Division 9.3 PCR test

Prepare a suspension of approximately 10⁶ cells per ml in ultra pure water (UPW).

Heat 100 µl of the cell suspension in closed tubes in a heating block or boiling waterbath at 100 °C for four minutes. If required, addition of freshly-prepared NaOH to a final concentration of 0,05M may assist cell lysis. The samples may then be stored at -16 to -24 °C until required.

Apply appropriate PCR procedures to amplify C. m. subsp. sepedonicus specific amplicons (e. g. Pastrik, 2000; see Appendix 4; Li and de Boer, 1995; Mills et al., 1997; Pastrik and Rainey, 1999; Schaad et al., 1999.

A positive identification of C. m. subsp.sepedonicus is achieved if the PCR amplicons are the same size and have the same restriction fragment length polymorphisms as for the positive control strain.