The MLST scheme used to characterize Listeria strains is based on the sequence analysis of the following seven housekeeping genes: acbZ (ABC transporter), bglA (beta-glucosidase), cat (catalase), dapE (Succinyl diaminopimelate desuccinylase), dat (D-amino acid aminotransferase), ldh (lactate deshydrogenase), and lhkA (histidine kinase). This MLST scheme was adapted from the MLST system proposed by Salcedo and colleagues [14] (link), with the following modifications. First, the template for gene ldh was extended from 354 to 453 nucleotides, thus improving strain discrimination. Second, gene templates were shortened because the extremities of the previous templates correspond to parts of the PCR primer sequences, thus possibly not corresponding totally to the genomic sequence of the isolates analyzed. Third, we incorporated universal sequencing tails to the PCR primers (Table 1), which allows to sequence PCR fragments of all genes using only two primers. DNA extraction was performed by the boiling method [41] (link). The PCR amplification conditions were as follows: an initial cycle of 94°C for 4 min; 25 amplification cycles, each consisting of 94°C for 30 s, 52°C for 30 s (except for bglA which has an annealing temperature of 45°C), and 72°C for 2 min; and a final incubation at 72°C for 10 min. The PCR products were purified by ultrafiltration (Millipore, France) and were sequenced on both strands with Big Dye v.1.1 chemistry on an ABI3730XL sequencer (Applied BioSystems).
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