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Christensen JJ, Andresen K, Justesen T, Kemp M. Ribosomal DNA sequencing: experiences from use in the Danish National Reference Laboratory for Identification of Bacteria. APMIS 2005;113:621–8. Diagnostic tools for identification of bacteria have developed dramatically in the last decade. Sequencing of genes coding for rRNA has led to revolutionary insights into the phylogeny and taxonomy of bacteria, and to new demands on the service provided by national reference laboratories for identification of bacteria. At the Danish Reference Laboratory for Identification of Bacteria, partial 16S rDNA sequencing has been used since 2001 to identify “difficult” strains submitted for taxonomic elucidation. Experiences relating to phenotypic as well as 16S rDNA sequencing of the first 175 strains examined are presented. Approximately 2/3 of the strains were Gram-positive and 1/3 Gram-negative. One fifth of the strains were anaerobic, while 4/5 were either facultatively anaerobic or aerobic. Methodological agreement was seen for most strains at species and/or genus level. Methodological disagreement was relatively rare. In 1/6 of the strains valuable information was obtained from sequencing results, while for some strains identification was based primarily on the phenotypic results. Only a few strains could not be clearly identified by either method. A very large number of strains representing taxons ranging from facultatively anaerobic to aerobic and anaerobic species and genera, Gram-positive as well as Gram-negative, were successfully examined. Of the submitted strains many have only rarely been encountered as human pathogens. Thus, genotypic identification may result in recognition of hitherto seldom recognized or unrecognized bacteria as human pathogens, which will lead to a better understanding of the nature of human infections. It is self evident that we should focus on slowly growing, fastidious or ‘difficult’ organisms when using sequencing for national reference purposes. Short sequences (450–650 base pairs) seem sufficient for most identifications. Molecular bacterial identification is a powerful tool for national reference laboratories, enhancing both the speed and validity of examinations performed.