Development and validation of a real-time PCR assay to detect Cannabis sativa in food

Regarding the prospective investigation of food authenticity and adulteration the aim of the present study was the development and validation of a real-time PCR assay to identify hemp (Cannabis sativa) which has gained increasing importance as a valuable food ingredient. The assay targets a specific spacer DNA sequence in Cannabis sativa chloroplasts and detects 1.5 pg hemp DNA, which is equivalent to 18 copies/mu L. Corresponding to the very low LOD (0.00031 ng/mu L) the method allows the detection of hemp even in the infinitesimal concentration of contaminants. Due to a SNP in position 603, hemp can be identified unequivocally and discriminated from its closest relative hops (Humulus lupulus). The PCR method shows no cross-reactivity with 39 of 46 tested plant species. Low cross-reactivity with mulberry, stinging nettle, lavender, cornflower, wine, figs and hops can be neglected, because the Delta Ct-values are>14, and the obtained Ct-values are beyond the cut-off for a positive assessment (Ct-values <= 33). Moreover, the suitability of the method to identify hemp as a food ingredient was proved by analysing diverse food products such as chocolate or cookies.

Automated summary

In this study, a real-time PCR assay was developed to accurately identify hemp as a food ingredient, targeting a specific DNA sequence to detect hemp in even trace contaminants and distinguish it from its closest relative. The method showed no cross-reactivity with the majority of tested plant species, making it a reliable tool for hemp detection in food products.