Division II of In Vitro Diagnostics for Infectious Diseases, Institute for In Vitro Diagnostics Control, National Institutes for Food and Drug Control, Beijing 100050, China.
Department of Biomedical Devices, Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510320, China.
Anal Chem. 2021 Jan 19;93(2):715-721. doi: 10.1021/acs.analchem.0c03996. Epub 2020 Dec 8.
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 ± 6.5 × 10 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.
新型冠状病毒病 2019（COVID-19）的爆发是由严重急性呼吸系统综合症冠状病毒 2（SARS-CoV-2）引起的，已在全球范围内传播。为满足对感染个体进行筛选和诊断的紧急和大量需求，世界各地的监管机构已紧急批准了许多使用核酸检测（NAT）的体外诊断检测方法。对于检测限（LoD）的研究，具有明确浓度或效价的参考标准至关重要，而检测限是诊断检测的关键特征。尽管已经公布了几种质粒或合成 RNA 的参考标准，但仍需要具有准确浓度的灭活病毒颗粒的参考标准来评估完整的程序。在这里，我们使用多个商业化平台上的数字 PCR（dPCR）进行了一项合作研究，以估计 NAT 可检测单位作为灭活全病毒 SARS-CoV-2 参考标准候选物的病毒基因组等效数量（GEQ）。定量结果的中位数（4.6×10±6.5×10GEQ/mL）被视为参考标准中病毒颗粒的共识真实 GEQ 值。然后，将该参考标准用于挑战六种官方批准的诊断检测方法的 LoD。我们的研究表明，通过 dPCR 定量的灭活全病毒可作为参考标准，并为检测方法的开发、质量控制和法规监测提供了统一的解决方案。
