Computational protocol: Foodborne Listeria monocytogenes: A Real Challenge in Quality Control

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Protocol publication

[…] Genomic DNA was isolated from cultures of 1.5 mL of brain heart infusion broth (LAB M Limited, UK) or Tryptic Soy Broth (OXOID, UK) containing inoculated frankfurter products, using the Wizard Genomic DNA Purification Kit (Promega, USA) according to the instructions of the manufacturer. The integrity of the genomic DNA was tested by analyzing the samples on 0.8% agarose gel stained with ethidium bromide. Purity and concentration of the DNA were determined spectrophotometrically. Primers were designed with the Oligo Explorer software (version 1.2; Gene Link, http://www.genelink.com/), evaluated with the OligoAnalyzer (version 1.1.2., Teemu Kuulasma) software, and purchased from Integrated DNA Technologies (USA). Oligonucleotides used in the PCR reactions were the hlyQF/hlyQR primers of Rodríguez-Lázaro et al. [] and the following designed primer pair: hly F: 5′-CACTCAGCATTGATTTGCCA-3′, hly R: 5′-CATACGCCACACTTGAGATA-3′.PCR reactions for the hlyF/hlyR primers were run under the following conditions: the final volume of the 50 μL PCR solution contained 2–4 μL of genomic DNA, 5 μL of 10x Taq buffer, 5 μL 25 mM of MgCl2, 2-2 μL of each 10 μM primer, 4 μL of 10 mM dNTP mixture, and 2.5 U Taq polymerase (Fermentas, Germany). In the case of the hlyQF/hlyQR primers, PCR reaction conditions were determined by Rodríguez-Lázaro et al. []. The annealing temperature in the case of our hlyF/hlyR primers was 55°C. The amplification conditions were 3 min denaturation at 96°C, 35 cycles each consisting of 30 s at 94°C, 45 s at the corresponding annealing temperature, and 1.3 min at 72°C followed by a final extension for 10 min at 72°C. We applied L. monocytogenes NCAIM B 01934 genomic DNA template as a positive control in each PCR run.hly F: 5′-CACTCAGCATTGATTTGCCA-3′,hly R: 5′-CATACGCCACACTTGAGATA-3′.Validation of the PCR protocol was carried out with artificially contaminated food samples. 25 g portions of commercial pasteurized frankfurter were suspended in 225 mL TSB (Tryptic Soy Broth; OXOID, UK) and homogenized in a Stomacher homogenizer (IUL Instruments, Spain) for 1 minute. The suspensions were inoculated with decimal dilutions of exponential phase culture of the L. monocytogenes NCAIM B 01934 reference strain suspension from 1 to 105 cells/mL concentrations, mixed well, and samples were taken immediately after inoculation. Following DNA isolation, hlyF/hlyR primers were applied in the PCR reactions. PCR products were sequenced in order to test their identity (data not shown). Reference sequences were gained from the National Center for Biotechnology Information Database (http://www.ncbi.nlm.nih.gov/) and aligned with the sequenced PCR products using the BioEdit Biological Sequence Alignment Editor Program []. […]

Pipeline specifications

Software tools OligoAnalyzer, BioEdit
Application qPCR
Organisms Listeria monocytogenes, Homo sapiens