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

Human monkeypox has attracted attention recently. Monkeypox virus (MPXV) keeps evolving as it spreading around the world rapidly, which may threaten the health of more and more people. Here, we have developed a high order reference method based on digital PCR (dPCR) for MPXV detection, of which the limits of quantification (LoQ) and detection (LoD) are 38 and 6 copies/reaction, respectively. Pseudovirus reference materials (RM) containing the conserved F3L gene has been developed, and the homogeneity assessment showed that the RM was homogeneous. The reference value with its expanded uncertainty determined by the established dPCR is (2.74 +/- 0.46) x 10(3) copies/mu L. Six different MPXV test kits were accessed by the RM. Four out of six test kits cannot reach their claimed LoDs. The poor analytical sensitivity might cause false-negative results, which lead to incorrect diagnosis and treatment. The establishment of a high order reference method of dPCR and pseudovirus RM is very useful for improving the accuracy and reliability of MPXV detection.

Automated summary

A high order reference method based on digital PCR (dPCR) is developed for the detection of monkeypox virus (MPXV), with limits of quantification (LoQ) and detection (LoD) of 38 and 6 copies/reaction, respectively. A pseudovirus reference material (RM) containing the conserved F3L gene is developed and found to be homogeneous. Using this method, the reference value for MPXV is determined to be (2.74 +/- 0.46) x 10(3) copies/μL. Four out of six MPXV test kits tested with this RM fail to reach their claimed LoDs, which can lead to false-negative results and incorrect diagnosis and treatment. The establishment of a high order reference method using dPCR and pseudovirus RM is crucial for improving the accuracy and reliability of MPXV detection.