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Development of oligonucleotide probes and PCR primers for detecting phylogenetic subgroups of sulfate-reducing
Kristian Daly1, Richard J Sharp2, Alan J McCarthy1
1School of Biological Sciences, Life Sciences Building, University of Liverpool, Liverpool L69 7ZB, UK1.
Abstract:
PCR primer sets for the 16S rRNA gene of six phylogenetic groups of sulfate-reducing bacteria (SRB) were designed. Their application in conjunction with group-specific internal oligonucleotide probes was used to detect SRB DNA in samples of landfill leachate. Six generic/suprageneric groups could be differentiated: DESULFOTOMACULUM:; DESULFOBULBUS:; DESULFOBACTERIUM:; DESULFOBACTER:; DESULFOCOCCUS:-DESULFONEMA:-DESULFOSARCINA:; DESULFOVIBRIO:-DESULFOMICROBIUM: The predicted specificities of the PCR primer and oligonucleotide probe combinations were confirmed with DNA from reference strains. In all cases, the PCR primers and probes were specific, the only exception being that the Desulfococcus-Desulfonema-Desulfosarcina (group 5) PCR primers were able to amplify DNA from DESULFOBACTERIUM: (group 3) reference strains but these groups could nevertheless be differentiated with the internal oligonucleotide probes. The proliferation of SRB in landfill sites interferes with methanogenesis and waste stabilization, but relatively little is known about the composition of SRB populations in this environment. DNA was extracted from samples of landfill leachate from several municipal waste landfill sites and used as template in PCR reactions with SRB group-specific primer sets. Group-specific oligonucleotide probes were then used to confirm that the PCR products obtained contained the target SRB 16S rDNA. Both 'direct' and 'nested' PCR protocols were used to amplify SRB 16S rDNA from landfill leachates. Three of the six SRB groups could be detected using the 'direct' PCR approach (DESULFOTOMACULUM:, DESULFOBACTER: and Desulfococcus-Desulfonema-Desulfosarcina). When 'nested' PCR was applied, an additional two groups could be detected (DESULFOBULBUS: and DESULFOVIBRIO:-DESULFOMICROBIUM:). Only DESULFOBACTERIUM: could not be detected in any leachate samples using either direct or nested PCR. The SRB-specific 16S rDNA primers and probes described here can be applied to investigations of SRB molecular ecology in general, and can be further developed for examining SRB population composition in relation to landfill site performance.