TABLE 3.
Real-time PCR limits of detection for three recovery methods using DNA isolated from Bacillus anthracis-spiked swabs
| Recovery methoda | LOD (CFU/swab [genome copy numbers])b |
|||||
|---|---|---|---|---|---|---|
| QIAamp |
MagNA Pure Compact |
MagNA Pure LC |
||||
| PCR 1 | PCR 2 | PCR 1 | PCR 2 | PCR 1 | PCR 2 | |
| SETS | 500 (8.2 × 102) | 500 (8.2 × 102) | 500 (9.0 × 102) | 500 (9.0 × 102) | 500 (6.2 × 102) | 500 (6.2 × 102) |
| Sonication | 500 (5.1 × 102) | 500 (5.1 × 102) | 500 (7.0 × 102) | 500 (7.0 × 102) | 500 (4.9 × 102) | 5,000 (4.9 × 103) |
| Vortex | 5,000 (4.6 × 103) | 500 (4.6 × 102) | 500 (6.5 × 102) | 500 (6.5 × 102) | 5,000 (5.0 × 103) | 5,000 (5.0 × 103) |
Nylon-flocked swabs were spiked in triplicate with 10 μl of 10-fold dilutions of virulent B. anthracis Ames strain cells at a starting concentration of 107 CFU/ml and then underwent the three swab recovery methods. DNA was isolated by the use of three different DNA purification methods, the QIAamp manual method and the MagNA Pure Compact and MagNA Pure LC automated methods.
Real-time PCR was performed in triplicate using two different assays, the TaqMan assay described by Hoffmaster et al. (PCR 1) (16) and the molecular beacon assay described by Hadjinicolaou et al. (PCR 2) (12). The limit of detection (LOD) (in numbers of CFU/swab) was calculated based on numbers of CFU/ml of the spiking concentration, and genome equivalents (genome copy numbers) were calculated for the lowest concentration that resulted in three out of three replicates with positive results for all targets in each real-time PCR assay.