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Development of highly accurate digital PCR method and reference material for monkeypox virus detection.

Jiayi Yang1, Ruohui Guo1,2, Huijie Li3

  • 1Center for Advanced Measurement Science, National Institute of Metrology, Beijing, 100029, China.

Analytical and Bioanalytical Chemistry
|January 21, 2023
PubMed
Summary

A new digital PCR (dPCR) method and reference materials improve monkeypox virus (MPXV) detection accuracy. This validated assay helps ensure reliable MPXV testing, crucial for public health surveillance and accurate diagnosis.

Keywords:
Digital PCR (dPCR)Monkeypox virus (MPXV)Reference material (RM)qPCR kits

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Area of Science:

  • Virology
  • Molecular Biology
  • Biotechnology

Background:

  • Human monkeypox (MPXV) is a growing global health concern due to rapid spread and viral evolution.
  • Accurate detection of MPXV is critical for effective diagnosis, treatment, and public health response.
  • Existing MPXV detection methods may lack sufficient analytical sensitivity, potentially leading to false negatives.

Purpose of the Study:

  • To develop a high-order reference method for monkeypox virus (MPXV) detection using digital PCR (dPCR).
  • To establish and characterize pseudovirus reference materials (RM) for MPXV quantification.
  • To evaluate the performance of commercially available MPXV test kits using the developed reference materials.

Main Methods:

  • Development of a digital PCR (dPCR) assay for MPXV detection with established limits of quantification (LoQ) and detection (LoD).
  • Creation and homogeneity assessment of pseudovirus reference materials (RM) containing the conserved F3L gene.
  • Quantification of MPXV RM using the dPCR method to determine a reference value with expanded uncertainty.
  • Performance evaluation of six commercial MPXV test kits against the established RM.

Main Results:

  • The dPCR method achieved an LoQ of 38 copies/reaction and an LoD of 6 copies/reaction.
  • The pseudovirus RM demonstrated homogeneity and a reference value of (2.74 ± 0.46) × 10^3 copies/μL.
  • Four out of six evaluated MPXV test kits failed to meet their claimed limits of detection (LoDs).
  • Poor analytical sensitivity in some test kits may result in false-negative diagnoses.

Conclusions:

  • The establishment of a high-order dPCR reference method provides a reliable tool for MPXV detection.
  • The developed pseudovirus RM is valuable for validating and standardizing MPXV diagnostic assays.
  • Improving the accuracy and reliability of MPXV detection is essential for controlling viral spread and ensuring patient care.