Table 1.

Methods and reagents used for emm typing of Streptococcus pyogenes.

Anchorage		Winnipeg		Edmonton
Reagents	per Reaction	Reagentsa	per Reaction	Reagents	per Reaction
AccuPrime™ Super MixIIb	10.0 µL	5× buffer	7.0 µL	10× buffer + MgCl2	4.0 µL
Primer 1 (70 pM/µL)c	1.0 µL	5 mM MgCl2	3.2 µL	dNTPs (2.5 mM)	4.0 µL
Primer 2 (70 pM/µL)c	1.0 µL	dNTPs (5 mM)	1.4 µL	Primer 1 (10 µM)	5.6 µL
dH2O	6.0 µL	Primer 1 (100 µM)	0.5 µL	Primer 2 (10 µM)	5.6 µL
Template	2.0 µL	Primer 2 (100 µM)	0.5 µL	HotStar Taqd (5 U/µL)	0.6 µL
Total	20 µL	GoTaq® (5 U/µL)	0.2 µL	dH2O	18.2 µL
		dH2O	20.2 µL	Template	2.0 µL
		Template	2.0 µL	Total	40 µL
		Total	35 µL
Cycling conditions		Cycling conditions		Cycling conditions
94 °C 1 min 1 cycle		94 °C 5 min 1 cycle		94 °C 15 min 1 cycle
94 °C 15 sec 10 cycles		94 °C 1 min 40 cycles		94 °C 15 sec 10 cycles
46.5 °C 30 sec 10 cycles		47.5 °C 1 min 40 cycles		46.5 °C 30 sec 10 cycles
72 °C 1 min 10 cycles		72 °C 2 min 40 cycles		72 °C 1 min 10 cycles
94 °C 15 sec 20 cycles		72 °C 7 min 1 cycle		94 °C 15 sec 20 cycles
46.5 °C 30 sec 20 cycles		4 °C Hold		46.5 °C 30 sec 20 cycles
72 °C 1 min 15 sec with a 10 sec increment for each subsequent 19 cycles 10 cycles				72 °C 1 min 15 sec with a 10 sec increment for each subsequent 19 cycles
				10 cycles
72 °C 10 min 1 cycle				72 °C 10 min 1 cycle
4 °C Hold				4 °C Hold

^aPCR Master Mix (Go Taq® Flexi PCR Kit, Promega, Madison, WI).

^bLife Technologies, Grand Island, NY.

^cPCR primers from http://www.cdc.gov/streplab/protocol-emm-type.html.

^dHotStar Taq DNA Polymerase (Qiagen, Redwood City, CA).