Caterina Agrimonti

Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: A tool for disclosure of olive oil adulteration

Extra virgin olive oil is frequently subjected to adulterations with addition of oils obtained from plants other than olive. DNA analysis is a fast and economic tool to identify plant components in oils. Extraction and amplification of DNA by PCR was tested in olives, in milled seeds and in oils, to investigate its use in olive oil traceability. DNA was extracted from different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and pumpkin. Comparing the DNA melting profiles in reference plant materials and in the oils, it was possible to identify any plant components in oils and mixtures of oils. Real-Time PCR (RT-PCR) platform has been added of the new methodology of high resolution melting (HRM), both were used to analyse olive oils mixed with different percentage of other oils. Results showed HRM a cost effective method for efficient detection of adulterations in olive oils.

Applicability of SSR markers to the traceability of monovarietal olive oils

BACKGROUND To protect the features and authenticity of food products, the European Commission enforces two certification labels: Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI). EEC Regulation No. 510/2006 imposes criteria for labelling, production and commercialisation of olive oil. Since plant genotype is a major determinant in establishing the PDO and PGI labels, methods to ascertain the varieties present in a batch of olive oil are essential in validating product conformity. The traceability of olive oil can be assessed through simple sequence repeat (SSR) co-dominant markers targeted to specific regions of DNA from olive cultivars. RESULTS Twenty-one monovarietal olive oils were analysed with nine nuclear and two shortened SSRs. For each marker the correspondence of allelic profile with the reference cultivar, the reproducibility of profiles in different DNA extractions and the polymorphism information content were determined. CONCLUSION The results showed that using a panel of SSR markers such as those described in this paper allows one to make a reliable attribution of an olive oil to a specific cultivar.

Development of a multiplex real time PCR to detect thermophilic lactic acid bacteria in natural whey starters.

A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of those species during ripening of derived dairy products. A major increase in understanding the starter culture contribution to cheese ecosystem could be harnessed to control cheese ripening and flavor formation.

Silencing of G1-1 and A2-1 genes. Effects on general plant phenotype and on tuber dormancy in Solanum tuberosum L.

SummaryWe have evaluated the effect that the, silencing of two genes specifically expressed in conditions of dormancy (A2-1) and sprouting (G1-1) had on tuber dormancy. For this purpose potato plants (Solanum tuberosum L. cv. Désirée) were transformed with the antisense of the genes G1-1 and A2-1 under the control of constitutive 35S CaMV promoter. A first generation of transgenic plants was propagated from axenic stem cuttings and a second generation by tuber planting. The plants obtined were analyzed for the length of dormancy, plant morphology and agronomic characteristics. Statistical analysis of dormancy in lines obtained from the original transformants for the antisense of G1-1 gene showed a significant increase in length as compared with different types of control plants, with few effects on plant vegetative habit and tuber production. In contrast, results obtained on A2-1 antisense transformed plants did not reveal any significant change on the length of dormancy. Here we report small-scale field trials performed with the aim to select and regenerate commercially exploitable potato plants with a stable transgene-dependent phenotype, affecting the length of dormancy.

In vitro and in silico analysis of two genes (A2-1 and G1-1) differentially regulated during dormancy and sprouting in potato tubers.

SummaryTwo cDNAs, corresponding to genes differentially regulated during dormancy and sprouting in potato tubers, cultivar Désirée, were isolated: i) G1-1 corresponded to a gene that was turned off during dormancy and turned on during early phases of sprouting; ii) A2-1 corresponded to a gene activated during dormancy and strongly repressed during the transition from dormancy to sprouting. When induced, both genes were expressed at low level. Full-length cDNAs and genomic clones were isolated and characterized. G1-1 was a short gene, 452 bp long, containing an intronless open reading frame, coding for a putative protein of 64 aminoacids. Sequence analysis showed that G1-1 was homologous to an expressed sequence tag (EST) ofArabidopsis thaliana. A2-1 full-length cDNA was 1577 bp long and contained an open reading frame coding for a putative protein of 383 aminoacids, which contained a Walker box binding domain, common to a multifunctional family of intracellular ATPases.

A Real-Time PCR/SYBR Green I Method for the Rapid Quantification of Salmonella enterica in Poultry Meat

It has been developed a method for quantitative detection of Salmonella enterica in poultry meat based on real-time PCR (qtPCR) with species-specific primers and SYBR® GreenER™ chemistry. Two methods for bacterial DNA extraction were compared: one based on a commercial kit (AccuPrep®) and the other on silica–magnetite nanoparticles. Primers were designed on sequence of invA gene encoding for an inner membrane protein associated with invasiveness of Salmonella. Serial dilutions of DNA from pure cultures of Salmonella and from broiler breast samples spiked with serial dilutions of Salmonella were analyzed in different replicates and with different PCR equipments. Robustness of the method was evaluated and compared in terms of repeatability, reproducibility, and consistency with conventional plate count methods and for applicability to the different equipments. The matrix effect upon each reaction specificity was assessed with addition of DNA from a noncompetitive internal amplification control. The limit of detection (LOD) was determined between 10 and 40 colony-forming units (CFUs)/ml; whereas, the limit of quantification (LOQ) was 102 CFUs/ml. Quantification with qtPCR was in the same order of magnitude as enumeration with plate counting but with an overestimation.

Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products.

ABSTRACT Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in “Food Genomics;” a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or “end-point” PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA. This review describes qPCR applications to the analysis of microbiota, and to the identification of the animal species source of milk from which dairy products have been made. These are important aspects for ensuring safety and authenticity. The various applications of qPCR are discussed, as well as advantages and disadvantages in comparison with other analytical methods.