Eight-hour PCR-based procedure for the detection ofSalmonellain raw oysters

Abstract

The aim of this study was to evaluate the ability of a nested PCR system to detect Salmonella senftenberg in raw oysters. The specific primers of the PCR were derived from the invA gene sequence, essential for Salmonella invasiveness into epithelial cells. First, for the extraction of DNA, four methods (guanidine isothiocyanate, E.Z.N.A. Mollusc Kit, Chelex-100, and lysis with detergents) were compared. A nested PCR method combined with 3.5 h pre-enrichment in buffered peptone water (BPW) and DNA extraction by the resin Chelex-100 is proposed for the detection of S. senftenberg in oyster samples. The detection limit of the method is less than 0.1 CFU/ml (<1 CFU/g of oyster). This procedure is shown to be an excellent tool for the sensitive detection of S. senftenberg from naturally contaminated oysters, with results being obtained within 8 h.

A Simple Gold Nanoprobe Assay for the Identification of Staphylococcus Aureus, Listeria Monocytogenes and Salmonella Enteritidis in Food Specimens

“…The following oligonucleotides pairs (OLG) were used: (a) 5'-CTT-ACT-TAC-TGG-CTG-TAC-CTG-3' and 5'-ATG-TCG-CTT-GTT-ATG-TGC-3', corresponding to a 686 bp fragment of the femA gene of S. aureus, (b) 5'-CAT-TAG-TGG-AAA-GAT-GGA-ATG-3' and 5'-GTA-TCC-TCC-AGA-GTG-ATC-GA-3', corresponding to a 732 bp fragment of the hly gene encoding the pore-forming listeriolysin of L. monocytogenes and (c) 5΄-TAT-CGT-ACT-GGC-GAT-ATT-GGT-GTT-TA-3΄ and 5΄-GGA-CAA-ATC-CAT-ACC-ATG-GCG-AGT-CA-3΄, corresponding to a 540 bp fragment of the invA sequence of Salmonella spp. (Vannuffel et al, 1995;Gouws & Liedeman, 2005;Vasquez-Novel et al, 2005). All OLG were thiolated (modified with 10xdATP) in the 5'-end of the primer.…”

“…PCR was performed according to previously published protocols (Vannuffel et al, 1995;Gouws & Liedeman, 2005;Vasquez-Novel et al, 2005) in 50 μl final volume, using the GoTaq Hot Start Master Mix (Promega Gmbh, 68199, Germany), 1mM each of the primers and 10 μl of eluted DNA. PCR products were separated in a 2% agarose gel, stained with ethidium bromide (0.5 μg/ml) and documented under UV illumination.…”

Polymerase Chain Reaction for the Rapid Detection and Serovar Identification of Salmonella in Food and Feeding Stuff

“…Often it is sufficient to extract the bacterial DNA by thermal cell lysis in the presence of a chelating resin, e.g., 6% (w/v) Chelex 100 suspension. Many authors have shown the usefulness of this resin for DNA extraction (Vazquez-Novelle et al 2005;Malorny et al 2003bMalorny et al , 2004b. Chelex 100 is a chelating resin that has a high affinity for polyvalent metal ions.…”

The Use of Molecular Methods for Detecting and DiscriminatingSalmonellaAssociated with Foods — A Review

“…The authors concluded that the systemic variant isolates of S. Paratyphi B in the marine environment are of notable public health significance as a result of the potential of acquiring enteric fever linked to the consumption of raw shellfish. Vázquez-Novelle et al (2005) developed an 8 h nested PCR assay for the detection of Salmonella senftenberg in raw oysters. The outer primers SAO1/ SA02 (Table 2) (Table 2) amplified a 281-bp sequence.…”