TABLE 2.

PCR conditions for specific amplification of 16S rRNA gene fragments of six phylogenetic groups of bacteria

Conditionα-Proteobacteriaβ-ProteobacteriaActinobacteriaPlanctomycesFirmicutesFlavobacteria-Cytophaga
Fragment length (bp)a507430704896612588
Forward primer (μM)bGC-517f (0.2)Beta680f (1.0)GC-517f (1.0)8f (1.0)GC-LGC354f (1.0)GC-CFB319f (1.0)
Reverse primer (μM)Alf968r (0.2)GC-1055r (1.0)AB1165r (1.0)PLA886r (0.2)907r (1.0)907r (1.0)
Annealing10 cycles at 68°C for 20 s; 30 cycles at 62°C for 20 s10 cycles at 56°C for 1 min; 20 cycles at 51°C for 1 min10 cycles at 62°C for 45 s; 20 cycles at 57°C for 45 s10 cycles at 68°C for 30 s; 20 cycles at 63°C for 30 s10 cycles at 58°C for 45 s; 20 cycles at 53°C for 45 s10 cycles at 65°C for 45 s; 25 cycles at 60°C for 45 s
Total no. of cycles403030303025
SpecificitybFew Actinobacteria, γ- or δ-ProteobacteriaExclusively β-ProteobacteriaExclusively ActinobacteriaExclusively PlanctomycesAt decreased stringency: one subgroup of PlanctomycesFew β-Proteobacteria
Coveragec (%)84.756.49695.152.190
  • a Based on E. coli numbering.

  • b Determined by a combination of (i) database analysis using the RDP Probe_Match routine (http://rdp.cme.msu.edu/probematch/search.jsp) and (ii) the empirical detection of 16S rRNA partial gene sequences of nontarget phylogenetic groups as described in Results.

  • c As reported for target group using the specific primer as a FISH probe in the original publications (cited in Table 1) or in reference (32), or determined by the RDP Probe_Match routine.