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

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as template for PCR. In addition, a nested PCR based on the apxIVA gene is
required in order to attain an appropriate sensitivity of the test. The method
allows the detection of A. pleuropneumoniae bacteria in lung tissue and nasal
secretory fluid. For subtyping, however, it is necessary to isolate A.
pleuropneumoniae by culture (9).
3.2.1. Preparation of Template from Lung Tissue (see Note 4)
A. pleuropneumoniae can be detected directly from pathological lung lesions
by PCR. The preparation of the template DNA is done using the following method:
1. Excise 0.25 g lung tissue from the edge of a lesion.
2. Place in a sterile tube and homogenize by hand using a sterile pestle.
3. Add 0.5 mL PBS to facilitate homogenization.
4. Centrifuge 5 s at 10,000g to remove large debris.
5. Retain 200 µL of the supernatant, discard the rest.
6. Centrifuge the supernatant 5 min at 13,000g.
7. Keep the pellet.
8. Add 200 µL Instagene matrix and resuspend pellet.
9. Heat to 56°C for 30 min.
10. Vortex mix briefly.
11. Heat to 95°–97°C (or boil) for 8 min.
12. Vortex mix briefly.
13. Centrifuge 5 min at 13,000 g.
14. Keep the supernatant as template for PCR.
3.2.2. Preparation of Template from Nasal Secretory Fluid (see Note 4)
A. pleuropneumoniae can be detected by PCR directly from nasal secretory
fluid taken by simple cotton-tipped swabs:
1. Insert a cotton-tipped swab 2 cm into the nasal cavity of the pig.
2. Return swab immediately to its sterile plastic housing.
3. Keep on ice until further processing.
4. Immerse swab in 1 mL PBS for 30 min on ice.
5. Twirl for 1 min, then remove swab, and discard.
6. Vortex mix suspension.
7. Centrifuge 5 min at 13,000g.
8. Discard supernatant.
9. Add 200 µL Instagene matrix and resuspend pellet.
10. Heat to 56°C for 30 min.
11. Vortex mix briefly.
12. Heat to 95–97°C (or boil) for 8 min.
13. Vortex mix briefly.
14. Centrifuge 5 min at 13,000g.
15. Keep the supernatant as template .