Screening and Detecting in Different Food Matrices in Southern Tunisia Using a Combined Enrichment/Real-Time PCR Method: Correlation with Conventional Culture Method.

Journal: Frontiers in microbiology
Published Date: Dec 7, 2017

Abstract

A combined enrichment/ newly developed TaqMan real-time PCR (qPCR) method as a screening assay to detect spp. in 500 naturally food matrices is evaluated. DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysis-guanidine isothiocyanate method. Heterologous internal amplification control (IAC) was incorporated during qPCR assays and co-amplified with the gene of the target pathogen. qPCR exhibited 100% specificity when testing 94 strains (inclusivity) and 32 non- strains (exclusivity). The qPCR showed a consistent detection of two copies of the gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%). QPCR was sensitive and showed detection at 8.5 × 10 CFU mL of artificially spiked poultry meat -BWP solution in less than 40 cycles. When analyzing 500 different food matrices and comparing the results with the ISO 6579:2002 conventional culture method, the sensitivity and specificity were 100 and 76.6%, respectively. QPCR showed spp. DNA in raw poultry meat 27/45 (60%), milk 31/93 (33.3%), raw red meat 5/13 (38.5%), and fish 11/46 (23.9%) samples. The prevalence of spp. in cakes, dairy, cooked meals, charcuterie products using qPCR was 11/14 (26.8%), 5/22 (22.7%), 32/150 (21.3%), and 5/20 (25%), respectively, compared to 0% as demonstrated by culture. Anatum was the most common serovar found associated with red meat compared to kentucky isolated from fish and poultry meat. In conclusion, our study is the first to use a combined enrichment/ qPCR method as a screening assay to detect DNA in different types of commercialized food in Southern Tunisia. QPCR results indicate that contamination is common in milk and in other types of food samples.

Authors

  • Mariam Siala
    Department of Biology, Preparatory Institute for Engineering Studies of Sfax, University of Sfax, Sfax, Tunisia.
  • Amina Barbana
    Department of Life Sciences, Research Laboratory of Environmental Toxicology-Microbiology and Health (LR17ES06), Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia.
  • Salma Smaoui
    Regional Hygiene Care Laboratory, Department of Microbiology, Hedi-Chaker University Hospital, Sfax, Tunisia.
  • Salma Hachicha
    Regional Hygiene Care Laboratory, Department of Microbiology, Hedi-Chaker University Hospital, Sfax, Tunisia.
  • Chema Marouane
    Regional Hygiene Care Laboratory, Department of Microbiology, Hedi-Chaker University Hospital, Sfax, Tunisia.
  • Sana Kammoun
    Regional Hygiene Care Laboratory, Department of Microbiology, Hedi-Chaker University Hospital, Sfax, Tunisia.
  • Radhouane Gdoura
    Department of Life Sciences, Research Laboratory of Environmental Toxicology-Microbiology and Health (LR17ES06), Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia.
  • Férièle Messadi-Akrout
    Regional Hygiene Care Laboratory, Department of Microbiology, Hedi-Chaker University Hospital, Sfax, Tunisia.

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