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.
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 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 300nM 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.
authors.affiliation: LEPABE, Department of Chemical Engineering, Faculty of Engineering of the University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal; Centre of Biological Engineering, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; Biomode, Ed. GNRATION, Praça Conde Agrolongo no 123, 4700-312 Braga, Portugal. Electronic address: pdeqb1209967@fe.up.pt.
authors.forename: Rui
authors.initials: R
authors.lastname: Rocha
authors.affiliation: LEPABE, Department of Chemical Engineering, Faculty of Engineering of the University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal; Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Gent, Belgium; i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
authors.forename: Rita S
authors.initials: RS
authors.lastname: Santos
authors.affiliation: IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre n.° 823, 4150-180 Porto, Portugal; ICBAS-Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira n.° 228, 4050-313 Porto, Portugal; i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Madureira
authors.affiliation: LEPABE, Department of Chemical Engineering, Faculty of Engineering of the University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal; Centre of Biological Engineering, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; Biomode, Ed. GNRATION, Praça Conde Agrolongo no 123, 4700-312 Braga, Portugal.
authors.forename: Carina
authors.initials: C
authors.lastname: Almeida
authors.affiliation: LEPABE, Department of Chemical Engineering, Faculty of Engineering of the University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.
authors.forename: Nuno F
authors.initials: NF
authors.lastname: Azevedo
mesh_terms: D001419:Bacteria / Q000187:drug effects / Q000302:isolation & purification*; D015342:DNA Probes / Q000378:metabolism*; D016264:Dextran Sulfate / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008970:Molecular Weight; D020135:Peptide Nucleic Acids / Q000378:metabolism*
chemical_list: D015342:DNA Probes; D020135:Peptide Nucleic Acids; D016264:Dextran Sulfate
keywords: Dextran sulfate; Eubacteria; PNA EUB338; PNA-FISH; pH