Novel quantitative real-time PCR approach to determine safflower (Carthamus tinctorius) adulteration in saffron (Crocus sativus)

•DNA-based methods to detect and quantify safflower as an adulterant in saffron.•ITS region as a DNA marker for safflower detection.•Normalised quantitative real-time PCR to estimate safflower in the range of 0.1-20%.•Successful validation and application of method to 19 saffron/seasoning samples.•Safflower detected in 3 samples at levels suggesting cross-contamination.

This work intended to develop DNA-based methods to detect and quantify safflower as an adulterant in saffron. Species-specific PCR and real-time PCR with EvaGreen dye targeting the ITS region of Carthamus tinctorius L. (safflower) were successfully proposed. The assays allowed absolute and relative sensitivities of 2 pg of safflower DNA (∼1.4 DNA copies) and 0.1% of safflower in saffron (Crocus sativus L.), respectively. A normalised real-time PCR approach was also proposed in the range of 0.1-20% (w/w) of safflower in saffron, which was successfully validated and applied to commercial saffron samples (stigmas, powders and seasonings). From 19 samples, three were positive to safflower, though at levels below the limit of detection, suggesting cross-contamination rather than adulteration. In this work, specific, sensitive and accurate tools were proposed to authenticate saffron. To the best of our knowledge, this is the first successful attempt to quantify safflower by a DNA-based approach.