title: Genotyping bacterial and fungal pathogens using sequence variation in the gene for the CCA-adding enzyme.
abstract: BACKGROUND
  To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology.
  
  
  RESULTS
  Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced.
  
  
  CONCLUSIONS
  These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.
authors.affiliation: Institute for Biochemistry, Leipzig University, Brüderstr. 34, 04103, Leipzig, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Franz
authors.affiliation: Institute for Biochemistry, Leipzig University, Brüderstr. 34, 04103, Leipzig, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Betat
authors.affiliation: Institute for Biochemistry, Leipzig University, Brüderstr. 34, 04103, Leipzig, Germany. mario.moerl@uni-leipzig.de.
authors.forename: Mario
authors.initials: M
authors.lastname: Mörl
mesh_terms: D001230:Aspergillus / Q000737:chemistry / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D001424:Bacterial Infections / Q000382:microbiology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009181:Mycoses / Q000382:microbiology*; D010802:Phylogeny; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D014733:Vibrio / Q000737:chemistry / Q000145:classification / Q000201:enzymology / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase
keywords: CCA-adding enzyme; Genotyping; Infectious diseases; Pathogen detection; Sequence analysis; tRNA nucleotidyltransferase