Quantification of Desulfitobacterium frappieri strain PCP-1 and Clostridium-like strain 6 in mixed bacterial populations by competitive polymerase chain reaction

Competitive polymerase chain reaction (cPCR) was used to quantify two anaerobic, Gram-positive bacteria, Desulfitobacterium frappieri strain PCP-1 and Clostridium-like strain 6 in mixed bacterial populations. cPCR was done with specific primers targeting the respective 16S rRNA genes of these strains and an internal standard (IS). The IS for each strain had the same primer binding sites, size and sequence as the target DNA, except that a unique restriction site was introduced by PCR-mediated site-directed mutagenesis. To distinguish between the IS and the target DNA, appropriate restriction digestion was done, cPCR was performed on genomic DNA of both strains, from which, a close value of the number of genomes added in the PCR mixtures was obtained, cPCR was also carried out with DNA extracted from a soil inoculated with a known amount of strain PCP-1 cells (determined by a:plating method). The cell number of strain PCP-1 in the soil, determined by the cPCR, was similar to the number of CFU inoculated. Evaluation of the strain PCP-1 cell concentration was achieved in a pentachlorophenol (PCP)-degrading methanogenic consortium, from which strain PCP-1 was isolated. This consortium was used in a continuous flow activated sludge reactor to treat PCP-contaminated industrial effluents. cPCR revealed that strain PCP-1 was present at a concentration of 1.8x10(5) cells/mL in the reactor. The cell concentration of strain 6 was evaluated in a phenol-degrading methanogenic consortium, from which strain 6 was isolated. Strain 6 was estimated by cPCR to be 6.5x10(5) cells/mL in a 20-day-old consortium culture. This is the first time that these two anaerobic Gram-positive bacteria were quantified in mixed bacterial populations. (C) 1998 Elsevier Science B.V.