B. Joshi et Sk. Walia, DETECTION OF METAPYROCATECHASE HOMOLOGOUS GENES IN PETROLEUM HYDROCARBON CONTAMINATED GROUNDWATER BY POLYMERASE CHAIN-REACTION, Journal of microbiological methods, 27(2-3), 1996, pp. 121-128
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.