TABLE 1.

Primers and PCR amplification conditions

Primer set	Target group	Thermocycling programb,c
		Initial denaturation period (min)	Annealing temp (°C)	Extension time (s)
27f-Nbac-1050r	Nitrobacter (genus)	5	65.2	80
27f-Nspira-705r	Nitrospira (genus)	5	63	50
357f-GC-518ra	Eubacteria	2	55	30

^aA GC clamp was attached to the 5′ end of primer 357f-GC (45).

^bThe general thermocycling program used was as follows: 5 min at 94°C; 10 cycles of 30 s at 94°C, 30 s at the specified annealing temperature, and the specified extension time at 72°C; 25 cycles of 30 s at 92°C, 30 s at the specified annealing temperature, and the specified extension time at 72°C (increasing by 1 s per cycle); and a 10-min final extension at 72°C.

^cNitrobacter- and Nitrospira-specific amplifications were performed with a “hot start” by adding the Taq polymerase after the initial denaturation step. All nested second-stage nonspecific amplifications were started by placing cooled tubes containing the PCR mix into a preheated (94°C) PCR block.