The sample referred to the Government Chemist for advice and analysis was a commercially available paprika-based sample (assumed to mainly comprise Capsicum annuum), which was suspected of also containing material derived from Prunus species.

A representative panel of control materials were sourced from commercial suppliers. Standard/smoked ground paprika (Capsicum annuum), organic cumin seeds (Cuminum cyminum), ground mahaleb (Prunus mahaleb) and almond (Prunus dulcis) were purchased from online retailers; apricot stone (Prunus armeniaca, stone taken from whole fruit) and peach stone (Prunus persica, stone taken from whole fruit) were purchased from UK supermarket stores.

Species identity of these control materials were independently confirmed using PCR sequencing primers annealing to the internal transcribed spacer (ITS 2) and ribulose bisphosphate carboxylase long chain (rbcL) regions of the nuclear and chloroplast genomes respectively, followed by Sanger DNA sequencing of the resultant PCR products and sequence confirmation using the Barcode Of Life Database (BOLD) [24] and GenBank^® [25] databases (data not shown).

Unless pre-ground, the materials were ground to a fine powder using an IKA Tube Mill control (IKA^®-Werke GmbH & Co. KG, Germany) and DNA extracted from 100 mg samples using a modified CTAB method [26] . Extracted DNA was quantified using a NanoDrop^TM ND-1000 spectrophotometer (Thermo Scientific, UK).

A general Prunus species real-time PCR-based assay was adapted from a published Prunus mahaleb specific assay [14] targeting the conserved internal transcribed space (ITS) region of Prunus species using a Prunus mahaleb ITS sequence (#JQ776826) representative of the genus. The assay was developed using Primer Express 3.0.1 (Life Technologies, Paisley, UK) under standard “Taqman^® Quantitation” type assay design parameters and specificity evaluated using Primer-BLAST [27] .

Twenty-five μl reactions were prepared comprising 12.5 μl 2x Fast Plus EvaGreen^® qPCR Master Mix, High ROX™ (Biotium, Inc., Hayward, USA), 900 nM PRU_ITS_FWD1 (5’-TAGCAGAACGACCCGAGAAC- TAG-3’) forward and 900 nM PRU_ITS_REV1 (5’-CGCCGGTGTTCGTTTGTAC-3’) reverse primers. HPLC purified primers were sourced from Eurofins Genomics (Ebersberg, Germany). The reactions were made up to 20 μl with DNase/DNA-free water (Ambion brand, Life Technologies, Paisley, UK) and 5 μl of the template DNA/water control was added to each reaction as appropriate, to bring the total volume to 25 μl. Template DNA input was normalised (where possible) to 50 ng for each test sample.

Real-time PCR was performed under 2-step thermal cycling conditions (10 min/95˚C; 15 s/95˚C, 1 min/60˚C, 45 cycles) incorporating a melt analysis step (15 s/95˚C; 60˚C to 95˚C, 1.75˚C/minute ramp rate) using a 7900HT Fast Real-Time PCR System (Life Technologies, Paisley, UK) set to monitor FAM-based fluorescence. The data was analysed using SDS 2.4.1 software (Life Technologies, Paisley, UK) using automated baseline and threshold settings.

The melt analysis methodology was adapted from an in-house developed hydrolysis probe-based real-time PCR assay [14] targeting the Prunus mahaleb internal transcribed spacer (ITS) sequence (GenBank#JQ776826). GenBank^® [25] Entrez Nucleotide sequence database searches and ClustalW [28] alignments showed conserved and variable regions with related Prunus species which supported the application of the assay to species differentiation by melt analysis.

A preliminary evaluation study was conducted that challenged the DNA-binding dye-based real-time PCR assay with a panel of commonly found Prunus species, cumin and paprika control materials, and a paprika-based commercial sample referred to the UK Government Chemist for further analysis. Immunological methods comprising several ELISA commercial kits, indicated the presence of an unidentified Prunus species within the referred sample at an approximate concentration of 500 mg kg⁻¹ expressed as almond (data not shown).

Our results demonstrate that the real-time PCR assay described herein is capable of detecting the Prunus species mahaleb, almond, apricot and peach and generates melt profiles capable of distinguishing between these species (Figure 1(a) and Table 1). The paprika-based referred sample generated a positive amplification response as shown by ~24 Cq value (Table 1) and a well-defined multiple melt peak profile (Figure 1(d)). The second peak (~88˚C) most closely matched almond DNA due to the similarity of T_m values. Analysis of the 10% w/w mahaleb/almond in paprika DNA admixtures strongly supports this conclusion as the primary peak (consistent with almond) shows a mean T_m 88.07˚C (Table 1 and Figure 1(d)) which is identical to the minor peak observed in the referred paprika sample with overlapping confidence intervals.

However the lower T_m melt peak (~82.6˚C) does not appear to correlate with any of the tested Prunus control materials within this study and appears to represent a slightly higher concentration than that of the almond present. Hence, a primary unidentified peak has also been observed in the melt profile of the referred paprika sample that is indicative of a Prunus species that was unrecognised by immunological analysis, and which requires further study to fully characterise. In-silico analysis of the ITS region, also borne out through the experimental work, indicated that this primary peak is unlikely to arise as a result of multiple copies of the ITS region. Clonal sequencing of this primary peak may provide further information on the nature of the product and if this pertains to a particular Prunus species. However, this was not investigated further as part of the current study,

which had already shown the paprika-based sample to be non-compliant with relevant food labelling legislation as there was a very high likelihood that it contained almond which was not listed as an ingredient.

As is often observed in DNA-binding dye-based real-time PCR assays, the negative extraction and negative template controls showed late amplification (>37 cycles) and poor low T_m peaks (~75˚C) that are characteristic of primer artefact features, particularly primer-dimerisation (Figure 1(b)) [29] . These primer artefacts are absent when sufficient Prunus template DNA is provided in a sample, as evidenced by the controls of mahaleb, apricot, peach and almond, and the primer artefacts are also absent in the referred sample and the 10% w/w mahaleb/almond in paprika DNA sample.

Paprika and cumin control materials were included as comparators for the paprika-based referred sample and showed late amplification (Table 1). These paprika and cumin control samples also demonstrated some evidence for low melt peaks characteristic of primer artefacts (Table 1 and Figure 1(c)). The low level multiple peaks observed around the 85˚C and 88˚C regions were inconsistently formed and represent possible trace levels of amplifiable targets. These may be potentially due to either limited assay specificity issues or more likely the presence of trace Prunus species contamination in the control samples because of the manufacturing/processing techniques typically used in spice manufacture. The C_q values and the magnitude of these melt peaks (when observed) are lower in the paprika and cumin control samples compared to the Prunus control materials and the referred sample, further reinforcing the possible trace level detection of these elements. The peanut control material showed characteristic primer artefacts but no evidence of other melt peaks, further supporting the premise that a lack of Prunus template DNA often produces observable primer artefacts.

The results indicate that a melt profile has been generated from the paprika-based referred sample which is consistent with almond being present. A second Prunus like species has been detected (estimated to be at a higher concentration to the potential almond content), whose profile is not consistent with the control materials used to represent the species of mahaleb, almond, peach or apricot in the current study. In addition, the study highlights the potential trace contamination of spices with non-labelled Prunus species such as almond which may represent an allergenic risk to certain individuals and warrants further investigation to determine the scale of the issue.