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A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production

Sanchita Bhadra, Andre C. Maranhao, Andrew D. Ellington
doi: https://doi.org/10.1101/2020.03.29.013342
Sanchita Bhadra
Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, 78703
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Andre C. Maranhao
Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, 78703
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Andrew D. Ellington
Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, 78703
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  • For correspondence: ellingtonlab@gmail.com
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ABSTRACT

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed a RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase / DNA polymerase (RTX)1. Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use – (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs from Addgene or our lab and begin to develop their own assays, within whatever regulatory framework exists for them.

We lay out protocols for dye-based and TaqMan probe-based assays, in order to best compare with ‘gold standard’ reagents. These protocols should form the basis of further modifications that can simplify the assay to the use of overexpressing cells themselves as reagents.

Developing dye-based and TaqMan probe-based RT-qPCR assays with RTX

Footnotes

  • ↵1 Inquiries about manuscript to ellingtonlab{at}gmail.com

  • ↵2 Inquiries about vectors to a.maranhao{at}utexas.edu

  • The final concentration of dNTPs in reaction mixes has been corrected to 400 uM instead of 400 nM.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 09, 2020.
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A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production
Sanchita Bhadra, Andre C. Maranhao, Andrew D. Ellington
bioRxiv 2020.03.29.013342; doi: https://doi.org/10.1101/2020.03.29.013342
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A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production
Sanchita Bhadra, Andre C. Maranhao, Andrew D. Ellington
bioRxiv 2020.03.29.013342; doi: https://doi.org/10.1101/2020.03.29.013342

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