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GeneDisc multiplex-PCR and IMS-chromogenic media for detection of VTEC and Salmonella in lettuce, strawberries and basil

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n° 244994
Abstract
Introduction: VTEC have been increasingly recognized as food borne pathogen, also in fresh produce. Detection methods are primarily focused on E. coli O157:H7, but there is an increasing attention for non-O157 VTEC (1). Adequate detection methods are lacking for non-O157 VTEC, in particular for fresh produce with high levels of competing flora (2). Multiplex PCR methods are recommended for detection of VTEC virulence factors and have potential to detect multiple enteric pathogens or emerging virulence factors (e.g. aggR) in one run. Materials and methods: The aim of the present study is to validate an appropriate approach for high throughput multiscreening for VTEC and Salmonella in fresh produce focusing on the case studies of strawberries, lettuce and basil. Sample replicates were inoculated (1000, 100, 10 cfu/25 g) and analysed after 1 and 5 days storage at 7°C using in parallel GeneDisc Multiplex PCR (stx2, stx1, eae, aggR and Salmonella) and IMS-chromogenic media (CT-SMAC (O157), MAC (O26) and Brilliance Salmonella (Salmonella spp.)). Results and Discussion: Both methods enabled detection of the E. coli O157 and O26 and Salmonella Thompson strain at all inoculums if analysed after 24h although, in particular for basil, GeneDisc needed ½ dilution of DNA extract to overcome inhibition and competitive flora troubled reading of chromogenic media. After 5 days cold storage, pathogens were recovered for basil (stored at 10°C) whereas lower inoculums levels were more difficult to detect for strawberries (13/18 for VTEC and 10/18 for Salmonella) and lettuce (16/18 for VTEC and 18/18 for Salmonella). GeneDisc Multiplex PCR was shown to be a suitable screening method for simultaneous sensitive detection of VTEC and Salmonella in fresh produce although overcoming PCR inhibition by DNA dilution was needed in particular after 5 days storage and for the leafy vegetables. IMS combined with chromogenic media was shown to be a cumbersome method in particular for VTEC to differentiate from competing flora in leafy vegetables. When using CHROMagar STEC as chromogenic media (without IMS) better and clearer results were obtained.
Keywords
IMS, Strawberries, Salmonella, Lettuce, VTEC, GeneDisc, Chromogenic media, Basil

Citation

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Chicago
Delbeke, Stefanie, Kevin Holvoet, Eric Samuels, and Mieke Uyttendaele. 2012. “GeneDisc multiplex-PCR and IMS-chromogenic Media for Detection of VTEC and Salmonella in Lettuce, Strawberries and Basil.” In FoodMicro 2012, Abstracts.
APA
Delbeke, S., Holvoet, K., Samuels, E., & Uyttendaele, M. (2012). GeneDisc multiplex-PCR and IMS-chromogenic media for detection of VTEC and Salmonella in lettuce, strawberries and basil. FoodMicro 2012, Abstracts. Presented at the 23rd International ICFMH symposium FoodMicro 2012 : Global issues in food microbiology.
Vancouver
1.
Delbeke S, Holvoet K, Samuels E, Uyttendaele M. GeneDisc multiplex-PCR and IMS-chromogenic media for detection of VTEC and Salmonella in lettuce, strawberries and basil. FoodMicro 2012, Abstracts. 2012.
MLA
Delbeke, Stefanie, Kevin Holvoet, Eric Samuels, et al. “GeneDisc multiplex-PCR and IMS-chromogenic Media for Detection of VTEC and Salmonella in Lettuce, Strawberries and Basil.” FoodMicro 2012, Abstracts. 2012. Print.
@inproceedings{3080947,
  abstract     = {Introduction: VTEC have been increasingly recognized as food borne pathogen, also in fresh produce. Detection methods are primarily focused on E. coli O157:H7, but there is an increasing attention for non-O157 VTEC (1). Adequate detection methods are lacking for non-O157 VTEC, in particular for fresh produce with high levels of competing flora (2). Multiplex PCR methods are recommended for detection of VTEC virulence factors and have potential to detect multiple enteric pathogens or emerging virulence factors (e.g. aggR) in one run.
Materials and methods: The aim of the present study is to validate an appropriate approach for high throughput multiscreening for VTEC and Salmonella in fresh produce focusing on the case studies of  strawberries, lettuce and basil. Sample replicates were inoculated (1000, 100, 10 cfu/25 g) and analysed after 1 and 5 days storage at 7{\textdegree}C using in parallel GeneDisc Multiplex PCR (stx2, stx1, eae, aggR and Salmonella) and IMS-chromogenic media (CT-SMAC (O157), MAC (O26) and Brilliance Salmonella (Salmonella spp.)).
Results and Discussion: Both methods enabled detection of the E. coli O157 and O26 and Salmonella Thompson strain at all inoculums if analysed after 24h although, in particular for basil, GeneDisc needed {\textonehalf} dilution of DNA extract to overcome inhibition and competitive flora troubled reading of chromogenic media. After 5 days cold storage, pathogens were recovered for basil (stored at 10{\textdegree}C) whereas lower inoculums levels were more difficult to detect for strawberries (13/18 for VTEC and 10/18 for Salmonella) and lettuce (16/18 for VTEC and 18/18 for Salmonella). GeneDisc Multiplex PCR was shown to be a suitable screening method for simultaneous sensitive detection of VTEC and Salmonella in fresh produce although overcoming PCR inhibition by DNA dilution was needed in particular after 5 days storage and for the leafy vegetables. IMS combined with chromogenic media was shown to be a cumbersome method in particular for VTEC to differentiate from competing flora in leafy vegetables. When using CHROMagar STEC as chromogenic media (without IMS) better and clearer results were obtained.},
  author       = {Delbeke, Stefanie and Holvoet, Kevin and Samuels, Eric and Uyttendaele, Mieke},
  booktitle    = {FoodMicro 2012, Abstracts},
  language     = {eng},
  location     = {Istanbul, Turkey},
  title        = {GeneDisc multiplex-PCR and IMS-chromogenic media for detection of VTEC and Salmonella in lettuce, strawberries and basil},
  year         = {2012},
}