Utilize este identificador para referenciar este registo: https://hdl.handle.net/1822/41403

TítuloOptimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration
Autor(es)Rocha, Rui
Santos, Rita S.
Pedro, Madureira
Almeida, Carina
Azevedo, Nuno F.
Palavras-chavePNA-FISH
Eubacteria
Dextran sulfate
pH
PNA EUB338
DataMai-2016
EditoraElsevier B.V.
RevistaJournal of Biotechnology
CitaçãoRocha, R.; Santos, Rita S.; Pedro, Madureira; Almeida, Carina; Azevedo, Nuno F., Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration. Journal of Biotechnology, 226, 1-7, 2016
Resumo(s)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.
TipoArtigo
URIhttps://hdl.handle.net/1822/41403
DOI10.1016/j.jbiotec.2016.03.047
ISSN0168-1656
e-ISSN0168-1656
Versão da editorahttp://www.sciencedirect.com/science/article/pii/S0168165616301638
Arbitragem científicayes
AcessoAcesso aberto
Aparece nas coleções:CEB - Publicações em Revistas/Séries Internacionais / Publications in International Journals/Series

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