Apr 16, 2020

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

BioRxiv : the Preprint Server for Biology
David G. RobinsonJohn D. Storey


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). 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.

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Mentioned in this Paper

Real-Time Polymerase Chain Reaction
System Process
Saccharomyces cerevisiae allergenic extract
Technology, Health Care
Gene Expression
Saccharomyces cerevisiae
Silo (Dataset)

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