Viral detection and identification in 20 min by rapid single-particle fluorescence in-situ hybridization of viral RNA

Hepp, Christof; Shiaelis, Nicolas; Robb, Nicole C.; Vaughan, Alison; Matthews, Philippa C.; Stoesser, Nicole; Crook, Derrick W.; Kapanidis, Achillefs N.

doi:10.1038/s41598-021-98972-z

Cited by 23 publications

(19 citation statements)

References 51 publications

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“…A major strength of this system is that aptamers may be selected via SELEX 34,35 for any surface antigen, and RNA FISH probes may be designed to target different viral strains 20,27 . These properties will allow the system to be rapidly customized and applied to emerging pathogens and any desired viral vector without the need for fluorescent transgenes that are either not present in natural pathogens or not permitted in the minimum payload for gene therapies 36,37 .…”

Section: Discussionmentioning

confidence: 99%

Paired aptamer capture and FISH detection of individual virions enables cell-free determination of infectious titer

Liu

Potts

Nian

et al. 2022

Preprint

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Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titer is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and reverse transcription polymerase chain reaction (RT-PCR)-based (sensitive but not rapid). Current viral titer methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid-aptamer FISH or raptamer FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are infectious, thus serving as a more consistent proxy of infectious titer. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer, then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH) thus, it is selective for infectious particles (i.e., positive for coat protein and positive for genome).

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Section: Discussionmentioning

confidence: 99%

Paired aptamer capture and FISH detection of individual virions enables cell-free determination of infectious titer

Liu

Potts

Nian

et al. 2022

Preprint

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“…Despite the advantages claimed for the Accula test, the widespread availability of the test is still a limitation − however, the inability to conduct large sample testing has limited their applicability in clinical practice. Further, the immunohistochemistry results are dependent on the type of antibody used to perform the test …”

Section: Nucleic Acid Amplification Test (Naat)mentioning

confidence: 99%

Diagnostic Approaches For COVID-19: Lessons Learned and the Path Forward

Alafeef

Pan

2022

ACS Nano

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Coronavirus disease 2019 (COVID-19) is a transmitted respiratory disease caused by the infection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although humankind has experienced several outbreaks of infectious diseases, the COVID-19 pandemic has the highest rate of infection and has had high levels of social and economic repercussions. The current COVID-19 pandemic has highlighted the limitations of existing virological tests, which have failed to be adopted at a rate to properly slow the rapid spread of SARS-CoV-2. Pandemic preparedness has developed as a focus of many governments around the world in the event of a future outbreak. Despite the largely widespread availability of vaccines, the importance of testing has not diminished to monitor the evolution of the virus and the resulting stages of the pandemic. Therefore, developing diagnostic technology that serves as a line of defense has become imperative. In particular, that test should satisfy three criteria to be widely adopted: simplicity, economic feasibility, and accessibility. At the heart of it all, it must enable early diagnosis in the course of infection to reduce spread. However, diagnostic manufacturers need guidance on the optimal characteristics of a virological test to ensure pandemic preparedness and to aid in the effective treatment of viral infections. Nanomaterials are a decisive element in developing COVID-19 diagnostic kits as well as a key contributor to enhance the performance of existing tests. Our objective is to develop a profile of the criteria that should be available in a platform as the target product. In this work, virus detection tests were evaluated from the perspective of the COVID-19 pandemic, and then we generalized the requirements to develop a target product profile for a platform for virus detection.

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“…The seal design with 2xATTO647N was chosen over the ATTO647N-DABCYL pair for further experiments, since the presence of two fluorophores on the seals lead to brighter fluorescence upon binding compared with a singly fluorophore labelled seals; the 2xATTO647N is also associated with slightly higher operational range of concentrations. The use of contact-mediated quenching should also be useful in other techniques relying on DNA-binding, such as DNA PAINT (15), FISH (25)(26)(27), and molecular beacons (22).…”

Section: Concept Of Linking Single-molecule Phenotypes To Dna Sequenc...mentioning

confidence: 99%

Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

Andrews

Steuer

El‐Sagheer

et al. 2022

Preprint

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Protein interactions with nucleic acids are central to all genetic processes and many biotechnological applications. While many sequence-dependent protein-DNA interactions have been studied in detail using single-molecule methods, there is no standard high-throughput way to link the complex single-molecule kinetics of protein-DNA interactions with the DNA sequence of a single molecule. Here we provide the missing link by introducing a single-molecule imaging method (Gap-Seq) that interrogates DNA sequences via transient binding of short fluorescent DNA to a single DNA molecule previously used to characterise a protein-DNA interaction. In Gap-Seq, we identify a base by the degree of binding of 6-9 nt-long DNAs to surface-immobilised DNA substrates featuring a short single-stranded gap. To facilitate detection, we also developed a fluorescence quenching strategy that allows single-molecule detection at up to 500 nM of unbound fluorescent DNA. We link single-base differences on single DNA molecules to the kinetics of protein-DNA interactions by studying the interaction of a transcription activator with its cognate site. Finally, we show that our assay can address mixed sequences by distinguishing between two different sequences immobilised on the same field of view, paving the way for interrogation of sequence libraries for both mechanistic work and biotechnological applications.

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Viral detection and identification in 20 min by rapid single-particle fluorescence in-situ hybridization of viral RNA

Cited by 23 publications

References 51 publications

Paired aptamer capture and FISH detection of individual virions enables cell-free determination of infectious titer

Paired aptamer capture and FISH detection of individual virions enables cell-free determination of infectious titer

Diagnostic Approaches For COVID-19: Lessons Learned and the Path Forward

Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

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