Resumo (PT):
Abstract (EN):
Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables
and the way they interact with each other. This is translated into a wide variability of FISH procedures
found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for
the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH
parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species
(Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe
concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between
the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx.
10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species
and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v])
for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study
shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according
to the target bacteria.
Idioma:
Inglês
Tipo (Avaliação Docente):
Científica
Nº de páginas:
7