Genomic DNA from each colony was prepared as follows. One ml of
stationary-phase broth cultures was centrifuged, the pellet was
washed twice with Tris–Acetate EDTA buffer 1× and re-suspended
in 1 ml of distilled sterile water. The cell suspension was sonicated
(500kHz) and heat treated at 99 ° C for 10 min in order to facilitate
the DNA release from the cells. One or 5 μl of the suspensions was
used as DNA template in the PCR reactions. PCR primers and cycling
conditions and the positive and negative controls were as those
described in Tables 3 and 4.