Systematic Optimization of the Detection of Single Genes in Microorganisms by Fluorescence In Situ Hybridization (geneFISH)

Culture-independent techniques have played an increasingly important role in microbial ecology. Fluorescence in situ Hybridization (FISH) is a well-established method for the identification and quantification of microorganisms. Among the FISH-based techniques, geneFISH allows the linkage of particular genes to the microbial identity at the single cell level. The protocol involves ribosomal RNA-targeted Catalyzed Amplification Reported Deposition (CARD) for cell identification. For gene detection, digoxigenin-labeled polynucleotide probes are applied, to which specific antibodies conjugated with horseradish-peroxidases can bind. So far, low gene detection efficiencies have prevented a quantitative determination of the cell fraction carrying the target gene. Additionally, the CARD step hinders the sub-cellular signal quantification, increases DNA degradation and cell damage and makes the protocol long and labor intensive. Therefore, the main aim of this thesis was to systematically improve the geneFISH protocol by increasing the gene detection efficiency, simplifying the method and achieving a quantification of gene number per cell