Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.
File(s)
Accepted version
Author(s)
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.
Date Issued
2021-08
Date Acceptance
2021-04-21
Citation
Journal of Virological Methods, 2021, 294, pp.1-7
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/
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/33984396
PII: S0166-0934(21)00113-0
Subjects
COVID-19
RNase P
RT-qPCR
SARS-CoV-2
Viral burden
Publication Status
Published
Coverage Spatial
Netherlands
Date Publish Online
2021-05-10