TABLE 1.
SYBR green real-time PCR assay characteristicsa
| Parameter | Assay value for indicated gene
|
|||
|---|---|---|---|---|
| ltrBE1 | ltrB | ltrA | oppD | |
| PCR efficiency determined by usingb: | ||||
| Recombinant plasmid DNA | 1.97 | 1.96 | 1.91 | 1.96 |
| cDNA templates | 1.92 | 1.95 | 1.98 | 1.94 |
| Melting temp (°C) | 78.5 | 79.0 | 77.0 | 78.0 |
| Variationc (%) | ||||
| Intra-assay | 1.7 | 1.3 | 1.6 | 3.9 |
| Interassay | 3.0 | 3.9 | 2.7 | 4.2 |
a
PCR efficiency (E) is calculated according to the following equation: E = 10(−1/slope). The maximal efficiency of PCR is 2 where every PCR product is replicated every cycle.
b
PCR efficiency was determined by using recombinant plasmid DNA containing target genes as templates and by using cDNA templates generated from in vitro-transcribed total L. lactis ML3 RNA.
c
Intra-assay variations were determined in three repeats within one real-time PCR run. Interassay variations were determined in four different experiment runs in 4 days. Intra-assay and interassay variations were determined by using recombinant plasmid DNA containing target genes as templates.