Nucleic acid extraction and mpox q-PCR
After a heat inactivation step (12 minutes at 70°C), nucleic acids
(500µL of samples) were extracted using MagNA Pure LC 2.0 Instrument
(Roche, Meylan, France) and eluted in 100µL. Mpox virus specific
real-time PCR assay (qPCR) validated by both Centers for Disease Control
& Prevention (CDC)9 and French Orthopoxvirus
national reference center10 was
performed11. An exogenous internal extraction and
amplification control (IEAC) was added to each sample before extraction
(Simplexa™ Extraction and Amplification Control Set, DiaSorin, Saluggia,
Italy)12. Negative controls were added to each
extraction batch and a positive control (DNA extracted from a MXPV cell
culture given by the French Orthopoxvirus national reference center) was
tested in each PCR run.