A SARS-CoV-2 coronavirus nucleocapsid protein antigen-detecting lateral flow assay

Grant, Benjamin D.; Anderson, Caitlin; Alonzo, Luis F.; Garing, Spencer; Williford, John R; Baughman, Ted A.; Rivera, Roberto; Glukhova, Veronika; Boyle, David S.; Dewan, Puneet; Weigl, Bernhard H.; Nichols, Kevin P.

doi:10.1371/journal.pone.0258819

Cited by 24 publications

(22 citation statements)

References 9 publications

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“…The nucleocapsid protein (N) is a major contributor to the structure and humoral response of the virus ( Smits et al, 2021 ) but is also a key structure for diagnostic purposes such as rapid antigen tests ( Grant et al, 2021 ). In this context, an in-solution ELISA was used to detect the cell-free synthesized nucleocapsid wild type (WT) protein as well as two early onset mutants (SER343, ASN202), which were detected in April 2020, in order to validate cell-free synthesis for testing antibodies as well as providing viral antigens for the diagnostic use.…”

Section: Resultsmentioning

confidence: 99%

The Potential of Eukaryotic Cell-Free Systems as a Rapid Response to Novel Zoonotic Pathogens: Analysis of SARS-CoV-2 Viral Proteins

Ramm

Dondapati

Trinh

et al. 2022

Front. Bioeng. Biotechnol.

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The ongoing pandemic caused by the novel coronavirus (SARS-CoV-2) has led to more than 445 million infections and the underlying disease, COVID-19, resulted in more than 6 million deaths worldwide. The scientific world is already predicting future zoonotic diseases. Hence, rapid response systems are needed to tackle future epidemics and pandemics. Here, we present the use of eukaryotic cell-free systems for the rapid response to novel zoonotic diseases represented by SARS-CoV-2. Non-structural, structural and accessory proteins encoded by SARS-CoV-2 were synthesized by cell-free protein synthesis in a fast and efficient manner. The inhibitory effect of the non-structural protein 1 on protein synthesis could be shown in vitro. Structural proteins were quantitatively detected by commercial antibodies, therefore facilitating cell-free systems for the validation of available antibodies. The cytotoxic envelope protein was characterized in electrophysiological planar lipid bilayer measurements. Hence, our study demonstrates the potential of eukaryotic cell-free systems as a rapid response mechanism for the synthesis, functional characterization and antibody validation against a viral pathogen.

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Section: Resultsmentioning

confidence: 99%

The Potential of Eukaryotic Cell-Free Systems as a Rapid Response to Novel Zoonotic Pathogens: Analysis of SARS-CoV-2 Viral Proteins

Ramm

Dondapati

Trinh

et al. 2022

Front. Bioeng. Biotechnol.

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“…The anti-N capture antibody is biotinylated and is itself captured in the test region by pre-immobilized polystreptavidin R (PR). 27,28 For conventional unamplified lateral flow assays, the protein target and anti-target signal antibodies flow to the test region along a single membrane channel. For our amplified HCR lateral flow assay, we leverage prior work that explored the use of multi-channel lateral flow assays.…”

Section: Resultsmentioning

confidence: 99%

HCR Lateral Flow Assays for Amplified Instrument-Free At-Home SARS-CoV-2 Testing

Schulte

Huang

Pierce

2022

Preprint

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The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 minutes. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/μL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/μL, 1000 copies/μL, 2000 copies/μL, 2000 copies/μL, and 20,000 copies/μL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious disease. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection.

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“… 4 , 5 Common methods for SARS‐CoV‐2 detection mainly include lateral flow assay (LFA)‐based antigen detection and reverse transcription polymerase chain reaction (RT‐PCR)‐based nucleic acid detection. 6 , 7 , 8 , 9 The SARS‐CoV‐2 antigen LFA detection method is simple and rapid, which has emerged as a tool widely used for SARS‐CoV‐2 field test or self‐test. 10 However, its performance is unsatisfactory in terms of sensitivity and accuracy, especially for samples with low pathogen concentrations.…”

Section: Introductionmentioning

confidence: 99%

“…As the impact of the pandemic continues, efficient and accurate diagnosis of the pathogen is crucial for the rapid identification of infected patients and control of COVID‐19 transmission 4,5 . Common methods for SARS‐CoV‐2 detection mainly include lateral flow assay (LFA)‐based antigen detection and reverse transcription polymerase chain reaction (RT‐PCR)‐based nucleic acid detection 6–9 . The SARS‐CoV‐2 antigen LFA detection method is simple and rapid, which has emerged as a tool widely used for SARS‐CoV‐2 field test or self‐test 10 .…”

Section: Introductionmentioning

confidence: 99%

Highly sensitive and rapid detection of SARS‐CoV‐2 via a portable CRISPR‐Cas13a‐based lateral flow assay

Liu¹,

Chang

Chen³

et al. 2022

Journal of Medical Virology

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To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and control the spread of coronavirus disease (COVID‐19), there is an urgent need for highly sensitive on‐site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein (Cas)‐based molecular diagnostic method was developed for this purpose. Here, a CRISPR system‐mediated lateral flow assay (LFA) for SARS‐CoV‐2 was established based on multienzyme isothermal rapid amplification, CRISPR‐Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/μl in both fluorescence‐ and immunochromatography‐based assays. To enhance the quality control of the CRISPR‐based LFA method, glyceraldehyde‐3‐phosphate dehydrogenase was detected as a reference using a triple‐line strip design in a lateral flow strip. In total, 52 COVID‐19‐positive and 101 COVID‐19‐negative clinical samples examined by reverse transcription polymerase chain reaction (RT‐PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT‐PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR‐mediated LFA and provides a crucial tool for on‐site detection of SARS‐CoV‐2.

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A SARS-CoV-2 coronavirus nucleocapsid protein antigen-detecting lateral flow assay

Cited by 24 publications

References 9 publications

The Potential of Eukaryotic Cell-Free Systems as a Rapid Response to Novel Zoonotic Pathogens: Analysis of SARS-CoV-2 Viral Proteins

The Potential of Eukaryotic Cell-Free Systems as a Rapid Response to Novel Zoonotic Pathogens: Analysis of SARS-CoV-2 Viral Proteins

HCR Lateral Flow Assays for Amplified Instrument-Free At-Home SARS-CoV-2 Testing

Highly sensitive and rapid detection of SARS‐CoV‐2 via a portable CRISPR‐Cas13a‐based lateral flow assay

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