Design of primers and probes for extensive detection of aromatic compound-degrading bacteria

For the extensive detection of the aromatic compound-degrading bacteria, two PCR primer sets to amplify specific fragments from a variety of catechol 1,2-dioxygenase (C12O) genes (C12O primers) and catechol 2,3-dioxygenase (C23O) genes (C23O primers) were designed from the homologous regions of 11 C12O and 17 C23O genes, respectively. Oligonucleotide probes (C12Op and C23Op) were also designed on the internal homologous regions. The C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 tested strains used for their design, and C12Op detected positive signals from 4 of the 5 amplified fragments by hybridization The C23O primers amplified those from all the 11 tested bacterial strains, while the C23Op detected 9 strains. The applicability of them was evaluated using phenol- and/or benzoate-degrading bacteria newly isolated from various environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 isolates tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 isolates. These results suggest that our primer and probe systems can detect a considerable portion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.