Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

Graham, Thomas G.W.; Dugast-Darzacq, Claire; Dailey, Gina M; Nguyenla, Xammy; Dis, Erik Van; Esbin, Meagan; Abidi, Abrar; Stanley, Sarah A.; Darzacq, Xavier; Tjian, Robert

doi:10.1101/2020.09.16.20193466

Cited by 10 publications

(10 citation statements)

References 48 publications

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“…This will facilitate the implementation of widespread testing. Furthermore, they can be readily deployed in other testing frameworks and complement open-source solutions for one- and two-step RT-qPCR diagnostics 41–44 .…”

Section: Discussionmentioning

confidence: 99%

McQ – An open-source multiplexed SARS-CoV-2 quantification platform

Vonesch

Bredikhin

Dobrev

et al. 2020

Preprint

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McQ is a SARS-CoV-2 quantification assay that couples early-stage barcoding with high-throughput sequencing to enable multiplexed processing of thousands of samples. McQ is based on homemade enzymes to enable low-cost testing of large sample pools, circumventing supply chain shortages.Implementation of cost-efficient high-throughput methods for detection of RNA viruses such as SARS-CoV-2 is a potent strategy to curb ongoing and future pandemics. Here we describe Multiplexed SARS-CoV-2 Quantification platform (McQ), an in-expensive scalable framework for SARS-CoV-2 quantification in saliva samples. McQ is based on the parallel sequencing of barcoded amplicons generated from SARS- CoV-2 genomic RNA. McQ uses indexed, target-specific reverse transcription (RT) to generate barcoded cDNA for amplifying viral- and human-specific regions. The barcoding system enables early sample pooling to reduce hands-on time and makes the ap-proach scalable to thousands of samples per sequencing run. Robust and accurate quantification of viral load is achieved by measuring the abundance of Unique Molecular Identifiers (UMIs) introduced during reverse transcription. The use of homemade reverse transcriptase and polymerase enzymes and non-proprietary buffers reduces RNA to library reagent costs to 92 cents/sample and circumvents potential supply chain short-ages. We demonstrate the ability of McQ to robustly quantify various levels of viral RNA in 838 clinical samples and accu-rately diagnose positive and negative control samples in a test-ing workflow entailing self-sampling and automated RNA ex-traction from saliva. The implementation of McQ is modular, scalable and could be extended to other pathogenic targets in future.

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Section: Discussionmentioning

confidence: 99%

McQ – An open-source multiplexed SARS-CoV-2 quantification platform

Vonesch

Bredikhin

Dobrev

et al. 2020

Preprint

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“…Next, the silicon dioxide particulate was pelleted via centrifugation and the eluted RNA was separated and concentrated via isopropanol precipitation, as previously described. 30,29 4S-column and 4S-Milk-of-Silica reagent costs are listed in Supplementary Table 6.…”

Section: Methodsmentioning

confidence: 99%

Sewage, Salt, Silica and SARS-CoV-2 (4S): An economical kit-free method for direct capture of SARS-CoV-2 RNA from wastewater

Whitney

Kennedy

Fan

et al. 2020

Preprint

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Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods’ sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled “Sewage, Salt, Silica and SARS-CoV-2” (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol and silica RNA capture matrices to recover 6-fold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, both suitable as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and could be performed in approximately 3 hours. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic.SYNOPSISThe 4S method for measuring SARS-CoV-2 in wastewater is promising for effective, economical, and accessible wastewater-based epidemiology.ABSTRACT ART

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“…An unprecedented demand for RT-PCR diagnostics has put a strain on every aspect of conducting these laboratory-based assays. Despite the availability of quickly developed protocols (e.g., [ 2 , 3 , 4 , 5 ]), shortages in materials (e.g., swabs, reagents, and consumables) and of infrastructure (e.g., approved facilities, technicians, and equipment) have prevented the efficient testing, tracing, and isolation of infectious individuals. These shortages have led to a global public health crisis of unforeseen consequences, which is further exacerbated in the global south.…”

Section: Introductionmentioning

confidence: 99%

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

Bektaş¹,

Covington²,

Aidelberg

et al. 2021

Viruses

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The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on 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 scalable but suffer from high false negative rates. To address this problem, 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) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification 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 multiplexing 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 labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (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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Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

Cited by 10 publications

References 48 publications

McQ – An open-source multiplexed SARS-CoV-2 quantification platform

McQ – An open-source multiplexed SARS-CoV-2 quantification platform

Sewage, Salt, Silica and SARS-CoV-2 (4S): An economical kit-free method for direct capture of SARS-CoV-2 RNA from wastewater

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

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