TABLE 1.
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 |
A GC clamp was attached to the 5′ end of primer 357f-GC (45).
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.
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.