Yuki OHTSUKA
The detection by high sensitive FISH of the apsA mRNA expression microorganism group in the liquid waste treatment reactor
Takashi YAMAGUCHI
Simultaneous fluorescence in situ hybridization (FISH) of mRNA of a key enzyme and rRNA is one of ideal tools for linking microbial metabolic activities and phylogeny.
Recently several protocols for mRNA FISH have been introduced in microbiology. In these studies, digoxigenin-labeled polyribonucleotide probes and Catalyzed reporter deposition (CARD) was employed, but in order to detect mRNA under different taxonomic levels with higher specificity than polyribonucleotide probes, use of oligonucleotide probes is one of the choices.
In this study, we simultaneously detected mRNA and rRNA by in situ hybridization with oligonucleotide probes as a case study.
We chose an apsA gene as a model microorganism and used the sludge of the UASB reactor, which processed artificial wastewater for a sample. In this study, we detected apsA mRNA from sludge sample by in situ hybridization with oligonucleotide probes. In addition, sulfate-reducing bacteria, which belonged to a Deltaproteobacteia detected it by FISH method. However, a result of both did not accord at all. On this account I was able to detect 16S rRNA and the mRNA of the function gene by FISH method by this experiment at the same time, but was not able to link phylogenetic identity to metabolic trait of microorganisms in a condition of single cell level and in situ at the same time. Therefore, I analyzed a microorganism having the apsA gene in sludge by Cloning method to investigate the cause why both signals did not accord. As a result, the microorganism, which is closely related to sulfur oxidation bacteria were detected from sludge.
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