TABLE 1.

Primers and conditions of nested PCR used for limiting-dilution assays by the three laboratories participating in phase 2 of the study

PCR and laboratory	Gene region	Position		Annealing temp (°C)	No. of cycles	Sequence reference no.
		Forward primera	Reverse primer
PCR 1
1a	S	392–416	750–726	65–57.4b	30	14
1b	X	1275–1302	1577–1551	67.9–60.4b	30	14
2	pre-S and S	2718–2738	1305–1285	55	35	8
8	S	194–213	706–687	55	40	27
PCR 2
1a		427–449	694–672	60.1/58.1b	20/20
1b		1305–1328	1542–1522	63.5/61.5b	20/20
2		2821–2843	191–168	60	35
8		305–324	433–414	55	40

^aNumbering starts at the unique EcoRI site at position 3221/0. 

^bLaboratory 1 used a touchdown program for annealing temperatures from 5°C above the melting temperature of the primer down to −2.5°C for 15 cycles and then 15 further cycles at the lower temperature.