DNA Arrays and Membrane Hybridization Methods for Screening of Six Lactobacillus Species Common in Food Products
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DNA Arrays and Membrane Hybridization Methods for Screening of Six Lactobacillus Species Common in Food Products Palmiro Poltronieri & Oscar Fernando D’Urso & Giuseppe Blaiotta & Maria Morea
Received: 2 October 2007 / Accepted: 14 January 2008 / Published online: 7 March 2008 # Springer Science + Business Media, LLC 2008
Abstract Dot blot and deoxyribonucleic acid (DNA) array hybridization assays for the traceability of Lactobacillus species in food have been developed to monitor and validate typical food products. A primer set was designed to amplify the 540-bp region located at +157 of the tuf (Elongation factor Tu) gene of the Lactobacillus genus. An oligonucleotide array, containing 73 Lactobacillus speciesspecific tuf sequences representing 21 species, was developed and tested for identifying L. paracasei, L. rhamnosus, L. plantarum, and L. buchneri. We also tested a rapid screening method for monitoring the species present in airy samples. Dot blot hybridization identified polymerase chain reaction amplicons immobilized on nylon membranes, using six tuf-based cyanine-3-labeled 18-mer oligonucleotides, specific for L. paracasei, L. zeae, L. fermentum, L. plantarum, L. rhamnosus, and L. buchneri. This method discriminates between multiple species of Lactobacilli isolated directly from cheese samples, simultaneously. The P. Poltronieri : M. Morea ISPA-CNR, Institute of Sciences of Food Productions, National Research Council, Bari, Italy O. F. D’Urso Biotecgen srl, Ecotekne, Lecce, Italy G. Blaiotta Department of Food Science, University of Napoli ‘Federico II’, Portici, Italy P. Poltronieri (*) ISPA-CNR, via Monteroni km 7, 73100 Lecce, Italy e-mail: [email protected]
tuf gene sequences, verified here with the DNA array method and used in dot blot hybridization, were shown to be a reliable tool for the simultaneous detection and differentiation of four Lactobacillus species. The hybridization techniques developed in this study may be useful in food processing and the analysis of food origin traceability. Keywords Lactobacillus Species . Dairy Products . Traceability . Elongation Factor Tu (tuf) . PCR Amplification . Fluorescent Probes . DNA Hybridization
Introduction Lactobacillus species, widespread in food and the environment, are important in technological applications as starter or adjunct nonstarter strains in food productions. The simultaneous presence of various lactic acid bacteria (LAB), expressing different species-specific enzyme activities, is essential for the development of flavor and specific quality traits of cheese (Baruzzi et al. 2000, 2005; Morea et al. 1998; Cappello et al. 2001). There is a need to characterize regional dairy products and to monitor the natural adjuvant nonstarter strains responsible for the production process. Many Lactobacillus species are associated with fermented foods and often may characterize products with traditional designation of origin. L. plantarum strains are found in fermented products as olives and other pickled vegetables and in sourdoughs (Co
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