TABLE 2.

Primers used for qPCR assays, annealing temperatures, target regions, and the specificity of the amplicons cloned from qPCR assays with soil DNA^a

Target group	Forward primer	Reverse primer	Approximate amplicon length (bp)	Annealing temp (°C)	% of soil clones belonging to the target groupc
All Bacteria	Eub338	Eub518	200	53	100
α-Proteobacteria	Eub338	Alf685	365	60	75
β-Proteobacteria	Eub338	Bet680	360	60	96
Actinobacteria	Actino235	Eub518	300	60	60
Firmicutes	Lgc353	Eub518	180	60	100
Bacteroidetes	Cfb319	Eub518	220	65	100
Acidobacteria	Acid31	Eub518	500	50	100
All Fungi	5.8s	ITS1f	300b	53	100
Basidiomycota	ITS4b	5.8sr	500b	55	100

^aThe bacterial qPCR assays target 16S rRNA genes, while the fungal assays target the internal transcribed spacer region found in rRNA genes.

^bThe length of the targeted ITS region can vary significantly between different fungal strains.

^cThe percentage includes only those clones that were nonchimeric and exceeded the 80% confidence threshold for taxonomic assignment using the RDP classifier program.