Interlaboratory assessment of quantification of SARS-CoV-2 RNA by reverse transcription digital PCR

Abstract

Graphical abstract The pandemic of the novel coronavirus disease 2019 (COVID-19) has caused severe harm to the health of people all around the world. Molecular detection of the pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), played a crucial role in the control of the disease. Reverse transcription digital PCR (RT-dPCR) has been developed and used in the detection of SARS-CoV-2 RNA as an absolute quantification method. Here, an interlaboratory assessment of quantification of SARS-CoV-2 RNA was organized by the National Institute of Metrology, China (NIMC), using in vitro transcribed RNA samples, among ten laboratories on six different dPCR platforms. Copy number concentrations of three genes of SARS-CoV-2 were measured by all participants. Consistent results were obtained with dispersion within 2.2-fold and CV% below 23% among different dPCR platforms and laboratories, and Z′ scores of all the reported results being satisfactory. Possible reasons for the dispersion included PCR assays, partition volume, and reverse transcription conditions. This study demonstrated the comparability and applicability of RT-dPCR method for quantification of SARS-CoV-2 RNA and showed the capability of the participating laboratories at SARS-CoV-2 test by RT-dPCR platform. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03680-2.

Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks

“…dPCR-quantified reference materials have also been found to give reproducible results across different laboratories, even when different dPCR instruments are used ( 70 , 71 ). For example, an interlaboratory assessment of in vitro -transcribed (IVT) RNA reference materials was conducted by the National Institute of Metrology, China (NIMC), using 6 different platforms and 10 participating laboratories.…”

“…Currently, several companies have developed commercial reference materials that can be used to validate these assays. Additionally, in their studies, Zhou et al ( 70 ) and Niu et al ( 71 ) have shown that a reference candidate can be developed for head-to-head comparisons of different dPCR instruments and methods for SARS-CoV-2 detection. With the introduction of new variants, the analysis of novel PP sets that can readily detect the new SARS-CoV-2 variants will be of great importance.…”

“…Reference materials have played an important role in the molecular diagnosis of SARS-CoV-2 since the beginning of the pandemic. The establishment of stable, well-characterized reference materials helps not only in the validation of test results but also in the analysis of assays, workflows, devices, and SARS-CoV-2 diagnostic kits ( 70 , 71 , 79 – 81 ). Quantification by qPCR requires a sample of a known quantity to establish a standard curve, which in turn is used for relative quantification ( 27 , 30 , 72 ).…”

“…These results were also found to be acceptable as all three targets had a Z score of ≤2. The slight variations between different laboratories and instruments were also noted to arise from differences in PCR assays, partition volumes, and reverse transcription conditions ( 71 ). Similarly, Zhou et al developed a reference standard from whole SARS-CoV-2 cultured in a cell and quantified by RT-dPCR ( 70 ).…”

Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR

“…Nevertheless, compared with DNA quantification by dPCR, big variations always occurred for RNA quantification [12][13][14]. The involvement of the reverse transcription (RT) process is the major difference between DNA and RNA quantification.…”