TABLE 2.
Effects of 16 PCR facilitators on amplification capacity of rTth DNA polymerase in the presence of feces and meat
| Facilitator and facilitator concnc | PCR resultsa with the following at the indicated concn (% [vol/vol])b:
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Feces
|
Meat
|
|||||||||
| 20 | 4 | 2 | 0.4 | 0.2 | 20 | 4 | 2 | 0.4 | 0.2 | |
| Acetamide | ||||||||||
| 0 | −, − | −, − | −, − | +, ± | +, + | −, − | −, − | ±, − | +, ± | +, ± |
| 1 | −, − | −, − | −, − | +, − | +, ± | −, − | −, − | −, − | +, ± | +, + |
| 2.5 | −, − | −, − | −, − | +, − | +, ± | −, − | −, − | −, − | +, − | +, ± |
| 4 | −, − | −, − | −, − | ±, − | +, − | −, − | −, − | −, − | ±, − | ±, − |
| Betaine | ||||||||||
| 0 | −, − | −, − | −, − | +, − | +, + | −, − | −, − | −, − | ±, + | ±, + |
| 5.9 | −, − | −, − | −, − | +, − | +, + | −, − | ±, − | +, − | ±, + | +, + |
| 8.8 | −, − | −, − | −, − | +, ± | +, + | −, − | −, ± | ±, ± | +, + | +, + |
| 11.7 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | +, − | +, + | +, + |
| BSA | ||||||||||
| 0 | −, − | −, − | −, − | −, + | +, + | −, − | −, − | ±, − | +, + | +, + |
| 0.2 | −, − | +, − | ±, ± | −, + | +, + | +, + | +, + | +, + | +, + | +, + |
| 0.4 | −, − | +, ± | +, + | +, + | +, + | +, + | +, + | +, + | +, + | +, + |
| 0.6 | −, − | −, ± | −, + | +, + | +, + | +, + | +, + | +, + | +, + | +, + |
| Dextran 40 | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | ±, − | +, + | +, + |
| 1 | −, − | −, − | −, − | +, + | +, + | −, − | +, − | ±, + | +, + | +, + |
| 2.5 | −, − | −, − | +, − | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 4 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | +, + | +, + | +, + |
| Dextran 500 | ||||||||||
| 0 | −, − | −, − | −, + | +, + | +, + | −, − | −, − | +, ± | +, + | +, + |
| 1 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | ±, − | +, + | +, + |
| 2.5 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | ±, − | +, + | +, + |
| 4 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | ±, + | +, + |
| Dimethyl sulfoxide | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | ±, − | ±, + | +, + |
| 2.5 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | ±, + | +, + |
| 5 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | −, ± | +, + |
| 7.5 | −, − | −, − | −, − | +, − | +, − | −, − | −, − | −, − | −, − | ±, − |
| Formamide | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | ±, ± | ±, + | +, + |
| 0.25 | −, − | −, − | −, − | +, − | +, + | −, − | ±, − | ±, − | ±, + | +, + |
| 0.5 | −, − | −, − | −, − | +, − | +, + | −, − | −, − | ±, − | +, − | +, + |
| 1 | −, − | −, − | −, − | +, − | +, − | −, − | −, − | ±, − | ±, − | +, − |
| Glycerol | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 5 | −, − | −, − | −, − | +, + | +, + | −, − | −, ± | −, ± | +, + | +, + |
| 7.5 | −, − | −, − | −, − | +, + | +, + | −, − | −, ± | +, ± | +, + | +, + |
| 10 | −, − | −, − | −, − | +, + | +, + | −, − | −, ± | −, + | +, + | +, + |
| gp32 | ||||||||||
| 0 | −, − | −, − | +, + | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 0.01 | −, − | +, + | +, + | +, + | +, + | −, − | +, + | +, + | +, + | +, + |
| Nonidet P-40 | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, ± | +, + | +, + |
| 0.1 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 0.25 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 0.5 | −, − | −, − | −, ± | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| Polyethylene glycol 400 | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 1 | −, − | −, − | −, ± | +, + | +, + | −, − | −, − | −, − | −, + | ±, + |
| 2 | −, − | −, − | −, ± | +, + | +, + | −, − | −, − | −, − | −, + | ±, + |
| 4 | −, − | −, − | −, ± | +, + | +, + | −, − | −, − | −, − | −, ± | −, + |
| Polyethylene glycol 4000 | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 1 | −, − | −, − | −, ± | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 2.5 | −, − | −, − | −, + | +, + | +, + | −, − | −, − | −, − | −, + | +, + |
| 4 | −, − | −, − | −, ± | +, + | +, + | −, − | −, − | −, − | ±, ± | ±, + |
| Proteinase inhibitord | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | ±, − | +, + | +, + |
| 0.25× | −, − | −, − | +, ± | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 0.5× | −, − | −, − | +, ± | +, + | +, + | −, − | −, − | ±, − | +, − | +, + |
| 1× | −, − | +, ± | +, ± | +, + | +, + | −, − | −, − | −, − | ±, − | +, + |
| TMAC | ||||||||||
| 0 | −, − | −, − | −, − | +, ± | +, + | −, − | −, − | −, − | ±, + | +, + |
| 2.8 × 10−4 | −, − | −, − | −, − | +, ± | +, ± | −, − | −, − | −, − | +, + | +, + |
| 5.5 × 10−4 | −, − | −, − | −, − | +, ± | +, + | −, − | −, − | −, − | ±, + | +, + |
| 1.1 × 10−3 | −, − | −, − | −, − | +, ± | +, + | −, − | −, − | −, − | ±, + | +, + |
| Tween 20 | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | ±, ± | +, + |
| 0.1 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | ±, − | ±, ± | +, + |
| 0.25 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | +, ± | +, + |
| 0.5 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | ±, + | +, + |
| Tween 80 | ||||||||||
| 0 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | −, − | +, + | +, + |
| 0.1 | −, − | −, − | −, − | +, + | +, + | −, − | −, − | ±, − | +, ± | +, + |
| 0.25 | −, − | −, − | −, ± | +, + | +, + | −, − | −, − | ±, − | +, + | +, + |
| 0.5 | −, − | −, − | −, + | +, + | +, + | −, − | −, − | ±, − | +, + | +, + |
a
Results are of two independent PCR results; +, PCR product of high yield; ±, PCR product of low yield; −, no PCR product. The results were recorded in the order in which the PCRs were done.
b
Percentage (vol/vol) of homogenates of feces and meat in PCR mixture for rTth containing 1 ng of L. monocytogenes DNA.
c
Percentage (wt/vol) of 16 amplification facilitators in the reaction mixtures. The PCR sensitivity in the presence of the concentrations of these facilitators was the same as that with a solution of pure water with DNA.
d
One tablet of Proteinase Inhibitor Complete Mini cocktail (EDTA-free; Roche Molecular Biochemicals) was dissolved in 2 ml of deionized water. This concentration was five times greater than that recommended by the supplier.