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

Abstract

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/µL. Corresponding to the very low LOD (0.00031 ng/µL) 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 Δ 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.

Figure 1

Intraspecific variability of hemp varieties with the numbers 1 and 21–101 (A) and with the numbers 4–20 and 107–128 (B) according to Table 1, obtained with 5 ng/µl DNA per tube. Results of Grubbs outlier and Shapiro-Wilks test (C).

Figure 1

Intraspecific variability of hemp varieties with the numbers 1 and 21–101 (A) and with the numbers 4–20 and 107–128 (B) according to Table 1, obtained with 5 ng/µl DNA per tube. Results of Grubbs outlier and Shapiro-Wilks test (C).

Figure 2

Results of determination of limit of detection (LOD) with digital droplet PCR, using DNA extracts from Novosadska seeds in concentrations from 2.5 ng/µL to 4.77 × 10–6 ng/µL.

Figure 3

Results of cross-reactivity tests obtained with 5 ng/µl DNA per tube. Cross-reacting species: mulberry, stinging nettle, lavender, cornflower, wine, figs and hops. The cross-reactivity is negligible, because the ∆ Ct values are > 14 and the Ct values are beyond the determined limit of detection.

Figure 4

Results of analysing diverse foodstuff obtained with 5 ng/µl DNA per tube. Hempseeds were analysed as reference material.

Figure 5

(A) Position of primers and probe presented in the spacer DNA sequence between the trnL 3´exon and the trnF gene in Cannabis sativa chloroplasts. The probe HempS_19, marked in red, discriminates hemp from hops based on a SNP in position 603. The primer pair Hemp_19 Fw/Rv is marked in green. (B) Amplification curves obtained with tested primer and probe concentrations by analysing Monoica seeds with DNA concentrations of 100 ng/µl, 25 ng/µl, 6.25 ng/µl and 1.5625 ng/µl. (1) Purple: 0.5 µmol/L per primer, 0.1 µmol/L probe. (2) Light blue: 0.5 µmol/L per primer, 0.25 µmol/L probe. (3) Rose: 0.25 µmol/L per primer, 0.25 µmol/L probe. (4) Green: 0.25 µmol/L per primer, 0.1 µmol/L probe.