DGGE analysis was carried out on each PCR amplicon using a DCode™ Universal Mutation Detection System , following the procedure described previously Electrophoresis was performed in 1-mm polyacrylamide gel containing a 30–60% urea–formamide denaturing gradient , increasing in the electrophoretic run direction. The gel was subjected to a constant voltage of 150 V for 4 h at 60 °C in TAE Buffer 1X (50X TAE stock solution consisting in 2 M Tris base, 1 M glacial acetic acid, 50 mM EDTA). After electrophoresis, the DGGE gels were stained in 1X TAE solution containing ethidium bromide for 15 min and photographed under a UV illumination using a BioDoc Analyze system