Detection of metapyrocatechase homologous genes in petroleum hydrocarbon contaminated groundwater by polymerase chain reaction

Abstract

PCR assay was developed and used for rapid detection of metapyrocatechase (MPC) homologous gene sequences among hydrocarbon-degrading bacterial 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 published sequences. The primary primers, PF313 and PR313 were located 882 bp apart and were able to amplify the conserved region of the MPC homologous gene sequences under standardized PCR conditions. The nested primers, NF313 and NR313 amplified a confirmatory internal fragment of 506 bp from within the 882 bp region of MPC gene. Specific amplification of the unique 506 bp nested fragment was also obtained using DNA extracted from nine naturally occurring hydrocarbon degrading bacteria while, no amplification was observed when the DNAs extracted from 18 unrelated bacterial strains were used as template. The specificity and identity of the amplified 506 bp nested DNA fragment from hydrocarbon-degrading bacterial strains was confirmed by restriction digestion with EcoRI and by southern hybridization using digoxigenin-labeled internal probe. The MPC homologous gene sequences were also amplified when DNA directly extracted from petroleum hydrocarbon contaminated groundwater samples was used as template. Humic substances present in the groundwater samples did not inhibit the amplification reaction when the DNA extracted directly from groundwater was used for the PCR assay without further purification.