title: Multiplex real-time PCR and culture methods for detection of Shiga toxin-producing Escherichia coli and Salmonella Thompson in strawberries, a lettuce mix and basil.
abstract: An appropriate approach of high throughput multi-screening was verified for Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp. in strawberries, lettuce and basil. Sample replicates were inoculated with STEC O157 or O26 and Salmonella Thompson (ca. 10-70, 100-700 and 1000-7000 cfu/25 g) and analysed after 1 and 5 days of storage (strawberries and lettuce at 7 °C and basil at 10 °C). After 18-24 h of enrichment at 37 °C in buffered peptone water, detection was performed using the GeneDisc multiplex PCR (stx1, stx2, eae and iroB genes) and selective culture media for isolation of STEC (with immunomagnetic separation (IMS)) and Salmonella spp. in parallel. After 1 day, the pathogenic strains were recovered from all samples for all inoculum levels, whereas reduced detection rates of STEC O157 and S. Thompson were observed after 5 days of storage in case of strawberries, in particular for the lowest inoculums level, suggesting superior survival potential for STEC O26. Overall, this study indicates the ability of PCR based screening methods for reproducible multi-detection of low numbers (10-70 cfu/25 g) of STEC and Salmonella in this type of foods. However, for the basil samples, PCR needed twofold dilution of the DNA extract to overcome inhibition. It was noted that on several occasions growth of competitive microbiota obstructed finding presumptive colonies on the selective agar media, whereas the use of an additional agar medium such as CHROMagar STEC (without IMS) improved recovery rate of STEC.
authors.affiliation: Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Electronic address: stefanie.delbeke@ugent.be.
authors.forename: S
authors.initials: S
authors.lastname: Delbeke
authors.affiliation: Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Ceuppens
authors.affiliation: Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium; Research Group EnBiChem, Department of Industrial Engineering and Technology, University College West-Flanders (Howest), Graaf Karel de Goedelaan 5, B-8500 Kortrijk, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Holvoet
authors.affiliation: Pall GeneDisc Technologies, 1 rue du Courtil, Centre CICEA, 35170 Bruz, France.
authors.forename: E
authors.initials: E
authors.lastname: Samuels
authors.affiliation: Research Group EnBiChem, Department of Industrial Engineering and Technology, University College West-Flanders (Howest), Graaf Karel de Goedelaan 5, B-8500 Kortrijk, Belgium.
authors.forename: I
authors.initials: I
authors.lastname: Sampers
authors.affiliation: Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Uyttendaele
mesh_terms: D003470:Culture Media; D005516:Food Microbiology / Q000379:methods*; D031985:Fragaria / Q000382:microbiology*; D018189:Immunomagnetic Separation; D018545:Lactuca / Q000382:microbiology*; D060885:Multiplex Polymerase Chain Reaction; D018649:Ocimum basilicum / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors
chemical_list: D003470:Culture Media
keywords: Culture media; Fresh produce; GeneDisc PCR detection; STEC O157; STEC O26; Salmonella Thompson