CCQM-P199b: Interlaboratory comparability study of SARS-CoV-2 RNA copy number quantification

Published

March 27, 2025

Author(s)

Alison Devonshire, Eloise Busby, Gerwyn Jones, Denise O'Sullivan, Ana Fernandez-Gonzalez, Laura Hernandez-Hernandez, Xinhua Dai, Lianhua Dong, Chunyan Niu, Jie Xie, Xia Wang, Xiaoting Qiao, Xiang Fang, Clare Morris, Neil Almond, Megan Cleveland, Peter Vallone, Esther Castro Galván, Melina Pérez Urquiza, Mercedes Guadalupe Herrera López, Arifa Khan, Sandra Fuentes, Elsa Baumeister, John Emerson Leguizamon Guerrero, Sergio Luis Davila Gonzalez, Andres Felipe León Torres, Aurea Folgueras-Flatschart, Marcelo Neves de Medeiros, Antonio Marcos Saraiva, Roberto Becht Flatschart, Carla Divieto, Mattia Pegoraro, Massimo Zucco, Laura Revel, Marco Mazzara, Philippe Corbisier, Gerhard Buttinger, Young-Kyung Bae, Alexandra Bogožalec Košir, Mojca Milavec, Malcolm Hawkins, A. Pia Sanzone, Phattarapornn Morris, Sasithon Temisak, David Lynch, Jacob McLaughlin, Michael Forbes-Smith, Felicity Hall, Daniel Burke, Sachie Shibayama, Shin-ichiro Fujii, Megumi Kato, Samreen Falak, Rainer Macdonald, Andreas Kummrow, Andrey Komissarov, Sema Akyurek, Muslum Akgoz, Maxim Vonsky, Andrey Runov, Elena Kulyabina, Denis Rebrikov, Jim Huggett

Abstract

RNA is the analyte targeted by nucleic acid amplification tests for SARS-CoV-2 virus, the causative agent of the COVID-19 pandemic. RNA concentration measured in respiratory specimens can vary by over nine orders of magnitude (1, 2). While RNA abundances are not generally used to guide treatment, quantitative thresholds have been proposed to better stratify patients in terms of clinical relevance (3, 4) and quantitative RNA measurements are needed to demonstrate conformity of an in vitro diagnostic solution to stipulated limit of detection targets (5-8). In the absence of an established reference measurement system, these measures were often based on a variety of units with unclear traceability making comparison between different tests difficult (9). Routes to standardization include the implementation of the WHO International Standard for SARS-CoV-2 RNA and the use of reverse transcription-digital PCR (RT-dPCR) as a candidate reference measurement procedure (RMP), which was employed during the COVID-19 pandemic in External Quality Assurance schemes and for value assignment of Reference Materials. Interlaboratory study CCQM P199b "SARS-CoV-2 RNA copy number quantification" was designed to test the fitness-for-purpose of developed candidate RMPs for SARS-CoV-2 genomic targets, and was conducted under the auspices of the Consultative Committee for Amount Substance to evaluate the measurement comparability of national metrology institutes (NMIs) and designated institutes (DIs), thereby supporting international standardization (10). Twenty-one laboratories participated in CCQM P199b and were requested to report the RNA copy number concentration, expressed in number of copies per microliter, of the SARS-CoV-2 nucleocapsid (N) gene partial region (NC_045512.2: 28274-29239) (Measurand 1, all four Study Materials) and envelope (E) gene (NC_045512.2: 26245-26472) (optional Measurand 2, Study Materials 1-2 only) in samples consisting of in vitro transcribed RNA (Study Materials 1, 3 and 4) or purified RNA from lentiviral constructs (Study Material 2). Materials were provided in two categories: lower concentration Study Materials 1-3 (101-104 /L in aqueous solution containing human RNA background) and high concentration Study Material 4 (109 /L in aqueous solution without any other RNA background). For the measurement of N gene concentration in the lower concentration Study Materials 1-3, the majority of laboratories (n = 17) used one-step RT-dPCR, with three laboratories applying two-step RT-dPCR and one laboratory RT-qPCR. Sixteen laboratories submitted results for Measurand 2. Reproducibility (%CV or equivalent) for RT-dPCR ranged from 19% to 31% (all materials/measurands). Measurements of the high concentration Study Material 4 by orthogonal methods (isotope dilution mass spectrometry and single molecule flow cytometry) and lower concentration Study Material 3 (prepared by gravimetric dilution from Study Material 4) were in a good agreement, suggesting a lack of overall bias in RT-dPCR measurements. However methodological factors such primer and probe (assay) sequences, RT-dPCR reagents and dPCR partition volume were found to be potential sources of interlaboratory variation which needs to be controlled when applying this technique. This study demonstrates that the accuracy of RT-dPCR is fit for purpose as a RMP for viral RNA quantification and highlights where metrological approaches such as the use of in vitro transcribed controls, orthogonal methods and measurement uncertainty evaluation can support standardization of molecular methods.

Citation

bioRxiv

Pub Type

Journals

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Keywords

dPCR, SARS-CoV-2, CCQM

Citation

Devonshire, A. , Busby, E. , Jones, G. , O'Sullivan, D. , Fernandez-Gonzalez, A. , Hernandez-Hernandez, L. , Dai, X. , Dong, L. , Niu, C. , Xie, J. , Wang, X. , Qiao, X. , Fang, X. , Morris, C. , Almond, N. , Cleveland, M. , Vallone, P. , Castro Galván, E. , Pérez Urquiza, M. , Herrera López, M. , Khan, A. , Fuentes, S. , Baumeister, E. , Leguizamon Guerrero, J. , Davila Gonzalez, S. , León Torres, A. , Folgueras-Flatschart, A. , Neves de Medeiros, M. , Saraiva, A. , Becht Flatschart, R. , Divieto, C. , Pegoraro, M. , Zucco, M. , Revel, L. , Mazzara, M. , Corbisier, P. , Buttinger, G. , Bae, Y. , Bogožalec Košir, A. , Milavec, M. , Hawkins, M. , Sanzone, A. , Morris, P. , Temisak, S. , Lynch, D. , McLaughlin, J. , Forbes-Smith, M. , Hall, F. , Burke, D. , Shibayama, S. , Fujii, S. , Kato, M. , Falak, S. , Macdonald, R. , Kummrow, A. , Komissarov, A. , Akyurek, S. , Akgoz, M. , Vonsky, M. , Runov, A. , Kulyabina, E. , Rebrikov, D. and Huggett, J. (2025), CCQM-P199b: Interlaboratory comparability study of SARS-CoV-2 RNA copy number quantification, bioRxiv, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=957041 (Accessed May 18, 2025)