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Background <p>Viral antigen rapid detection tests (Ag-RDTs) and PCR-based nucleic acid amplification tests (NAATs) are essential for diagnosing infections like SARS-CoV-2. However, unlike nucleic acids which can be quantified precisely in copies, viral antigens lack a standardized unit of measurement, hindering precise analytical evaluation of Ag-RDTs and comparison with NAATs.</p> Methods <p>We established a universal national standard for SARS-CoV-2 antigen and nucleic acid based on an Omicron BA.1 strain, inactivated using β-propiolactone (BPL). Its concentration was assigned via multi-laboratory digital PCR (dPCR). Clinical samples were tested with Ag-RDTs and NAATs. We also compared the impact of heat and BPL inactivation on detection using dPCR, qPCR, and sequencing. The standard was then used to evaluate the limits of detection (LoDs) of commercial Ag-RDTs and NAATs using a common unitage.</p> Results <p>Clinical samples’ results showed that antigen positivity was correlated with higher nucleic acid titers. Both BPL inactivation and heat inactivation maintained comparable nucleic acid titers, but BPL inactivation preserved better antigen activity. The national standard concentration was assigned as 1.04 × 10<sup>8</sup> Unit/mL (standard uncertainty: 3.48 × 10<sup>6</sup> Unit/mL). Using this standard, NAATs exhibited lower LoDs than Ag-RDTs, though some Ag-RDT sensitivity approached NAAT levels. Most commercial assays met or exceeded their claimed LoDs, with all claimed NAAT LoDs falling within 2-fold of measured values. The standard enabled direct cross-format LoD comparison.</p> Conclusion <p>We established and validated a BPL-inactivated universal national standard specifically designed to overcome the difficulties in quantifying antigen and enabling direct sensitivity comparison between SARS-CoV-2 Ag-RDTs and NAATs.</p>

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The establishment of a universal standard for both viral antigen and nucleid acid detection based on digital PCR

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月15日

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  • Manyu Li,
  • Siyuan Yang,
  • Tingting Ma,
  • Alimire Abulikemu,
  • Chunlei Zhang,
  • Xun Cheng,
  • Jing Li,
  • Jia Lu,
  • Pengfei Wang,
  • Yong Guo,
  • Donglai Liu,
  • Linghang Wang,
  • Haiwei Zhou

摘要

Background

Viral antigen rapid detection tests (Ag-RDTs) and PCR-based nucleic acid amplification tests (NAATs) are essential for diagnosing infections like SARS-CoV-2. However, unlike nucleic acids which can be quantified precisely in copies, viral antigens lack a standardized unit of measurement, hindering precise analytical evaluation of Ag-RDTs and comparison with NAATs.

Methods

We established a universal national standard for SARS-CoV-2 antigen and nucleic acid based on an Omicron BA.1 strain, inactivated using β-propiolactone (BPL). Its concentration was assigned via multi-laboratory digital PCR (dPCR). Clinical samples were tested with Ag-RDTs and NAATs. We also compared the impact of heat and BPL inactivation on detection using dPCR, qPCR, and sequencing. The standard was then used to evaluate the limits of detection (LoDs) of commercial Ag-RDTs and NAATs using a common unitage.

Results

Clinical samples’ results showed that antigen positivity was correlated with higher nucleic acid titers. Both BPL inactivation and heat inactivation maintained comparable nucleic acid titers, but BPL inactivation preserved better antigen activity. The national standard concentration was assigned as 1.04 × 108 Unit/mL (standard uncertainty: 3.48 × 106 Unit/mL). Using this standard, NAATs exhibited lower LoDs than Ag-RDTs, though some Ag-RDT sensitivity approached NAAT levels. Most commercial assays met or exceeded their claimed LoDs, with all claimed NAAT LoDs falling within 2-fold of measured values. The standard enabled direct cross-format LoD comparison.

Conclusion

We established and validated a BPL-inactivated universal national standard specifically designed to overcome the difficulties in quantifying antigen and enabling direct sensitivity comparison between SARS-CoV-2 Ag-RDTs and NAATs.