TABLE 1.

Primers and PCR amplification conditions

Primer setTarget groupThermocycling programb,c
Initial denaturation period (min)Annealing temp (°C)Extension time (s)
27f-Nbac-1050rNitrobacter (genus)565.280
27f-Nspira-705rNitrospira (genus)56350
357f-GC-518raEubacteria25530
  • a A GC clamp was attached to the 5′ end of primer 357f-GC (45).

  • b The 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.

  • c Nitrobacter- 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.