A digital PCR approach to assess the purity of oregano

Abstract

Herbs and spices are food categories known to be at high risk of adulteration. Presence of undeclared foreign plant species has often been reported in oregano and may have a direct impact on its organoleptic quality and potentially the safety of this aromatic herb. A droplet digital PCR approach was developed to assess the purity of oregano by quantifying the DNA copies of oregano versus the total plant DNA copies. Nuclear single-copy genes were selected by targeting the terpene synthase 5 gene from oregano and the plant phosphoenolpyruvate carboxylase 2 gene. The reactions were specific to the Origanum genus and plant materials respectively, whereas trueness and precision data confirmed the reliability of the method to quantify oregano. The applicability of the method was further verified on proficiency test samples before being applied on commercial oregano samples.

Keywords: DNA; Digital PCR; Herbs; Oregano; Purity.

Fig. 1

Snapshots of nucleotide alignments from plant terpene synthase (A) and phosphoenolpyruvate carboxylase (B) genes obtained with the BLASTn MSA viewer when submitting O. vulgare TPS5 mRNA sequence (GenBank Accession no. GU385971) and Salvia splendens PEPC2 mRNA sequence (GenBank Accession no. XM_042164716), respectively. Conserved nucleotides are indicated with grey dots, whereas nucleotide polymorphisms are highlighted in red. Primers are indicated with arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Gradient ddPCR between 55 °C and 60 °C on pure oregano leaf DNA (A) and amplification at 58 °C on various plant species (B) using Oreg-TPS5 (upper graph) and Plant-PEPC2 (lower graph) primers. Positive and negative droplets are indicated in blue and grey, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Linear correlation between the theoretical number of plant pepc2 and oregano tps5 copies in oregano haploid genome (X-axis) and the number of copies measured by ddPCR (Y-axis). Plant pepc2 and oregano tps5 data are represented with blue squares and orange circles, respectively. All dilutions were tested in triplicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)