A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits

Mascuch, Samantha J.; Fakhretaha-Aval, Sara; Bowman, Jessica C.; H., Minh Thu; Thomas, Gwendell; Bommarius, Bettina; Ito, Chieri; Zhao, Liangjun; Newnam, Gary P.; Matange, Kavita; Thapa, Hem R.; Barlow, Brett M.; Donegan, Rebecca K.; Nguyen, Nguyet A.; Saccuzzo, Emily G.; Obianyor, Chiamaka T.; Karunakaran, Suneesh C.; Pollet, Paméla; Rothschild-Mancinelli, Brooke; Mestre-Fos, Santi; Guth-Metzler, Rebecca; Bryksin, Anton V.; Petrov, Anton S.; Hazell, Mallory; Ibberson, Carolyn B.; Penev, Petar I.; Mannino, Robert G.; Lam, Wilbur A.; Garcı́a, Andrés J.; Kubanek, Julia; Agarwal, Vinayak; Hud, Nicholas V.; Glass, Jennifer B.; Williams, Loren Dean; Lieberman, Raquel L.

doi:10.1074/jbc.ra120.015434

Cited by 33 publications

(24 citation statements)

References 47 publications

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“…However, the high cost of commercial master mixes could be prohibitive for widespread testing in resource-limited settings. Master mixes assembled using homemade enzymes may help to address this need [ 33 – 35 ].…”

Section: Introductionmentioning

confidence: 99%

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

et al. 2021

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Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.

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Section: Introductionmentioning

confidence: 99%

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

et al. 2021

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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][42][43][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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“…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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A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits

Cited by 33 publications

References 47 publications

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

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

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

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