Specific detection of an oil-degrading bacterium, Corynebacterium sp IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences

Abstract

A species-specific PCR technique to detect an oil-degrading bacterium, Corynebacterium sp. IC10, released into sand microcosms is described. PCR primers, specific to strain IC10, were designed based on 16S rRNA gene sequences and tested against both closely and distantly related bacterial strains using four primer combinations involving two forward and two reverse primers. Two sets of them were specific to the strain IC10 and Corynebacterium variabilis and one set was selected for further analysis. The PCR amplification was able to detect 1 pg template DNA of strain IC10 and 1.2x10(4) c.f.u. of IC10 ml wet sand(-1) in the presence of 3x10(8) Escherichia coli cells. In non-sterile sand microcosms seeded with the strain IC10, the sensitivity of detection decreased to 9.6x10(5) c.f.u. ml wet sand(-1). The detection sensitivity thus depends on the complexity of background heterogeneous DNA of environmental samples. The assay is suitable for detection of Corynebacterium sp. IC10 in laboratory microcosms, however, cross reaction with non-oil degrading coryneforms may prohibit its use in uncharacterized systems.