Table 1.

Reagents used to detect MPXV nucleic acids by qPCR and to confirm target specificity by amplicon sequencing. Primers and probes were based on CDC published sequences with some modifications. The forward primer was modified to increase the melting temperature to improve amplification efficiency. Other changes were to account for sequence differences in primers between the CDC set and that of virus isolated in Massachusetts in 2022 (GenBank accession number ON563414) that was used to generate qPCR standards. Primers listed for sequencing were used to generate the amplicon and prime the sequencing in both directions (Centers for Disease Control, and Prevention Poxvirus, and Rabies Branch, C. P, n.d.).

Assay		Primers/probe sequences	Binding site
qPCR	Forward	TCTTGCTATCACATAATCTGRAAGCGTA	2588–2615
	Reverse	GATATAGCACCACATGCACCA	2708–2687
	Probe	5HEX-AAGCCGTAA/ZEN/TCTATGTTGTCTATCGTGTCC-3IABkFQ	2655–2626
Sequencing	Forward	GCTATCACATAATCTGRAAGCGTA	2592–2615
	Reverse	GATATAGCACCACATGCACCA	2708–2687