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

Abstract

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) × 10 3 copies/μL. 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. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-023-04518-9.

Evaluation of a ddPCR Commercial Assay for the Absolute Quantification of the Monkeypox Virus West Africa in Clinical Samples

“…As of today, a few previous studies have reported the use of ddPCR for the detection of poxviruses [ 20 , 21 , 22 ]. Two recent works reported data obtained using in-house MPXV dPCRs for the quantification of in vitro cultured viruses [ 23 ] and of clinical samples [ 24 ]. In particular, Colavita et al [ 24 ] reported the follow-up laboratory investigation of three MPXV cases infected in May-June 2022 from diagnosis to disease resolution, and the viral DNA widely varied in the different biological matrices investigated but was higher in oropharyngeal swabs, saliva, and stools compared to semen and urine.…”

Establishment of reference measurement procedure and reference material for Treponema pallidum

“…To ensure the integrity of the oil-in-water droplets during the reaction, the ramp rate in the dPCR reaction program was set to 2 °C s −1 . 19…”

“…To ensure the integrity of the oil-in-water droplets during the reaction, the ramp rate in the dPCR reaction program was set to 2 °C s −1 . 19 Poisson statistics were used to calculate the absolute copy number concentration of the TP tpp47 gene in each sample. 20 To distinguish between positive (blue) and negative (gray) droplets, 21 a suitable threshold line was set and the nal concentration of the template was the value calculated with SightPro soware (V0.3.2, Sniper, China) multiplied by the dilution of the template in the reaction system.…”

Review of virological methods for laboratory diagnosis and characterization of monkeypox virus (MPXV): lessons learned from the 2022 Mpox outbreak

“…During the Mpox 2022 outbreak, dPCR was used for the absolute quantification of MPXV in clinical samples (46), to analyze the kinetics of viral DNA in body fluids during the acute phase of Mpox (49), and to quantify virus growth in vitro (50). A recent study demonstrated a strong correlation between MPXV DNA levels measured by dPCR and Ct levels determined by qPCR (51).…”