Table. PCR primers and conditions used to differentiate M1_global and M1_UK Streptococcus pyogenes lineages in study of emerging invasive group A Streptococcus M1_UK lineage detected by allele-specific PCR, England, 2020*.

Target gene	Primer type†	Sequences‡	PCR cycle conditions	Product, bp
rofA	WT sequence	TGTTAATTGCTTGGTTAAATCA	30 cycles of 95°C for 3 min, 45 s; 59.2°C for 30 s; 72°C for 1 min (final cycle: 5 min)	278
	Forward-SNP	5′-TGTTAATTGCTTGGTTAAAGtA-'3
	Forward-WT	5′-TGTTAATTGCTTGGTTAAAGCA-'3
	Reverse	5′-GCTCATCTCCTAACGGATTCTT-'3
gldA	WT sequence	AGATGGGTTAGCAACATGG	30 cycles of 95°C for 3 min, 45 s; 61.8°C for 30 s;72°C for 1 min (final cycle: 5 min)	292
	Forward-SNP	5′-AGATGGGTTAGCAACAAaG-'3
	Forward-WT	5′-AGATGGGTTAGCAACAAGG-'3
	Reverse	5′-GAATAGCACCTGTCAGCG-'3
pstB	WT sequence	GATAAATCAATCTTAGACCA	30 cycles of 95°C for 3 min, 45 s; 50°C for 30 s; 72°C for 1 min (final cycle: 5 min)	287
	Forward-SNP	5′-GATAAATCAATCTTAGATaA-'3
	Forward-WT	5′-GATAAATCAATCTTAGATCA-'3
	Reverse	5′-CGTGAGGCTTGCTGCATTGAG-'3

*SNP, single-nucleotide polymorphism; WT, wild-type. †Forward primers were designed to detect either the targeted SNP (M1_UK) or WT (M1_global) sequences. ‡Lowercase bold letters in primer sequences denote the base complementary to the targeted SNP in the M1_UK sequence. Underlined uppercase letters indicate an additional mismatched base introduced into primer sequences to increase primer specificity.