An Update and Optimisation of Oligonucleotide Probes Targeting Methanogenic Archaea for Use in Fluorescence in Situ Hybridisation (FISH)

Fluorescence in situ hybridisation (FISH) is a common and popular method used to investigate microbial populations in natural and engineered environments. DNA oligonucleotide probes require accurate determination of the optimal experimental conditions for their use in FISH. Oligonucleotides targeting the rRNA of methanogenic Archaea at various taxonomic levels have previously been published, although when applied in FISH, no optimisation data has been presented. In this study, 3000 Euryarchaeota 16S rRNA gene sequences were phylogenetically analysed and previously published oligonucleotides were evaluated for target group accuracy. Where necessary, modifications were introduced or new probes were designed. The updated set of probes was optimised for use in FISH for a more accurate detection of methanogenic Archaea.