A SARS-CoV-2 Reference Standard Quantified by Multiple Digital PCR Platforms for Quality Assessment of Molecular Tests

Abstract

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 10 5 ± 6.5 × 10 4 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.

Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs

“…Indeed, in the last few months, several works have underlined the benefits of using the RT-ddPCR for the analysis of samples with a low SARS-CoV-2 load or derived from patient's saliva, comparing both different platforms (i.e., QX200 system from Bio-Rad, TD-1 system from TargetingOne, or Naica system from Stilla Technologies) and using different available kits [ 21 – 23 , 25 , 26 ]. Despite the diverse approaches, all of them are in agreement with our data in considering RT-ddPCR a highly robust technique for SARS-CoV-2 detection in clinical applications, to be preferred during patients' follow-up, as the monitoring of the correct viral load is essential to contain the infections.…”

SARS-CoV-2/COVID-19 laboratory biosafety practices and current molecular diagnostic tools

“…Owing to its advantages, more should be done to include RT-ddPCR in routine diagnostic procedures. Its ability to perform absolute quantification can be leveraged to generate reference standards for validation of other SARS-CoV-2 NAATs [36] .…”

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

“…However, there is not much research to assess the comparability of RT-dPCR between different laboratories and platforms. Inactivated virus particles had been used as a reference standard quantified by RT-dPCR on multiple commercialized platforms, and a CV% of about 40% was observed [ 26 ]. In order to exclude the effect of the complex extraction step of virus which may introduce extra dispersion to the comparison results, in this study, an interlaboratory assessment on quantification of in vitro transcribed SARS-CoV-2 RNA by RT-dPCR of six different platforms has been organized and analyzed by the National Institute of Metrology, China (NIMC).…”