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CDC/ Alissa Eckert, MSMI; Dan Higgins, MAMS

National Measurement Institutes demonstrate high accuracy reference measurement system for SARS-CoV-2 testing

Twenty-one National Metrology Institutes and expert laboratories from sixteen countries have demonstrated that highly accurate measurements of the amount of the SARS-CoV-2 viral RNA can be achieved worldwide using reverse transcription-digital PCR (RT-dPCR). The ability to accurately measure the amount of the viral cause of COVID-19 with global equivalence will considerably improve testing confidence and support countries in effectively tackling the pandemic.

The comparison study (CCQM-P199.b) organized by the CCQM Working Group on Nucleic Acid Analysis (CCQM-NAWG), and coordinated by the National Measurement Laboratory at LGC (UK), NIM (China), NIBSC (UK) and NIST (US), required quantification of the same viral genetic sequences targeted by many of the diagnostic tests. The RT-dPCR results were found to agree very well with each other and different SI-traceable non-molecular orthogonal methods; most values were within +/-40% of mean. The reproducibility of the method is unprecedented for absolute molecular measurements, where orders of magnitude of spread in reported copy numbers can be found using conventional molecular diagnostic methods. This work, made possible by over a decade of CCQM led collaborative efforts within the bio-metrology community, represents the most comprehensive example of highly reproducible and sensitive measurement of RNA and opens the possibility for SI-traceable quantification of viral genes. The methods and results are already being used by National Metrology Institutes to value assign reference materials that underpin the quality of SARS-CoV-2 diagnostic tests. The comparison was conducted under an accelerated timeline, with the worldwide comparison completed in under six months, and the final report expected in early 2021.

In the longer term, these capabilities have far wider implications as they can provide a global foundation for ensuring the accuracy of associated molecular methods whether applied to COVID-19, as preparation for any future global pandemic or for wider diagnostic uses such as in testing for antibiotic resistance or cancer.

Results from part of the study demonstrating that laboratories performing molecular RT-dPCR methods are able to perform with good agreement with each other and the SI-traceable orthogonal methods (isotope-dilution mass spectrometry and single molecule flow cytometry), the latter providing the basis for the RT-dPCR method's claimed accuracy. The RT-dPCR results were compared with orthogonal methods using different concentrations of the same RNA material. The different concentrations were prepared using gravimetric dilution. Diamonds show individual laboratories' results with solid black line (mean) and dotted green lines +/-40% of mean value.