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Author(s): Rui Rocha
Rita Santos
Pedro Madureira
Carina Almeida
Nuno F. Azevedo
Title: 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
Issue Date: 2016
Abstract: 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 variablesand the way they interact with each other. This is translated into a wide variability of FISH proceduresfound 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 forthe Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISHparameters 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 probeconcentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction betweenthe 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 speciesand 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 studyshows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted accordingto the target bacteria.
Related Information: info:eu-repo/grantAgreement/Autoriadade de Gestão do Programa Operacional Regional do Norte/Programas Integrados de IC&DT/NORTE-07-0124-FEDER-000025/(Bio) Chemical Engineering: Multi-Scale Approaches for Sustainable Environment and Health/LEPAE/CEFT - RL2
info:eu-repo/grantAgreement/FCT - Fundação para a Ciência e Tecnologia/Projectos de Investigação Clínica/PIC/IC/82815/2007/Desenvolvimento e aplicação de mímicos de DNA para a rápida identificação de microrganismos patogénicos/DNA mimics
info:eu-repo/grantAgreement/FCT - Fundação para a Ciência e Tecnologia/Projetos Estratégicos/UID/EQU/00511/2013 - POCI-01-0145-FEDER-006939/Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia/LEPABE
Document Type: Artigo em Revista Científica Internacional
Rights: restrictedAccess
Appears in Collections:FEUP - Artigo em Revista Científica Internacional

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