PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

Identification of B. abortus by PCR 99
2.2. Sample Preparation
1. Nutrient agar plates such as tryptose agar (Difco no. 264300, Becton Dickinson,
Sparks, MD, USA), trypticase soy agar (BBL no. 211043; Becton Dickinson), or
Brucella agar (BBL no. 211086), containing 5% serum (bovine, calf, or fetal calf).
2. Saline: 0.85% (w/v) NaCl in sterile water.
3. CO2 (10%) incubator or jar.
4. Inoculating loops.
5. Spectrophotometer (e.g., DU 650; Beckman Instruments, Fullerton, CA, USA).
6. Disinfectant, e.g., 1% Lysol IC , (Reckitt & Colman, Wayne, NJ, USA).
The following items are needed for the methanol preservation of cells (optional):
7. Methanol.
8. Screw cap tubes, 20 × 125 mm.
9. Trypticase soy broth with 5% bovine serum (30 mL per 250-mL screw cap flask).
10. Shaking (rotating) water bath set at 37°C.
11. Screw cap centrifuge tubes (50 mL).
2.3. PCR Amplification
1. Thermal cycler (see Note 2).
2. Disposable 200-µL thin-walled tubes.
3. (Optional) A repeating pipet such as the Eppendorf ® Repeater Model 4780 (Cat.
no. 22-26-000-6; Eppendorf AG, Westbury, NY, USA) fitted with a 1.25-mL
combitip set to dispense 25 µL (see Note 3).
4. PCR-grade water (see Note 4).
5. FastStart Taq DNA Polymerase (Cat. no. 2-032-902; Roche Molecular
Biochemicals, Indianapolis, IN, USA) (see Note 5).
6. 10X Reaction buffer without MgCl 2 (included with FastStart Taq DNA Polymerase)
(see Note 6).
7. 25 mM MgCl 2 (see Note 6).
8. 10 mM dNTP mixture: 2.5 mM each of dATP, dCTP, dGTP, and dTTP (e.g., Cat.
no. R725-01; Invitrogen, Carlsbad, CA, USA).
9. Oligonucleotide primers (see Table 1 and Subheading 3.1.1.).
10. (Optional) GC Rich Enhancer (included with FastStart Taq DNA Polymerase),
(see Note 7).
11. TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA; can be stored for at least 2 yr at room
temperature.
2.4. Detection of Amplification
1. Electrophoresis equipment with power supply (e.g., Horizon 11-14 Gel Electrophoresis
Apparatus, cat. no. 11068-012; Life Technologies/Gibco-BRL,
Rockville, MD, USA).
2. Electrophoresis grade agarose (e.g., SeaKem ® GTG agarose, cat. no. 50071;
FMC/BioWhittaker, Rockland, ME, USA).
3. 6X Loading dye (e.g., cat. no. E190; AMRESCO, Solon, OH, USA).