147.30—Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae.
(a) DNA isolation.
Isolate DNA from 1 mL of eluate from tracheal swabs in PBS or 1 mL of broth culture by a non-phenolic procedure. Centrifuge samples at 14,000 x g for 5 to 10 minutes. Decant supernatant and wash the pellet with 1 mL of PBS. Centrifuge as above and re-suspend the pellet in 25-50 μl of 0.1 percent DEP (Diethyl Pyrocarbonate; Sigma) water. Boil at 120 °C for 10 minutes followed by 10 minutes incubation at 4 °C. Centrifuge as above and transfer the supernatant DNA to a nuclease-free tube. Estimate the DNA concentration and purity by spectrophotometric reading at 260 nm and 280 nm.
(b) Primer selection.
(1) M. gallisepticum.
The primer for M. gallisepticum should consist of the following sequences:
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(c) Polymerase chain reaction.
(1)
Treat each sample (100 to 2000 ng/5 μl) with one of the following 45 μl PCR cocktails:
(i)
5 μl 10x PCR buffer, 1 μl dNTP (10 mM), 1 μl of Reverse primer (50 μM), 1 μl of Forward primer (50 μM), 4 μl MgCl2 (25 mM), 1 μl taq-polymerase (5 U), 32 μl DEP water.
(ii)
18 μl water, 25 μl PCR mix (Promega), 1 μl Reverse primer (50 μM), 1 μl Forward primer (50 μM).
(2)
Perform DNA amplification in a Perkin-Elmer 9600 thermocycler or in a Hybaid PCR Express thermocycler. 20 The optimized PCR program is as follows:
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Footnote(s): 20 Trade names are used in these procedures solely for the purpose of providing specific information. Mention of a trade name does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement over other products not mentioned.
| Temperature ( °C) | Duration | Cycles |
|---|---|---|
| 94 | 30 seconds | 30-40. |
| 55 | 30 seconds | 30-40. |
| 72 | 1 minute | 30-40. |
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| 72 | 5 minutes | 1 (final extension). |
(d) Electrophoresis.
Mix PCR products (5 to 10 μl) with 2 μl loading buffer (Sigma) and electrophorese on a 2 percent agarose gel containing 0.5 μg/mL ethidium bromide in TAE buffer (40 mM tris; 2 mM EDTA; pH 8.0 with glacial acetic acid) for 30 minutes at 80 V. M. gallisepticum (185 bp) and M. synoviae (214 bp) amplicons can be visualized under an ultraviolet transilluminator along with the PCR marker (50 to 2000 bp; Sigma).