Short communication Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: A tool for disclosure of olive oil adulteration Michelangelo Vietina a,1 , Caterina Agrimonti b,1 , Nelson Marmiroli b,⇑ a SITEIA.PARMA Interdepartmental Centre, University of Parma, Parco Area Delle Scienze 181/A, 43124 Parma, Italy b Department of Life Sciences, University of Parma, Parco Area Delle Scienze 11/A, 43124 Parma, Italy article info Article history: Received 20 August 2012 Received in revised form 7 May 2013 Accepted 18 June 2013 Available online 28 June 2013 Keywords: Olive oil Seeds oil DNA extraction PCR high resolution melting (HRM) Traceability Adulteration abstract 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 amplifi- cation 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. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Extra virgin olive oil can certainly be considered a premium product because of its flavour, aroma and for its health properties, but it is frequently subjected to frauds and adulterations with addition of cheaper oils obtained from plants other than olive. Hazelnut oil, with a composition similar to olive oil, is the most fre- quently used, but maize, sunflower, sesame and grapevine oils are used as well. Adulteration of olive oil is not only a commercial fraud, but it can also be a threat to human health. In Spain in 1981, rapeseed oil, added with aniline and sold as olive oil, affected 20,000 persons with 300 deceases (Capote, Jiménez, & de Castro, 2007). Several papers showed how to fingerprint DNA in olive oil and to determine its plant varietal origin with molecular markers (Agri- monti, Vietina, Pafundo, & Marmiroli, 2011; Consolandi et al., 2008; Pafundo, Agrimonti, Maestri, & Marmiroli, 2007; Pafundo, Agrimonti, & Marmiroli, 2005; Pasqualone et al., 2007; Testolin & Lain, 2005; Vietina, Agrimonti, Bonas, Marmiroli, & Marmiroli, 2011). Presence of adulterants in the olive oil was assessed by Wu et al. (2008) with an olive specific sequence within the gene PIP coding for a membrane intrinsic protein, while Zhang et al., 2009 found that the gene MT3-B can differentiate palm oil from other oil samples. Spaniolas, Bazakos, Awad, and Kalaitzis (2008) identified a highly polymorphic region within the chloroplast gene trnL (UAA) intron that can be used to discriminate eight out of the ten oil-producing species, with a single PCR reaction. Wu et al. (2011) developed a PCR-CE-SSCP (Polymerase Chain Reaction-Capillary Electrophoresis-Single Strand Conformation Polymorphism) method to detect differences in DNA profile of se- ven oil species. High resolution melting (HRM) allows to discriminate DNA with only one base difference and constitutes an alternative to capillary electrophoresis for varietal fingerprinting, not only in plants but also in foods (Ganopoulos, Argiriou, & Tsaftaris, 2011; Mackay, Wright, & Bonfiglioli, 2008). HRM differs from standard SYBR Green dye melt curve analysis in three ways: (i) saturating and brighter double-stranded DNA binding dyes, in particular SYTO Ò 9, are used; in fact, with traditional dyes saturation of PCR products is not guaranteed because only limited concentrations of dye can be used before it inhibits the PCR; (ii) instruments performing HRM collect more data point than for standard melt curves; (iii) soft- ware uses new fluorescent normalisation algorithms and plots (Graham, Liew, Meadows, Lyon, & Wittwer, 2005; Reed, Kent, & Wittwer, 2007). This work describes the development and evaluation of a DNA based analytical method for identification of adulterant oils in olive oil using Real-Time PCR and HRM post PCR analysis. Both oils from single plant species and mixtures were successfully tested and resolved. 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.06.075 ⇑ Corresponding author. Address: Department of Life Sciences, University of Parma, Parco Area Delle Scienze 11/A, 43124 Parma, Italy. Tel.: +39 0521905606; fax: +39 0521905402. E-mail address: nelson.marmiroli@unipr.it (N. Marmiroli). 1 These authors contributed equally to this work. Food Chemistry 141 (2013) 3820–3826 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem