…”

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confidence: 99%

“…Solid-state nanopores have also been used to detect polymorphic insertion or deletion mutations in DNA, which determine the virulence or drug resistance of two different bacterial organisms, after cleavage with mutation site recognition enzymes. Conserved sequences of the HIV RNA genome have been detected using biotinylated ssDNA probes and monovalent streptavidin . DNA nanostructures were used to report the presence of a Zika virus gene by folding in its presence, as well as a microRNA biomarker for human lung cancer. − A loop-mediated isothermal amplification scheme was used to generate many amplicon copies that can be counted to develop qualitative and quantitative nucleic acid tests .…”

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confidence: 99%

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Screening for Group A Streptococcal Disease via Solid-State Nanopore Detection of PCR Amplicons

et al. 2022

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Single molecule detection methods are becoming increasingly important for diagnostic applications. Practical Early detection of disease requires sensitivity down to the level of single copies of the targeted biomarkers. Of the candidate technologies that can address this need, solid-state nanopores show great promise as digital sensors for single-molecule detection. Here, we present work detailing the use of solid-state nanopores as downstream sensors for a PCR-based assay targeting group A streptococcus (strep A) which can be readily extended to detect any pathogen that can be identified with a short nucleic acid sequence. We demonstrate that with some simple modifications to the standard PCR reaction mixture, nanopores can be used to reliably identify strep A in clinical samples. We also discuss methodological best practices both for adapting PCR-based assays to solid-state nanopore readout as well as analytical approaches by which to decide on sample status.Molecular methods for infectious disease diagnostic are gradually being introduced into clinical use, and this trend will only accelerate because of the Covid-19 pandemic. [1][2][3][4] Many of these molecular tests typically employ a DNA amplification method to generate many copies of the target sequence and then rely on downstream optical sensors to detect a fluorescent signal generated by the presence of amplified nucleic acid products (i.e., amplicons) in the sample.However, the optical components and the use of dyes as labels, required for fluorescence-based detection, can impose some limitations on the cost, size, and robustness of the instrument or

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Screening for Group A Streptococcal Disease via Solid-State Nanopore Detection of PCR Amplicons

et al. 2022

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“…Recent studies have developed a viral nucleic acid detection system using nanopores which holds great promise for a rapid detection system 10,12,13 . However, preamplification and enzymatic steps in preparation for nanopore detection limit the utility of such methods, although some approaches showed potential to omit these steps 32,33,42 .…”

Section: Discussionmentioning

confidence: 99%

Simultaneous identification of viruses and viral variants with programmable DNA nanobait

Bošković

Zhu

Tivony

et al. 2021

Preprint

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Respiratory infections are the major cause of death from infectious disease worldwide. The clinical presentation of many respiratory viruses is indistinguishable; therefore, diagnostic approaches that can identify multiple pathogens are essential for patient management. We aimed to address this challenge with self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid read-out via nanopore sensing. Here, we show this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus, influenza, and parainfluenza. Our nanobait could be reprogrammed to discriminate viral variants, and we identified several key SARS-CoV-2 variants with single-nucleotide resolution. We increased assay specificity with bespoke nanobait that could identify numerous short RNA targets in the same viral sample in a complex background of the human transcriptome. Notably, we found that the sequence position in the viral RNA secondary structure is critical for nanobait design. Lastly, we show that nanobait could discriminate between samples extracted from oropharyngeal swabs from negative and positive SARS-CoV-2 patients using programmable target cleavage without pre-amplification. Our system allows for multiplexed identification of native RNA molecules, providing a new scalable approach for diagnostics of multiple respiratory viruses in a single assay.

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“…As a joy to the passionate, besides the α-HL, a rich variety of nanopores were tested and proposed over the past years, in form of either protein-based nanopores, for example, aerolysin, [51][52][53][54] M. smegmatis porin A (MspA), [55] outer membrane protein G (OmpG), [56,57] fragaceatoxin C (FraC), [58] cytolysin A (ClyA), [59] 29 phage DNA packaging motor, [60] outer membrane protein F (OmpF), [61][62][63][64] solid-state nanopores manufactured in synthetic materials, [65][66][67][68][69][70][71][72][73][74][75][76][77] hybrid protein-solid state nanopores [78] or nanopores composed of DNA origami [79,80].…”

Section: Significance Statementmentioning

confidence: 99%

Single‐molecule, hybridization‐based strategies for short nucleic acids detection and recognition with nanopores

et al. 2021

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DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore-and hybridization-based DNA detection, and explores a range of state-ofthe-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.

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Direct Detection of Conserved Viral Sequences and Other Nucleic Acid Motifs with Solid-State Nanopores

Cited by 22 publications

References 52 publications

Screening for Group A Streptococcal Disease via Solid-State Nanopore Detection of PCR Amplicons

Screening for Group A Streptococcal Disease via Solid-State Nanopore Detection of PCR Amplicons

Simultaneous identification of viruses and viral variants with programmable DNA nanobait

Single‐molecule, hybridization‐based strategies for short nucleic acids detection and recognition with nanopores

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