A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR 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:10.1101/2020.07.29.20163949

Cited by 9 publications

(10 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, a dual bottleneck emerged early on in the pandemic in terms of sourcing the bio logical materials as well as sourcing the primary sources for manufacturing. The shortage of reagents and dis posables is one of the most obvious later stage problems once an outbreak becomes more widespread and ulti mately pandemic 80,81 . In such instances it may become mandatory for manufacturers to start sharing production processes and recipes for reagents 82 .…”

Section: Mass Production Of Testsmentioning

confidence: 99%

Considerations for diagnostic COVID-19 tests

et al. 2020

View full text Add to dashboard Cite

During the early phase of the coronavirus disease 2019 (COVID-19) pandemic, design, development, validation, verification and implementation of diagnostic tests were actively addressed by a large number of diagnostic test manufacturers. Hundreds of molecular tests and immunoassays were rapidly developed, albeit many still await clinical validation and formal approval. In this Review, we summarize the crucial role of diagnostic tests during the first global wave of COVID-19. We explore the technical and implementation problems encountered during this early phase in the pandemic, and try to define future directions for the progressive and better use of (syndromic) diagnostics during a possible resurgence of COVID-19 in future global waves or regional outbreaks. Continuous global improvement in diagnostic test preparedness is essential for more rapid detection of patients, possibly at the point of care, and for optimized prevention and treatment, in both industrialized countries and low-resource settings.

show abstract

Section: Mass Production Of Testsmentioning

confidence: 99%

Considerations for diagnostic COVID-19 tests

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Practices will vary by the source of false-positives, for example, using the NTC contamination example above, it may be useful to use an RT-PCR master mix formulation that includes a deoxynucleotide mix containing uracil instead of thymine along with a heatlabile uracil-N-glycosylase (UNG), which can be used to minimize potential carryover amplification contamination during the RT-PCR amplification of SARS-CoV-2 (Blanchard et al, 2014;Mascuch et al, 2020). For other sources of contamination, it may be advantageous to seek additional analyst training or to implement additional control measures such as an equipment or transport blanks.…”

Section: Plan a Course Of Actionmentioning

confidence: 99%

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Ahmed¹,

Simpson²,

Bertsch³

et al. 2021

Preprint

View full text Add to dashboard Cite

Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR test kits

Cited by 9 publications

References 50 publications

Considerations for diagnostic COVID-19 tests

Considerations for diagnostic COVID-19 tests

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

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

Contact Info

Product

Resources

About