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

168 Gallien
2. Materials
2.1. Bacterial Enrichment
1. Food samples: 25 g of food, e.g., raw minced beef, raw sausage containing beef,
soft cheese produced from unpasteurized milk or 25 mL of raw milk.
2. Sewage samples: 50 mL are used.
3. Stool samples and feces: use swab samples.
4. Nutrient medium for enrichment (for preparation procedure, see Subheading 3.1.4.):
a. Modified tryptose soy broth (mTSB) containing novobiocin pH 7.3: 33.0 g
mTSB containing Bile salts No. 3 and dipotassium hydrogen phosphate
(K2HPO4) (Mast Diagnostics, Merseyside, UK), 20 mg Novobiocin (Sigma,
Deisenhofen, Germany), make up to 1 L with distilled water.
b. MacConkey agar (Merck, Darmstadt, Germany): 17.0 g tryptone / peptone from
casein, 3.0 g Bacto-Peptone from meat, 10.0 g lactose · H2O, 1.5 g bile salts
No. 3, 5.0 g sodium chloride, 0.03 g neutral red, 0.001 g crystal violet, 10 to 15 g of
agar (depending on gelling properties), make up to 1000 mL with distilled water.
2.2. PCR, Electrophoresis, and Visualization
1. Water: autoclaved, free of DNase.
2. Primers: MK 1/MK 2, KS 7/KS 8, LP 43/LP 44, working solution 50 pmol/µL
(see Table 1).
3. Primers HB10/HB11 (see Note 1):
HB10 : 5' - att cca cac aac ata cga gcc g - 3', 50 pmol/µL,
HB 11: 5' - gtt tcg cca cct ctg act tga g - 3', 50 pmol/µL.
4. Deoxynucleoside triphosphate mixture (dNTP Mixture) stock solutions consisting
of: 10 mM 2'-Deoxy-adenosine-5'-triphosphate (dATP), 10 mM 2'-Deoxycytidine-5'-triphosphate
(dCTP), 10 mM 2'-Deoxy-guanosine-5'-triphosphate
(dGTP), 10 mM 2'-Deoxy-thymidine-5'-triphosphate (dTTP).
Dilute 1:10 in water before use. The working solution contains 10 µL of each
dNTP stock solution plus 360 µL of autoclaved distilled water.
5. Heat-stable DNA Polymerase: e.g., Gold Star, 5 U/µL (Eurogentech, Seraing,
Belgium), AmpliTaq ® LD, 5 U/µL (Perkin Elmer, Weiterstadt, Germany), or
Dynazyme, 5 U/µL (Biometra, Göttingen, Germany).
6. PCR buffer: supplied 10-fold concentrated with or without MgCl 2. Those buffers
containing MgCl 2 were optimized by the supplier for their particular polymerase.
7. MgCl 2: 25 mM.
8. TAE buffer: stock solution (50X) containing 242.0 g of Tris-[hydroxymethyl]aminomethane
(TRIS), 57.1 mL of glacial acetic acid, and 100.0 mL of 0.5 M
ethylene diamine tetraacetic acid, disodium salt (EDTA-Na 2). Make up to
1000 mL with distilled water.
9. Agarose: type A 6013 (Sigma). For analytical separation of polymerase chain
reaction (PCR) products prepare a 2% (w/v) suspension in TAE buffer and boil
for a few min. The solution must become clear and transparent. Cool down to
50°C and pour the solution on a gel frame.