Lara Tinacci

Two alternative multiplex PCRs for the identification of the seven species of anglerfish (Lophius spp.) using an end-point or a melting curve analysis real-time protocol

Anglerfish (Lophius spp.) is consumed worldwide and is an important economic resource though its seven species are often fraudulently interchanged due to their different commercial value, especially when sold in the form of fillets or pieces. Molecular analysis is the only possible mean to verify traceability and counteract fraud. We developed two multiplex PCRs, one end-point and one real-time with melting curve post-amplification analysis, which can even be run with the simplest two-channel thermocyclers. The two methods were tested on seventy-five reference samples. Their specificity was checked in twenty more species of those most commonly available on the market and in other species of the Lophiidae family. Both methods, the choice of which depends on the equipment and budget of the lab, provide a rapid and easy-to-read response, improving both the simplicity and cost-effectiveness of existing methods for identifying Lophius species.

Pentaplex PCR As Screening Assay for Jellyfish Species Identification in Food Products

Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market.

DNA barcoding for the verification of supplier’s compliance in the seafood chain: How the lab can support companies in ensuring traceability

Food Business Operators (FBOs) rely on laboratory analysis to ensure seafood traceability. DNA barcoding and Forensically Informative Nucleotide Sequencing may represent a support within self-checking programs finalized to suppliers’ qualification and products identity certification. The present study aimed at verifying the usefulness of a decisional procedure (decision tree) set up at the FishLab (Department of Veterinary Sciences, University of Pisa, Italy) for seafood species identification by DNA analysis, to cope with FBOs’ needs. The decision tree was applied to the analysis of 182 seafood (fish and molluscs) products, conferred to the FishLab by different FBOs between 2014 and 2015 as result of their self-checking activities. The analysis relied on a standard COI gene fragment eventually integrated by the analysis of alternative or supportive molecular targets (cytb and 16S rRNA). It also included a mini-DNA barcoding approach for processed products. Overall, 96.2% of the samples were unambiguously identified at species level using the elective target alone (92.4%) or a multi-target approach (3.8%). The lack of species identification (3.8%) was attributable to the absence of reference sequences or to the low resolution of the molecular targets. Nonetheless, all the molecular results were deemed adequate to evaluate the sample’s compliance to the label information. Non-compliances were highlighted in 18.1% of the products. The protocol was proven as an effective supportive tool for the seafood identity verification within the supply chain self-checking activities. In addition, a considerable fraud rate was confirmed and the species most frequently involved in substitution were pointed out.

Serum responsiveness to recombinant bovine somatotropin in buffalo: a three-month lactation study using an acid-stripping ELISA for screening

The misuse of recombinant bovine somatotropin (rbST) to increase milk yield involves buffalo not just cows. Screening methods to identify rbST-treated cattle have already been proposed. However, there have been no studies on prolonged periods with a high number of animals. In this study, we developed a new enzyme-linked immunosorbent assay (ELISA) to measure the serum responsiveness towards rbST, based on an acid-stripping procedure and relatively simple integral calculation dilution curves. We also applied the analysis to 640 serum and 96 milk samples collected from 16 buffalo treated with rbST and 16 controls, over a period of approximately three months. Its suitability as a screening method, in compliance with EU law, was also assessed. A bi-factorial approach was also evaluated, including the measurement of insulin-like growth factor 1 concentration in serum. Results showed that our ELISA could be used on its own for screening purposes, without the need to assess other biomarkers. Copyright