Division 9.3 PCR testF1

Prepare a suspension of approximately 10 ⁶ 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.