DETECTION OF METAPYROCATECHASE HOMOLOGOUS GENES IN PETROLEUM HYDROCARBON CONTAMINATED GROUNDWATER BY POLYMERASE CHAIN-REACTION

PCR assay was developed and used for rapid detection of metapyrocatech ase (MPC) homologous gene sequences among hydrocarbon-degrading bacter ial populations present in contaminated environments. The primers for the PCR assay were selected from the DNA sequence of MPC gene that has been cloned in Escherichia coli pAW313 after aligning with other publ ished sequences. The primary primers, PF313 and PR313 were located 882 bp apart and were able to amplify the conserved region of the MPC hom ologous gene sequences under standardized PCR conditions. The nested p rimers, NF313 and NR313 amplified a confirmatory internal fragment of 506 bp from within the 882 bp region of MPC gene. Specific amplificati on of the unique 506 bp nested fragment was also obtained using DNA ex tracted from nine naturally occurring hydrocarbon degrading bacteria w hile, no amplification was observed when the DNAs extracted from Is un related bacterial strains were used as template. The specificity and i dentity of the amplified 506 bp nested DNA fragment from hydrocarbon-d egrading bacterial strains was confirmed by restriction digestion with EcoRI and by southern hybridization using digoxigenin-labeled interna l probe. The MPC homologous gene sequences were also amplified when DN A directly extracted from petroleum hydrocarbon contaminated groundwat er samples was used as template. Humic substances present in the groun dwater samples did not inhibit the amplification reaction when the DNA extracted directly from groundwater was used for the PCR assay withou t further purification.