title: Detection of food spoilage and pathogenic bacteria based on ligation detection reaction coupled to flow-through hybridization on membranes.
abstract: Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific for Aeromonas spp., Pseudomonas spp., Shewanella spp., and Morganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.
authors.affiliation: Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: K
authors.initials: K
authors.lastname: Böhme
authors.affiliation: Institute of Agricultural Biology and Biotechnology, National Research Council, 26500 Lodi, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Cremonesi
authors.affiliation: Institute of Biomedical Technologies, Italian National Research Council, 20090 Segrate, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Severgnini
authors.affiliation: Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: Tomás G
authors.initials: TG
authors.lastname: Villa
authors.affiliation: Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: I C
authors.initials: IC
authors.lastname: Fernández-No
authors.affiliation: Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Barros-Velázquez
authors.affiliation: Institute of Agricultural Biology and Biotechnology, National Research Council, 26500 Lodi, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Castiglioni
authors.affiliation: Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Calo-Mata
mesh_terms: D001419:Bacteria / Q000302:isolation & purification*; D003101:Collodion; D015342:DNA Probes / Q000378:metabolism; D005516:Food Microbiology / Q000379:methods*; D008025:Ligases / Q000378:metabolism; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D015342:DNA Probes; D008567:Membranes, Artificial; D003101:Collodion; D008025:Ligases