Design of PCR primers and gene probes for the general detection of bacterial populations capable of degrading aromatic compounds via catechol cleavage pathways

For the general detection of bacterial populations capable of degrading aro matic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting prime r set (C12O primers) was designed based on the homologous regions of II C12 O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions t o identify the amplified fragments, The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C 12O and/or C23O genes used for the primer and probe design. Various authent ic bacterial strains carrying neither C12O nor C23O genes were used as nega tive controls. PCR with the C12O primers amplified DNA fragments of the exp ected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, a nd positive signals were obtained from 4 of the 5 amplified fragments on So uthern hybridization with the C12Op, The C23O primers amplified DNA fragmen ts of the expected size from all the 11 tested C23O-carrying bacterial stra ins used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments we re amplified from the negative controls. To evaluate the applicability of t he designed primers and probes for the general detection of aromatic compou nd-degrading bacteria, they were applied to wild-type phenol- and/or benzoa te-degrading bacteria newly isolated from a variety of environments. The C1 2O and/or C23O primers amplified DNA fragments of the expected sizes from 6 9 of the 106 wild-type strains tested, white the C12Op and/or C23Op detecte d positive signals in the amplified fragments from 63 strains. These result s suggest that our primer and probe systems can detect a considerable propo rtion of bacteria which can degrade aromatic compounds via catechol cleavag e pathways.