Affordable Access

deepdyve-link
Publisher Website

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

Authors
  • Whale, Alexandra S1
  • von der Heide, Eva K2
  • Kohlenberg, Max3
  • Brinckmann, Anja4
  • Baedker, Silke5
  • Karalay, Oezlem6
  • Fernandez-Gonzalez, Ana7
  • Busby, Eloise J8
  • Bustin, Stephen A9
  • Hauser, Heiko10
  • Missel, Andreas11
  • O'Sullivan, Denise M12
  • Huggett, Jim F13
  • Pfaffl, Michael W14
  • Nolan, Tania15
  • 1 National Measurement Laboratory, LGC, Queens Road, Teddington, Middlesex TW11 0LY, UK. Electronic address: [email protected]
  • 2 LGC Genomics GmbH, Research and Development, TGS Haus 8, Ostendstraße 25, 12459 Berlin, Germany. Electronic address: [email protected] , (Germany)
  • 3 LGC Genomics GmbH, Research and Development, TGS Haus 8, Ostendstraße 25, 12459 Berlin, Germany. Electronic address: [email protected] , (Germany)
  • 4 LGC Genomics GmbH, Research and Development, TGS Haus 8, Ostendstraße 25, 12459 Berlin, Germany. Electronic address: [email protected] , (Germany)
  • 5 QIAGEN GmbH, Research and Development, QIAGEN Strasse 1, 40724 Hilden, Germany. Electronic address: [email protected] , (Germany)
  • 6 QIAGEN GmbH, Research and Development, QIAGEN Strasse 1, 40724 Hilden, Germany. Electronic address: [email protected] , (Germany)
  • 7 National Measurement Laboratory, LGC, Queens Road, Teddington, Middlesex TW11 0LY, UK. Electronic address: [email protected]
  • 8 National Measurement Laboratory, LGC, Queens Road, Teddington, Middlesex TW11 0LY, UK. Electronic address: [email protected]
  • 9 Molecular Diagnostics Unit, Medical Technology Research Centre, Anglia Ruskin University, UK. Electronic address: [email protected]
  • 10 LGC Genomics GmbH, Research and Development, TGS Haus 8, Ostendstraße 25, 12459 Berlin, Germany. Electronic address: [email protected] , (Germany)
  • 11 QIAGEN GmbH, Research and Development, QIAGEN Strasse 1, 40724 Hilden, Germany. Electronic address: [email protected] , (Germany)
  • 12 National Measurement Laboratory, LGC, Queens Road, Teddington, Middlesex TW11 0LY, UK. Electronic address: [email protected]
  • 13 National Measurement Laboratory, LGC, Queens Road, Teddington, Middlesex TW11 0LY, UK; School of Biosciences & Medicine, Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK. Electronic address: [email protected]
  • 14 Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Munich, Germany. Electronic address: [email protected] , (Germany)
  • 15 LGC Genomics GmbH, Research and Development, TGS Haus 8, Ostendstraße 25, 12459 Berlin, Germany; Molecular Diagnostics Unit, Medical Technology Research Centre, Anglia Ruskin University, UK. Electronic address: [email protected] , (Germany)
Type
Published Article
Journal
Methods
Publisher
Elsevier
Publication Date
May 01, 2022
Volume
201
Pages
5–14
Identifiers
DOI: 10.1016/j.ymeth.2021.08.006
PMID: 34454016
Source
Medline
Keywords
Language
English
License
Unknown

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. Copyright © 2021 Elsevier Inc. All rights reserved.

Report this publication

Statistics

Seen <100 times