Objetivos e Atividades
Considering, the impact that NAMs have on FISH performance, our goal is to combine them with the two recent FISH procedures referred above in one technique designated by NAM-CLASI-FIVH; to our knowledge, there are no published results on this matter. First, to accomplish our goal, the NAMs probes used in NAM-CLASI-FIVH technique will be developed and optimized in conditions similar those found in stomach; then, the technique will be validated in gastric biopsies samples. Once this has been achieved, the developed protocol will be tested and adpated in a mouse model. Then, as a case study, we will analyse the microbial changes over time in a mouse model in terms of composition and spatial organization during H. pylori infection.
Resultados Esperados/Atingidos
An EUB LNA/2'OMe probe conjugated to one fluorophore were tested against the species; to rank the fluorophores, 8 versions of EUB LNA/2'OMe probe, each conjugated to one of 8 different fluorophores were tested against E. coli. Strong variations on the signal intensity were found between species and between fluorochromes However, this methodology allowed the establishment of balanced species/fluorochromes pairs and provided an unambiguous separation of up to eight fluorophores Validation tests with different proportions of bacteria labelled with the different fluorophores showed that the frequency of each bacterium in the mixture is equivalent to the frequency of each species in the individual images from our spectral imaging-FISH. In fact, the separate spectral imaging combined with FISH it works since the frequency of each label type in individual images and mixtures are similar.
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