Development of nucleic acid methods for the identification of sulphate-reducing bacteria
This study aimed to investigate the feasibility of using several genetic techniques for identification of sulphate-reducing bacteria. A group of eight type strains (seven of which were Desolfovibrio) were used as test group for every method except RSGP, for which an established set of oil field isolates was used. Ribotyping involves restriction fragment length polymorphism (RFLP) analysis of ribosomal RNA (rRNA) operons. Hybridisation of Eco RI-digested genomic DNA to a PCR-amplified (500 bp) rRNA gene product resulted in generation of discriminatory RFLP patterns for all but two of the eight test strains. Restriction digestion of PCR-amplified (1400 bp) rRNA gene products (PCR-robotyping) allowed differentiation between all eight strains for two of the seven enzymes tested. Random amplified polymorphic DNA (RAPD) analysis allows PCR-generation of distinct sets ("fingerprints") of amplification products from genomic DNA templates. Nine oligonucleotide decamers were tested as primers, two of which were found to generate discriminatory profiles for each of the eight type strains. Hybridisation of 32P-labelled RAPD products against genomic DNA from eleven sulphate-reducing strains was found to result in specific hybridisation of probes to their complementary genomic DNA. Reversal of the hybridisation procedure, using 32P-labelled genomic DNA against immobilised RAPD probes, was found to allow analysis of mixed genomic DNA samples in a single step. This reverse method is very similar in principle to RSGP, which allows analysis of mixed genomic DNA preparations by hybridisation against a master filter of genomic DNAs from a series of environmental bacterial standards. This study developed a method to allow quantitation of RSGP hybridisation signals, and went on to analyse biofilm samples from various locations in Western Canadian oil fields, resulting in identification of 21% (average) of each mixed DNA sample.