Table 1.
Quantitative PCR reaction composition, thermal cycling, source of calibration standards and primer references used in this study.
| Target gene | Reaction conditions | Source of calibration (standard curve) | Primer name and reference | ||||||
|---|---|---|---|---|---|---|---|---|---|
| F- and R-primer (pmol μl−1) | BSA (μg μl−1) | DMSO (μg μl−1) | Denaturation time at 95°C (s) | Annealing time and temperature | Elongation time at 72°C (s) | qPCR efficiency (%) | |||
| nifH | 0.26 | 0.6 | 0 | 60 | 27 s at 55°C | 60 | 94 | Bradyrhizobium liaoningense | FPGH19/ PolR (Simonet et al., 1991; Poly et al., 2001) |
| amoA (AOA) | 0.7 | 0.6 | 0 | 40 | 30 s at 56°C | 60 | 98 | Soil clone | Arch amoA-1F/Arch amoA-2R (Francis et al., 2005) |
| amoA (AOB) | 0.2 | 0.6 | 0 | 45 | 45 s at 60°C | 45 | 100 | Soil clone | amoA 1F/amoA 2R (Rotthauwe et al., 1997) |
| nirK | 0.3 | 0.6 | 30 | 15 | 30 s at 58°C | 30 | 99 | Pseudomonas fluorescens | nirK876/nirK5R (Braker et al., 1998; Henry et al., 2004) |
| nirS | 0.3 | 0.6 | 30 | 30 | 60 s at 57°C | 45 | 98 | Pseudomonas aeruginosa | nirS cd3af/nirS R3cd (Michotey et al., 2000; Throback et al., 2004) |
| nosZ clade I | 1 | 0.6 | 0 | 15 | 30 s at 60°C | 30 | 102 | Pseudomonas aeruginosa | nosZ2F/nosZ2R (Henry et al., 2006) |
| nosZ clade II | 2 | 0.6 | 0 | 30 | 30 s at 54°C | 45 | 107 | Gemmatimonas aurantiaca | nosZ-II-F/nosZ-II-R (Jones et al., 2013) |
| Total bacteria | 0.8 | 0.4 | 0 | 27 | 60 s at 62°C | 30 | 106 | Serratia plymuthica | FP16S/RP16S (Bach et al., 2002) |
| Total archaea | 0.3 | 0.4 | 0 | 20 | 30 s at 60°C | 27 | 104 | Soil clone | Arch-967F/Arch-1060R (Cadillo-Quiroz et al., 2006) |
AOB, ammonia-oxidizing bacteria; AOA ammonia-oxidizing archaea.