Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics

Whale, Alexandra S. and von der Heide, Eva K. and Kohlenberg, Max and Brinckmann, Anja and Baedker, Silke and Karalay, Oezlem and Fernandez-Gonzalez, Ana and Busby, Eloise J. and Bustin, Stephen A. and Hauser, Heiko and Missel, Andreas and O'Sullivan, Denise M. and Huggett, Jim F. and Pfaffl, Michael W. and Nolan, Tania (2021) Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics. Methods. ISSN 1095-9130

[img] Text
Accepted Version
Restricted to Repository staff only until 26 August 2022.
Available under the following license: Creative Commons Attribution Non-commercial No Derivatives.

Download (2MB)
[img] Text (Word version)
Accepted Version
Restricted to Repository staff only until 26 August 2022.
Available under the following license: Creative Commons Attribution Non-commercial No Derivatives.

Download (1MB)
Official URL: http://dx.doi.org/10.1016/j.ymeth.2021.08.006

Abstract

Coronavirus disease 2019 (COVID-19) is an infectious, acute respiratory disease caused mainly by person-to-person transmission of the coronavirus SARS-CoV-2. Its emergence has caused a world-wide acute health crisis, intensified by the challenge of reliably identifying individuals likely to transmit the disease. Diagnosis is hampered by the many unknowns surrounding this disease, including those relating to infectious viral burden. This uncertainty is exacerbated by disagreement surrounding the clinical relevance of molecular testing using reverse transcription quantitative PCR (RT-qPCR) for the presence of viral RNA, most often based on the reporting of quantification cycles (Cq), which is also termed the cycle threshold (Ct) or crossing point (Cp). Despite it being common knowledge that Cqs are relative values varying according to a wide range of different parameters, there have been efforts to use them as though they were absolute units, with Cqs below an arbitrarily determined value, deemed to signify a positive result and those above, a negative one. Our results investigated the effects of a range of common variables on Cq values. These data include a detailed analysis of the effect of different carrier molecules on RNA extraction. The impact of sample matrix of buccal swabs and saliva on RNA extraction efficiency was demonstrated in RT-qPCR and the impact of potentially inhibiting compounds in urine along with bile salts were investigated in RT-digital PCR (RT-dPCR). The latter studies were performed such that the impact on the RT step could be separated from the PCR step. In this way, the RT was shown to be more susceptible to inhibitors than the PCR. Together, these studies demonstrate that the consequent variability of test results makes subjective Cq cut-off values unsuitable for the identification of infectious individuals. We also discuss the importance of using reliable control materials for accurate quantification and highlight the substantial role played by dPCR as a method for their development.

Item Type: Journal Article
Keywords: dPCR, qPCR, Virus quantification, Cq value validity, MIQE and dMIQE guidelines, Sample matrix effects, Standard material, SARS-CoV-2
Faculty: COVID-19 Research Collection
Faculty of Science & Engineering
SWORD Depositor: Symplectic User
Depositing User: Symplectic User
Date Deposited: 08 Sep 2021 09:15
Last Modified: 22 Nov 2021 12:35
URI: https://arro.anglia.ac.uk/id/eprint/706907

Actions (login required)

Edit Item Edit Item