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Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.

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Title: Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.
Authors: Rowan, AG
May, P
Badhan, A
Herrera, C
Watber, P
Penn, R
Crone, MA
Storch, M
Garson, JA
McClure, M
Freemont, PS
Madona, P
Randell, P
Taylor, GP
Item Type: Journal Article
Abstract: There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4% of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.
Issue Date: Aug-2021
Date of Acceptance: 21-Apr-2021
URI: http://hdl.handle.net/10044/1/88654
DOI: 10.1016/j.jviromet.2021.114174
ISSN: 0166-0934
Publisher: Elsevier
Start Page: 1
End Page: 7
Journal / Book Title: Journal of Virological Methods
Volume: 294
Copyright Statement: © 2021 Elsevier Ltd. All rights reserved. This manuscript is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Licence http://creativecommons.org/licenses/by-nc-nd/4.0/
Keywords: COVID-19
RNase P
RT-qPCR
SARS-CoV-2
Viral burden
COVID-19
RNase P
RT-qPCR
SARS-CoV-2
Viral burden
0605 Microbiology
1108 Medical Microbiology
Virology
Publication Status: Published
Conference Place: Netherlands
Online Publication Date: 2021-05-10
Appears in Collections:Department of Immunology and Inflammation
Department of Infectious Diseases
Faculty of Medicine
Imperial College London COVID-19



This item is licensed under a Creative Commons License Creative Commons