Dipstick test for DNA-based identification of olive oil cultivar origin

Introduction: We describe the first DNA biosensors for the identification of the cultural variety of olive oil, which is an important agricultural product. Because the varietal origin affects the quality characteristics, monovarietal olive oils with protected designation of origin are the focus of consumers and producers. Contrary to other tedious and costly methods, such as real-time PCR with fluorometric detection and capillary electrophoresis, the proposed biosensors enable visual detection by the naked eye without sophisticated instrumentation. Also, the proposed method is based on unique DNA markers rather than small metabolites and is therefore not affected by environmental/climatic changes, geographic origin, soil composition, harvesting period and storage conditions.

Methods: We performed simultaneous genotyping of four single-nucleotide polymorphisms corresponding to eight alleles. The steps are DNA extraction, quadruplex PCR of cycloartenol synthase and lupeol synthase, octaplex genotyping by DNA polymerase (20 min) and application to the biosensor without prior purification. The biosensor is immersed in developing buffer and the detection requires 20 min.

Results: A red spot appears when an allele is present. The combination of red spots from eight alleles provides the genetic identity. The biosensors were applied, accurately and reproducibly, to the identification of seven olive varieties using DNA isolated from leaves and olive oil. The results were in full concordance with sequencing data. As low as 5 ng of extracted olive oil DNA can be used for PCR. The CVs were in the range of 0.5–9%.

Conclusions: Because of their simplicity and practicality, we anticipate that the proposed biosensors will find wide applicability in determining the authentication and traceability of olive oil.

Acknowledgements: This research has been co‐financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program ‘Competitiveness, Entrepreneurship and Innovation’, under the call ‘RESEARCH – CREATE – INNOVATE (project code: T2EDK-02637)’.