P3-156 Development of a Multiplex PCR Assay for Salmonella, Shigella, Listeria monocytogenes and Verocytoxogenic Escherichia coli Detection from Fresh Produce

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Sofia Arvizu-Medrano, Universidad Autónoma de Querétaro, Querétaro, Mexico
Montserrat Iturriaga, Universidad Autónoma de Querétaro, Mexico
Omar Hernandez Hernandez, Universidad Autónomade Queretaro, Queretaro, Mexico
Elisa Cabrera Díaz, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico
Jeannette Barba León, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico
Ramiro Pacheco-Aguilar, Universidad Autonoma de Queretaro, Queretaro, Mexico
Introduction: In recent years, the increased consumption of fresh produce, has been accompanied by an increased frequency of foodborne pathogen outbreaks associated to produce. Conventional detection of microbial pathogens typically requires 5 to 7 days. Techniques PCR-based are more sensitive, allow for shorter processing times, and enhance the likelihood of detecting bacterial pathogens. 

Purpose: The objective of this study was to design a multiplex PCR assay for the simultaneous detection of Salmonella, Shigella, Listeria monocytogenes and Verocytoxogenic Escherichia coli(VTEC) in fresh produce.

Methods: Two pre-enrichment media (TSB: tryptic soy broth and UPB: universal enrichment broth) were evaluated. A cocktail of 8 Salmonella, 7 Listeria monocytogenes, 4 Shigella and 2 VTEC strains, previously individually stressed on stainless steel chips (drying 2 h under laminar flowhood) was inoculated (10-100 cells each one) on carrots, lettuce, tomato, broccoli and jalapeño pepper and incubated at 35°C/18h in TSB and UPB. Pathogens quantification was performed along pre-enrichment in selective media for each pathogen supplemented with antibiotics (ASB+100 ppm of rifampicina, MOX+20 ppm of nalidixic acid, and XLD without antibiotics). Multiplex PCR was performed after 18h of pre-enrichment in both media and five produce. Volume of reaction was 20µl containing master mix (Quiagen) and specific primers for the following target genes: InvA (F:CGCGCTTGATGAGCTTTACC; R:CTCGTAATTCGCCGCCATTG), IpaH (F:CGCGCTCACATGGAACAATC; R: TCCCGACACGCCATAGAAAC), Hly (F:CTGCAAGTCCTAAGACGCCA; R: CTTCACTGATTGCGCCGAAG, and Stx1 (F:GATCAGTCGTACGGGGATGC; R: ATTGTGCGTAATCCCACGGA). 

Results: The maximum population (Log CFU/ml) in TSB and UPB, respectively, were: 6.68-7.76 and 6.39-7.64 for Shigella, 5.39-7.16 and 6.33-6.88 for Salmonella, 5.22-6.72 and 5.60-6.14 for Listeria monocytogenes and 7.52-8.16 and 7.02-8.28 for VTEC. The multiplex PCR assay was able to detect all pathogens after 18 h of pre-enrichment. 

Significance: These results can potentially lead to more rapid and sensitive methods for foodborne pathogen detection in fresh produce.