A one-enzyme rt-qpcr assay for sars-cov-2, and procedures for reagent production
Bio-protocol
; 11(2) (no pagination), 2021.
Article
in English
| EMBASE | ID: covidwho-2321994
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 an RTqPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson et al., 2016). 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 RTX. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs and begin to develop their own assays, within whatever regulatory framework exists for them.Copyright © 2021 Bio-protocol LLC. All Rights Reserved.
Coronavirus; Nucleic acid diagnostics; Quantitative RT-PCR; Reverse transcriptase; SARS-CoV-2; TaqMan RTqPCR; article; centrifugation; comparative study; controlled study; COVID-19 testing; molecular diagnosis; nonhuman; protein function; protein purification; real time polymerase chain reaction; Severe acute respiratory syndrome coronavirus 2; validation study; RNA directed DNA polymerase/ec [Endogenous Compound]; PCR assay kit; SARS coronavirus 2 nucleic acid test kit
Full text:
Available
Collection:
Databases of international organizations
Database:
EMBASE
Type of study:
Prognostic study
Language:
English
Journal:
Bio-protocol
Year:
2021
Document Type:
Article
Similar
MEDLINE
...
LILACS
LIS