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

PLoS One. 2022 Jan 20;17(1):e0262656. doi: 10.1371/journal.pone.0262656. eCollection 2022.

Abstract

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of 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 SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR 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 resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19 / diagnosis*
  • COVID-19 / epidemiology
  • COVID-19 / virology
  • COVID-19 Nucleic Acid Testing / methods*
  • Diagnostic Tests, Routine / methods*
  • Genes, Viral
  • Germany / epidemiology
  • Humans
  • RNA, Viral / genetics*
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • SARS-CoV-2 / genetics*
  • Viral Load / methods*

Substances

  • RNA, Viral

Grants and funding

This project was financially supported by the German Federal Ministry of Health based on Chapter 1503 title 68403 for the financial year 2020. As a result, the laboratories were able to receive the reference materials free of charge for this project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.