CCQM-K181 SARS-CoV-2 RNA copy number quantification

Main text Nucleic acid amplification tests for SARS-CoV-2, the virus responsible for the COVID-19 pandemic, primarily target RNA as the analyte. These tests detect the presence of SARS-CoV-2 specific RNA sequences, confirming infection through in vitro diagnostic methods. However, the lack of a standardized reference measurement system has led to varied units and unclear traceability in reporting RNA content quantities, complicating comparisons between different tests [1-5]. To address this challenge, a pilot study CCQM-P199b was initiated during the pandemic, to establish traceability to SARS-CoV-2 RNA quantification. Subsequently the present comparison study (CCQM-K181), coordinated by NIM, LGC, NIBSC and NIST was conducted as a follow up to CCQM-P199b. The key comparison CCQM-K181 aims to support participants in establishing calibration measurement capability (CMC) claim of SARS-CoV-2 RNA quantification. Sixteen NMIs/DIs laboratories participated in the CCQM-K181 "SARS-CoV-2 RNA copy number quantification". Participants were requested to evaluate the copy number concentration, expressed in µL -1 , of the RNA molecule containing the SARS-CoV-2 open reading frame 1ab (ORF1ab; partial region) coding region (NC_045512.2: 13201-15600), the nucleocapsid (N) coding region (NC_045512.2: 28274-29533) and envelope (E) coding region (NC_045512.2: 26245-26472). Materials were provided at two concentration levels: high concentration Study Material 1 (S1, at a nominal concentration of 105 µL -1 ) and low concentration Study Material 2 (S2, at a nominal concentration of 101 µL -1 ). An additional Study Material (S0, at a nominal concentration of 108 µL -1 in aqueous solution without any RNA background) was supplied upon request to be quantified by an orthogonal method, isotope dilution mass spectrometry (IDMS). S1 and S2 were gravimetrically diluted from S0 in an aqueous buffer solution containing yeast total RNA background. Fifteen laboratories reported results for S1 and 14 laboratories submitted results for S2. One laboratory applied 2-step RT-dPCR and the remainder of laboratories used one-step RT-dPCR. Three laboratories also performed IDMS measurement on S0, and two of them corrected their RT-PCR data with correction factors assuming an incomplete reverse transcription of the RNA molecules. Three laboratories with four independent measurements measured mass concentration in the high concentration Study Material S0 by IDMS, with two of the laboratories converting their results to copy number concentration values (three values in total). Consensus reference values and their uncertainties for the diluted S1 and S2 materials were calculated based on the IDMS results obtained for S0 and the gravimetric dilution factors applied. The KCRVs with their expanded uncertainties for S1 and S2 were determined to be (8.27±0.58)×105 µL -1 and (6.4±0.6)×101 µL -1 , respectively. Successful participation in CCQM-K181 demonstrates CMC for determining RNA copy number concentration range from 101 µL -1 to 106 µL -1 of defined SARS-CoV-2 target sequences in a non-target RNA matrix or as a single template in aqueous solution. This may include measurement capabilities such as: (1) value assignment of primary reference materials; (2) value assignment of calibration solutions; (3) measurement of RNA sequence copy number concentration using RT-dPCR. To reach the main text of this paper, click on Final Report . Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/ . The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).