Division I.3.2 Polymerase chain reaction (PCR)

Several PCR protocols have been developed and published.

PCR primers that target the internal transcribed spacer (ITS1) region shall be used as they are able to amplify only M. refringens.

PCR shall be carried out in a 50 μl volume. PCR mixtures shall contain buffer (500 mM KCl, 100 mM Tris/HCl [pH 9,0 at 25 °C] and 1 % Triton® X-100), 2,5 mM MgCl2, 0,2 mM dNTP mix, 1 μM forward and reverse primers, 0,02 units μl^{– 1} Taq DNA polymerase, and 10 to100 ng of extracted DNA. After denaturation of DNA at 94 °C for five minutes, 30 cycles shall be run as follows: denaturation at 94 °C for one minute, annealing at 55 °C for one minute, and elongation at 72 °C for one minute per kilo-base pair. A final elongation step of 10 minutes at 72 °C shall be performed. For the detection of M. refringens, PCR shall be performed with primers that target the ITS1 region (5′-CCG-CAC-ACG-TTC-TTC-ACT-CC-3′ and 5′-CTC-GCG-AGT-TTC-GAC-AGA-CG-3′).

Positive controls shall consist of genomic DNA from a highly infected host or plasmidic DNA including the target region.

Negative controls shall consist of genomic DNA from non-infected hosts and PCR reactives without target DNA.

A positive result shall be positive PCR amplification at the expected size (412 bp), with all negative controls being negative and all positive controls being positive.