1887

Abstract

species are fastidious bacterial pathogens that are difficult to culture by standard methods. A PCR-denaturing gradient gel electrophoresis (PCR-DGGE) technique for detection and identification of different species was developed and evaluated. The method involves PCR detection of DNA by genus-specific primers that target 16S rDNA and subsequent differentiation of PCR products by use of DGGE. Strains are identified by comparing mobilities of unknown samples to those determined for reference strains; sequence analysis can also be performed on purified amplicons. Sixteen DGGE profiles were derived from 44 type and reference strains of 20 species, indicating the potential of this approach for resolving infection of a single host by multiple species. Some more highly related species were not differentiated whereas in highly heterogeneous species, sequence divergence was observed and more than one PCR-DGGE profile was obtained. Application of the PCR-DGGE method to DNA extracted from faeces of zoo animals revealed the presence of DNA in 13 of 16 samples; a correlation was seen between the mobility of PCR products in DGGE analysis and DNA sequencing. In combination, this indicated that zoo animals are colonized by a wide range of different species; seven animals appeared to be colonized by multiple species. By this approach, presumptive identifications were made of and in a Nile crocodile, in a baboon and a red panda, and in a wolf and a Taiwan beauty snake. All of these PCR products (∼400 bp) showed 100 % sequence similarity to 16S rDNA sequences of the mentioned species. These results demonstrate the potential of PCR-DGGE-based analysis for identification of species in complex ecosystems, such as the gastrointestinal tract, and could contribute to a better understanding of the ecology of helicobacters and other pathogens with a complex aetiology.

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2003-09-01
2019-11-19
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