DNA analysis enables genotype fingerprinting with consequent identification of different organisms, thus allowing traceability along the food chain.1
Indeed, this technique can have interesting applications in the agro-food industry for the identification of species and cultivars of both raw materials and processed food, particularly when a protected designation of origin (PDO) has been assigned.2
Olive oil is a very important component of the so-called Mediterranean diet and extra-virgin olive oil is currently considered as a high quality product, with beneficial health properties related to its fatty acid balance and high polyphenolic content. PDO, protected geographical indication (PGI), and traditional specialty guaranteed (TSG) are important awards assigned to the quality of extra-virgin olive oil, recognized by the European Union,3
in relation to the quality and geographical origin of olive oils, establishing marketing standards and stating an obligatory regimen of origin designation for extra-virgin and virgin olive oils.4
Many studies have been published on the assessment of quality and authenticity of extra virgin olive oil,5
and a number of well-established methodologies are currently applied to control frauds and oil composition. However, distinction of oils having similar triglyceride composition requires complex and multiple-parameter approaches. For example, chemical analyses, based on the determination of specific metabolites, are able to detect the presence of hazelnut oil in olive oil, but only when its percentage exceeds 15–20%.6-9
As far as the possibility to distinguish the olive cultivars that have contributed to the oil blend, complications derive from the high number of cultivars of the species Olea europaea L., by the recent extension of cultivation of some traditional cultivars in new regions, and by the often occurring cases of synonymy.
DNA markers have been used for identification of olive cultivars, being independent from environmental fluctuations and on account of the high degree of polymorphism, which allows to effectively distinguish very similar cultivars and to solve homonymy cases. Molecular markers have been specially applied for cultivar discrimination,10
plant certification and collection management, whereas for defining olive oil composition new markers need to be developed based on single nucleotide mutations easily detectable at array level.11
At present, DNA analysis is more than a promising approach to distinguish the different cultivars from which the oil is produced,12
since it is not influenced by environmental and processing conditions in respect to other methods (i.e., metabolites). DNA extracted from olive oil has been studied by means of different techniques based on molecular markers. Amplified fragment length polymorphisms (AFLP),13
sequenced characterized amplified region (SCAR),14
simple sequence repeats (SSR, also referred to as microsatellites)15,16
have been used for the characterization of olive cultivars from olive oil. Detection of single nucleotide polymorphisms (SNPs) by ligation detection reaction (LDR)17
platform by using several olive SNPs has recently shown to be a very potent tool for olive oil cultivar characterization.
have demonstrated the negative effect of storage length on DNA present in the oil, showing a progressive degradation of the recovered DNA. Thus, methods enabling the detection of short DNA fragments are more suitable, being more robust and allowing the tracing of partially degraded DNA.
The detection of SNP markers can be best performed using specific DNA analogs, which are superior to oligonucleotides, in terms of sequence selectivity, as probes. Among them, peptide nucleic acids (PNAs), oligonucleotide mimics in which the sugar phosphate backbone has been replaced by a polyamide chain, constituted by N
-(2-aminoethyl)glycine units covalently linked to nucleobases through a carboxymethyl spacer (), have proven to be particularly suitable, showing higher affinity and higher single mismatch recognition than oligonucleotides. Modified PNAs, in particular chiral PNAs, were shown to be superior to unmodified PNAs in terms of sequence selectivity, a property particularly useful in biomedical applications.19,20
Figure 1. (A) Structure of a PNA, (B) C2-modified PNA containing an arginine monomer.
On account of these properties, PNAs have been used, in combination with different analytical techniques, in a wide variety of diagnostic methods for the detection of specific DNA tracts in biomedical and food research.21
In particular, PNA microarray technology proved to be very effective for the detection of genetically modified organisms (GMOs)22,23
or hidden allergens in food.24
A PNA microarray platform was also recently developed for the detection of SNPs related to tomato cultivars.25
PNAs have shown excellent properties also in the development of ultrasensitive techniques such as surface plasmon resonance (SPR) which allowed direct (PCR-free) detection of genomic DNA.26,27
Using PNA arrays, we have evaluated the possibility to detect and identify specific DNA sequences, which can be used for the assessment of the authenticity of olive oil, by revealing the presence of different oils or by fingerprinting the DNA of different olive cultivars, in a proof-of-principle approach. Thus, the aim of the present study was the application of the PNA microarray technology to the most challenging tasks of olive oil characterization: (1) the detection of hazelnut (Corylus avellana) oil adulteration, difficult to detect on the basis of the chemical composition and the amount of fatty acids being very similar in the two oils, and (2) the identification of olive oil cultivars by screening single nucleotide polymorphisms (SNPs). The former task requires the implementation of a method with high sensitivity, given the low amount of alien DNA in olive oil, and the latter needs highly selective probes.