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

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.

Keywords: Copy number; Quantification; RT-dPCR; SARS-CoV-2.

Fig. 1

Participants’ results for copy number concentration of SARS-CoV-2 RNA ORF 1ab gene, E gene, and N gene. Results of the five laboratories using dPCR platforms of Bio-Rad are indicated with red circle, and results of the five laboratories using other platforms with blue diamond. Solid and dashed horizontal lines indicate RV and expanded uncertainty (95% level of confidence, k = 2) of each gene

Fig. 2

Scatter diagram showing the copy number concentration of different gene targets obtained from all participants

Fig. 3

Z′ scores of the participants