Ultra-rapid detection of SARS-CoV-2 in public workspace environments

Yaren, Ozlem; McCarter, Jacquelyn; Phadke, Nikhil; Bradley, Kevin M.; Overton, Benjamin; Yang, Zunyi; Ranade, Shatakshi; Patil, Kunal; Bangale, Rishikesh; Benner, Steven A.

doi:10.1101/2020.09.29.20204131

Cited by 5 publications

(7 citation statements)

References 49 publications

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“…Our assay utilized a no-primer control to ensure that the reaction zones do not change color when heating. Other assays utilize a Human RNaseP mRNA control which increases diagnostic power ( Yang et al, 2021 ; Yaren et al, 2021 ). We screened primer sets in water using in-vitro transcribed RNA of the gene to assess performance and ability to dimerize.…”

Section: Discussionmentioning

confidence: 99%

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva

Davidson¹,

Wang²,

Maruthamuthu³

et al. 2021

Biosensors and Bioelectronics: X

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Herein, we describe the development of a paper-based device to detect nucleic acids of pathogens of interest in complex samples using loop-mediated isothermal amplification (LAMP) by producing a colorimetric response visible to the human eye. To demonstrate the utility of this device in emerging public health emergencies, we developed and optimized our device to detect SARS-CoV-2 in human saliva without preprocessing. The resulting device was capable of detecting the virus within 60 min and had an analytical sensitivity of 97% and a specificity of 100% with a limit of detection of 200 genomic copies/μL of patient saliva using image analysis. The device consists of a configurable number of reaction zones constructed of Grade 222 chromatography paper separated by 20 mil polystyrene spacers attached to a Melinex® backing via an ARclean® double-sided adhesive. The resulting device is easily configurable to detect multiple targets and has the potential to detect a variety of pathogens simply by changing the LAMP primer sets.

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Section: Discussionmentioning

confidence: 99%

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva

Davidson¹,

Wang²,

Maruthamuthu³

et al. 2021

Biosensors and Bioelectronics: X

View full text Add to dashboard Cite

show abstract

“…The two modifications are selected to be dark quencher-fluorophore pairs, and when the quenched FIP is incorporated into the LAMP product, subsequent amplification displaces the Fd oligo, releasing quenching and producing fluorescent signal specific to the label and target of interest. Other versions of this approach have been used, moving the quencher and fluorophore to a Loop primer[18, 19] or incorporated into the amplification products and detected after the reaction [20].…”

Section: Introductionmentioning

confidence: 99%

Development of Multiplexed RT-LAMP for Detection of SARS-CoV-2 and Influenza Viral RNA

Zhang

Tanner

2020

Preprint

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The ongoing COVID-19 pandemic has demonstrated the utility of widespread molecular testing for surveillance and diagnostic detection of SARS-CoV-2. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) has enabled testing outside of the standard clinical laboratory PCR infrastructure, with simple and rapid tests supplementing the existing, standard methods. However, current LAMP tests have detected single targets and required separate reactions for controls or multiple targets. As flu season arrives in the Northern Hemisphere the ability to screen for multiple viral targets will be increasingly important, and the ability to include internal control assays in the RT-LAMP test allows for decreased resource use and increased throughput. Here we describe a multiplexing approach to RT-LAMP with four targets (SARS-CoV-2, Influenza A, Influenza B, and internal control human RNA) in a single reaction using real-time and endpoint fluorescence detection. This increase to the functionality of RT-LAMP will, we hope, enable even broader adoption of this power molecular testing approach to aid in the global fight against this continuing public health threat.

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“…” Our Accessible LAMP-Enabled Rapid Test (ALERT) system packages the components of the well-established RT-LAMP reaction amongst layers of low-melting point waxes in order to provide room temperature and physical stability resulting in an easy to use “just-add-sample” solution for molecular diagnostics. Lyophilization is one method to ensure stable reagents and has been applied to RT-LAMP reagent mixes for SARS-CoV-2 [19] (Aidelberg 2021a submitted). Others have also shown that simple drying of reagents is sufficient [39].…”

Section: Discussionmentioning

confidence: 99%

Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

Bektaş¹,

Covington²,

Aidelberg

et al. 2021

Preprint

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The COVID-19 pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. PCR-based diagnostic tests are expensive, reliant on expensive centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests, that bind and detect the surface proteins of a virus, are rapid and inexpensive but suffer from high false negative rates. To address this problem, we have created an inexpensive, simple, and robust 60-minute Do-It-Yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μl) and specificity (>97% True Negative Rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP). Our workflow, ALERT (Accessible LAMP-Enabled Rapid Test), incorporates the following features: 1) Increased shelf-life and ambient temperature storage by using wax layers to isolate enzymes from reaction, 2) Improved specificity by using sequence-specific QUASR reporters, 3) Increased sensitivity through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal, 4) Quality control with a nasopharyngeal-specific mRNA target, and 5) Co-detection of other respiratory viruses, such as Influenza B, by duplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for centralized labs and hospitals. With minimal effort, SARS-CoV-2-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms or nucleic acid-based markers.

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Ultra-rapid detection of SARS-CoV-2 in public workspace environments

Cited by 5 publications

References 49 publications

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva

Development of Multiplexed RT-LAMP for Detection of SARS-CoV-2 and Influenza Viral RNA

Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

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