Biomolecular identification of fish species by PCR and analysis of microbiological risk linked to the consumption of ready to eat fishery products

• Main Author: DI TARANTO, PIETRO
• Other Authors: DI TARANTO, Pietro, NORMANNO, GIOVANNI GIUSEPPE
• Format: Doctoral or Postdoctoral Thesis
• Language: unknown
• Published: Università degli Studi di Foggia 2016
• Subjects: fishery products; food safety; frauds; PCR; Staphylococcus aureus; Listeria monocytogenes; Gadus morhua; Salmo salar
• Online Access: http://hdl.handle.net/11369/363186; https://doi.org/10.14274/di-taranto-pietro_phd2016

The present study regarded the isolation and the characterisation of Staphylococcus aureus and Listeria monocytogenes from ready to eat (RTE) fishery products and the development and the improvement of novel PCR protocols for the identification of fish species. For the detection of S. aureus and L. monocytogenes, 99 and 135 RTE samples, respectively, were collected at local retail outlets and analysed according to ISO procedures in the laboratories of Food Microbiology of Experimental Zooprophylactic Institute of Apulia and Basilicata located in Foggia. RTE fishery products for the isolation of S. aureus and L. monocytogenes consisted of 33 and 45 samples of marinated anchovies fillets (Engraulis encrasicolus), 33 and 45 samples of smoked salmon (Salmo salar), 33 and 45 samples of seafood salad, respectively. As regards the identification of fish species, novel species-specific primers were developed by the program "Primer Express 3.0" and by the software “Primer-BLAST” to amplify fragments of 200 bp, 250 bp, 300 and 562 bp, 350 bp, 400 bp and 522 bp within COI gene for Merluccius merluccius, Lates niloticus, Gadus morhua, Ruvettus pretiosus, Pangasius hypophthalmus, Epinephelus spp., respectively. Ten samples of each fish species of interest were obtained from wholesale fishery plants. DNA was extracted from individual sample and quantified. DNA isolates were subjected to end-point PCR analysis and PCR products were sequenced. Out of 33 samples of smoked salmon, S. aureus was isolated from one sample (3.03%). The S. aureus strains carried the icaA, seb and sec genes and were resistant to ampicillin and tetracycline. L. monocytogenes was isolated from 2 of 45 samples of smoked salmon (4.44%). The strains of L. monocytogenes, isolated from both samples, resulted to belong to the serovar 1/2a and to be susceptible to all antibiotics tested. Single PCRs were performed using DNA isolates and the developed primers for each fish species of interest. After sequencing, the isolates were compared with the selected ...