A digital PCR approach to assess the purity of oregano.

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.

A DNA Metabarcoding Workflow to Identify Species in Spices and Herbs.

BACKGROUND: Spices and herbs are food categories regularly cited as highly susceptible to be adulterated. To detect potential adulteration with undeclared species, DNA-based methods are considered the most suitable tools. OBJECTIVE: In this study, the performance of the ready-to-use Thermo Scientific™ NGS Food Authenticity Workflow (Thermo Fisher Scientific)-a commercial DNA metabarcoding approach-is described. The tool was further applied to analyze 272 commercial samples of spices and herbs. METHOD: Pure samples of spices and herbs were analyzed with the Thermo Scientific NGS Food Authenticity Workflow to assess its specificity, and spikings down to 1% (w/w) allowed evaluation of its sensitivity. Commercial samples, 62 and 210, were collected in Asian and European markets, respectively. RESULTS: All tested species were correctly identified often down to the species level, while spikings at 1% (w/w) confirmed a limit of detection at this level, including in complex mixtures composed of five different spices and/or herbs. The analysis of 272 commercial samples showed that 78% were compliant with the declared content, whereas the rest were shown to contain undeclared species that were in a few cases allergenic or potentially toxic. CONCLUSIONS: The Thermo Scientific NGS Food Authenticity Workflow was found to be suitable to identify food plant species in herbs and spices, not only when tested on pure samples, but also in mixtures down to 1% (w/w). The overall workflow is user-friendly and straightforward, which makes it simple to use and facilitates data interpretation. HIGHLIGHTS: The Thermo Scientific NGS Food Authenticity Workflow was found to be suitable for species identification in herbs and spices, and it allowed the detection of undeclared species in commercial samples. Its ease of use facilitates its implementation in testing laboratories.

A new real-time PCR assay to specifically detect crustaceans in vegan raw materials and vegan shrimps.

Contamination of vegan products and vegan shrimps with real shrimps and crustaceans must be avoided to comply with vegan claims, even more for preventing strong allergenic reactions. Therefore, the detection of crustaceans must be reliable and sensitive enough for authenticity, traceability, and food safety purposes. A new real-time PCR assay was developed targeting the mitochondrial 16S rRNA gene of crustaceans and was optimised to avoid critical mismatches with primers and probe. By testing several crustacean species and common food ingredients, the method was demonstrated to be specific to crustaceans only. To comply with the limit of non-vegan contamination established at 0.1% (w/w) by the European Vegetarian Union, dedicated cut-off CT values were determined on vegan raw materials and on vegan shrimps spiked with crustacean materials. The method reached a sensitivity ≤ 0.0005% (w/w), which was further confirmed on reference materials containing a similar amount of crustacean.

Performance assessment of digital PCR for the quantification of GM-maize and GM-soya events.

Accurate quantitative methods are needed to determine the amount of transgenic material in ingredients and comply with labelling GMO thresholds. Quantitative real-time PCR methods are usually applied for GMO quantification, but since a few years, digital PCR (dPCR) has been described as a potential alternative by quantifying DNA molecules directly without any standard curves. In this study, the performance of dPCR to quantify 9 GM-soya events and 15 GM-maize events was assessed. Following GMO validation guidelines, the trueness and precision were determined on high, medium and low levels of transgenic content. Results showed biases below ± 25% and satisfactory precision data. Limits of quantification were determined for each GM-event and were between 12 and 31 target copies. The reliability of GMO quantification by dPCR was further confirmed by analysing several proficiency test samples. Overall, dPCR showed accurate and precise GMO quantification on all the tested GM-events, from high to low transgenic amount. With its ease-of-use, dPCR was found to be an appealing alternative technology for routine GMO testing laboratories. Graphical abstract.

Two FAST multiplex real-time PCR reactions to assess the presence of genetically modified organisms in food.

Methods to detect the presence of genetically modified organisms (GMOs) are evolving constantly to comply with legislation and include new transgenic traits developed by biotechnology companies. Since traditional screening methods target only a limited number of markers, not all GM-events can be detected easily. To cover a broader range, a new GMO screening method was developed, based on two real-time PCR reactions, targeting i) six major GM-markers and ii) six GM-events that do not contain these markers. This method used the FAST PCR technology and allowed a further reduction in time-to-result. Using a wide array of reference materials and proficiency test samples, the method showed a broad screening capacity and the absolute limit of detection was consistently below 20 copies, thereby demonstrating the method is fit-for-purpose.

Food Adulteration: From Vulnerability Assessment to New Analytical Solutions.

Crises related to the presence of melamine in milk or horse meat in beef have been a wake-up call to the whole food industry showing that adulteration of food raw materials is a complex issue. By analysing the situation, it became clear that the risk-based approach applied to ensure the safety related to chemical contaminants in food is not adequate for food fraud. Therefore, a specific approach has been developed to evaluate adulteration vulnerabilities within the food chain. Vulnerabilities will require the development of new analytical solutions. Fingerprinting methodologies can be very powerful in determining the status of a raw material without knowing the identity of each constituent. Milk adulterated by addition of adulterants with very different chemical properties could be detected rapidly by Fourier-transformed mid-infrared spectroscopy (FT-mid-IR) fingerprinting technology. In parallel, a fast and simple multi-analytes liquid-chromatography tandem mass-spectrometry (LC/MS-MS) method has been developed to detect either high levels of nitrogen-rich compounds resulting from adulteration or low levels due to accidental contamination either in milk or in other sensitive food matrices. To verify meat species authenticity, DNA-based methods are preferred for both raw ingredients and processed food. DNA macro-array, and more specifically the Meat LCD Array have showed efficient and reliable meat identification, allowing the simultaneous detection of 32 meat species. While the Meat LCD Array is still a targeted approach, DNA sequencing is a significant step towards an untargeted one.

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.