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RNA reference materials with defined viral RNA loads of SARS-CoV-2 – A useful tool towards a better PCR assay harmonization



Laura Vierbaum, Nathalie Wojtalewicz, Vanessa Lindig, Ulf Dühring, Hans-Peter Grunert, Christian Drosten, Victor Corman, Daniela Niemeyer, Sandra Ciesek, Holger Rabenau, Annemarie Berger, Martin Obermeier, Andreas Nitsche, Janine Michel, Martin Mielke, Jim Huggett, Denise O'Sullivan, Simon Cowen, Megan Cleveland, Peter Vallone, Samreen Falak, Andreas Kummrow, Thomas Keller, Ingo Schellenberg, Heinz Zeichhardt, Martin Kammel


The outbreak and pandemic spread of SARS-CoV-2, the cause of the novel coronavirus disease (COVID-19), led to the need for a reliable detection method to track the circulation of this virus. After introducing PCR methods for genome detection of SARS-CoV-2 in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different target genes showed high variability depending on the test systems used by the individual laboratories. Especially in the context of clinical decisions, e.g. criteria for discharging patients from isolation, it became crucial to introduce reliable anchor samples to estimate the SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with estimated SARS-CoV-2 RNA loads of 107 copies/mL (RM 1) and 106 copies/mL (RM 2), respectively. Quantification was performed by RT-PCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This yielded 1,109 datasets. These results were differentiated with regard to test system and gene region. The two reference materials have proven to be a suitable tool for laboratories which enabled them to anchor their measured Ct values to specified SARS-CoV-2 RNA loads. However, our data analysis makes it clear that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that each individual laboratory relates its measured Ct values to the particular test system used and the targeted gene region.
Plos One


Vierbaum, L. , Wojtalewicz, N. , Lindig, V. , Dühring, U. , Grunert, H. , Drosten, C. , Corman, V. , Niemeyer, D. , Ciesek, S. , Rabenau, H. , Berger, A. , Obermeier, M. , Nitsche, A. , Michel, J. , Mielke, M. , Huggett, J. , O'Sullivan, D. , Cowen, S. , Cleveland, M. , Vallone, P. , Falak, S. , Kummrow, A. , Keller, T. , Schellenberg, I. , Zeichhardt, H. and Kammel, M. (2022), RNA reference materials with defined viral RNA loads of SARS-CoV-2 – A useful tool towards a better PCR assay harmonization, Plos One, [online],, (Accessed July 18, 2024)


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Created January 20, 2022, Updated November 29, 2022