Info

aPrimer sequences for MOIO1 and -2 and ER1 and -2 from refs. 6 and 8, respectively. ftGenBank/EMBL accession no. M23728. cGenBank/EMBL accession no. D64177.

5. 50X TAE buffer: add 121 g of Tris base, 23.6 mL glacial acetic acid, 50 mL 0.5 MEDTA, pH 8.0. Dissolve and make up to 500 mL with deionized water.

6. Electrophoresis buffer: add 40 mL 50X TAE buffer and 100 ^L ethidium bromide stock (10 mg/mL) to 1960 mL deionized water. Store at 2-8°C.

7. 6X Loading dye: add 1 g bromophenol blue and 160 g sucrose to 400 mL deionized water. Mix and store at 2-8°C.

8. TE buffer, pH 8.0: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA pH 8.0.

9. DNA Molecular Weight V III marker (Boehringer, Mannheim, Germany): For use, dilute 1:10 with TE buffer.

10. UV transilluminator.

11. Polaroid camera.

3. Methods (see Notes 1 and 2)

3.1. Preparation of PCR Master Mixes

Volumes given are based on 16 and 20 ^L per reaction mixture for the first- and second-round mix tubes, respectively. Thaw out the following reagents and keep them on ice at all times: 10X PCR buffer II, 25 mM MgCl2, 5 U/^L AmpliTaq Gold DNA polymerase, 100mM dNTP mix, and 10 ^M stocks of primers (MOIO1 and -2 and ER1 and -2).

3.2. Erysipelothrix spp. Master Mixes

1. Using aseptic techniques, add the following volumes of each reaction component to a sterile McCartney bottle to make 100 first-round mix tubes: 1134 ^L of DEPC water, 200 ^L of 10X PCR buffer II, 160 ^L of 25 mM MgCl2, 16 ^L of dNTP pool, 10 ^L of 5U/^L AmpliTaq Gold DNA polymerase, 40 ^L of 10 ^M MOIO1 and -2 primers.

2. To make 100 second-round mix tubes, add the following volumes: 1527 ^L of DEPC water, 204 ^L of 10X PCR buffer II, 163.2 ^L of 25 mM MgCl2, 16.3 ^L of dNTP pool, 10 ^L of 5 U/^L AmpliTaq Gold DNA polymerase, 40.8 ^L of 10 ^M MOIO1 and -2 primers.

3. Use the vortex mixer and gently but thoroughly mix the contents.

4. Place rows of sterile 0.2 mL microtubes into a microtube rack with the lids open.

5. Dispense 16 and 20 ^L of the reaction mixtures into the microtubes for the first- and second-round mix tubes, respectively.

6. Color code the tips of the microtubes for the first and second rounds, and make sure the lids are placed on firmly.

7. Place the rack with the microtubes in the -70°C freezer for 1 h.

8. Once the reaction mixtures are frozen, they can be removed from the rack and stored in boxes in the -70°C freezer until later required.

3.3. E. rhusiopathiae Master Mixes

1. Add the following volumes to a McCartney bottle for 100 first-round mix tubes: 1174 ^L of DEPC water, 200 ^L of 10X PCR buffer II, 120 ^L of 25 mM MgCl2, 16 ^L of dNTP pool, 10 ^L of 5U/^L AmpliTaq Gold DNA polymerase, 40 ^L of 10 ^M MOIO1 and -2 primers.

2. For 100 second-round mix tubes, add the following volumes to another McCartney bottle: 1565.7 ^L of DEPC water, 204 ^L of 10X PCR buffer II, 122.4 ^L of 25 mM MgCl2, 16.3 ^L of dNTP pool, 10 ^L of 5U/^L AmpliTaq Gold DNA polymerase, 40.8 ^L of 10 ^M MOIO1 and -2 primers.

3. Follow the same procedure as for Erysipelothrix master mixes from step 3, Subheading 3.2.

3.4. Preparation of Samples (see Note 4)

1. Dispense 10 mL sterile TSB/S into a McCartney bottle and inoculate the organism to be tested.

2. Use the vortex mixer to mix the contents thoroughly.

3. Incubate the broth for 48 h at 37°C.

4. Following the 48-h incubation, vortex the broth culture vigurously.

5. With a sterile plastic transfer pipet, remove 1.5 mL of the culture and transfer it to a sterile microcentrifuge tube.

6. Centrifuge the culture at 10,000g for 3 min to pellet the suspension.

7. Remove the supernatant and add 1.5 mL DEPC water to the pellet.

8. Wash the pellet by vortexing it into suspension.

9. Centrifuge the suspension once again at 10,000g for 3 min.

10. Remove the supernatant and resuspend the pellet in 100 ^L DEPC water.

3.5. Preparation of DNA Template

1. Boil the 100-^L suspensions at 100°C for 15 min.

2. Pellet the debris at 10,000g for 3 min.

3. Transfer the supernatant containing the DNA to a sterile microcentrifuge tube.

4. Store the microcentrifuge tube containing the DNA extract in the -70°C freezer until required.

3.6. PCR Amplification (see Note 5)

Inoculation of the mix tubes should be conducted in a laminar flow cabinet if possible, or if not, in a separate area or laboratory from where the mix tubes were prepared. Plastic sleeves should be worn while working in the cabinet. While inoculating the second-round mix tubes from the first, do not open them by hand; use the openers at all times.

3.6.1. First-Round Amplification

1. Thaw out the DNA extract from first-round Erysipelothrix and E. rhusiopathiae mix tubes, and then place them on ice.

2. Label mix tubes according to samples tested.

3. In a laminar flow cabinet, vortex the DNA extract and add 4 ^L to the corresponding Erysipelothrix and E. rhusiopathiae mix tubes.

4. Place the lid firmly on the microtubes and gently mix the contents (see Note 6).

5. Place the Erysipelothrix mix tubes in the thermal cycler and the E. rhusiopathiae mix tubes on ice.

6. For the Erysipelothrix mix tubes amplify the reaction by using the following cycle parameters: 94°C for 15 min and then 45 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 45 s, followed by an additional extension step at 72°C for 7 min and cooling to 4°C.

7. Once the amplification of the Erysipelothrix mix tubes is completed, place them in the fridge.

8. Gently mix the contents of the E. rhusiopathiae mix tubes and place them in the same thermal cycler.

9. For the E. rhusiopathiae mix tubes, amplify the reaction by using the following cycle parameters: heating at 94°C for 5 min and then 30 cycles of denaturation at 94°C for 1 min, annealing at 63°C for 30 s, and extension at 72°C for 1 min, followed by an additional extension step at 72°C for 7 min and cooling to 4°C.

10. Following amplification, also place the E. rhusiopathiae mix tubes in the fridge.

3.6.2. Double (Second-Round) Amplification

1. Place the second-round mix tubes on a rack in line with the first-round mix tubes.

2. Place all the equipment that is required in the cabinet and then put on plastic sleeves.

3. Use a microtube opener to open the first-round mix tube.

4. Remove 0.4 ^L from the first-round mix tube.

5. Use a clean opener to open the second-round mix tube.

6. Inoculate the 0.4 ^L from the first round into the second-round mix tube.

7. Remove the second-round mix tubes from the block, gently mix the contents, and place them in the thermal cycler.

8. Then amplify the second-round mix tubes using the same cycle parameters as for the first round for both Erysipelothrix and E. rhusiopathiae mix tubes.

9. Remove the samples from the thermal cycler and store them at 4°C.

Was this article helpful?

0 0

Post a comment