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In this study (open access) the researchers used a panel of three different DNA test protocols to verify the labelled species in dairy products sampled from Greek supermarkets over the winter of 2024.  They tested 74 samples in total encompassing cow, sheep and goat products. This included 15 different commercial brands of goat yoghurt, 7 brands of sheep yoghurts, 3 brands of goat kefir, and, samples of feta cheese and sheep-goat cheese from 16 to 11 different geographical origins and 7 brands of goat cheese were analysed.  The brands are anonymised within the publication.

They report widespread adulteration, particularly in goat yogurts (40 %) and cheeses (40 %), as well as in three kefirs and several mixed and whey-based cheeses. Notably, only 7 out of 17 - nominally goat - feta samples contained detectable goat DNA..

Image from the publication

In this survey a total of 107 tuna cans were collected between 2020 and 2022 from 7 different Portuguese commercial brands.  Samples were purchased from main supermarket chains, large-scale distributors and local stores.  Samples were categorised by the quarter of the year when the tuna was canned (inferred from the expiry date) with each sample was tested in triplicate.  Extracted DNA was purified and iteratively tested using molecular metabarcoding methods.

The researchers report (open access) that the occurrence of different species was observed only in products canned in brine or water (i.e. all products canned in oil were Skipjack Tuna). Skipjack tuna was predominant across all canning liquids and brands analysed. Nonetheless, other species like Thunnus obesus and T. albacares, or Auxis spp. (not considered true tuna) were also detected. The use of different species was limited to cans produced during the second quarter of the year, which could reflect differences in seasonal availability of different tuna species or in sourcing strategies/market preferences of each company. For four brands, multiple species were detected inside the same can.  This violates current European legislation.

The researchers conclude that these results provide the first broad assessment of species used in the Portuguese tuna canning industry and showed the inclusion of vulnerable species is limited.

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Species identification in canned tuna is much more challenging than for processed fish in most cooked foods.  This is because the DNA is substantively degraded during the canning process.

In this paper (purchase required) the authors present a protocol to increase concentration and purity of DNA extracted from canned samples. The experiment mainly consists of: (1) drying the canned tissue in paper filter, (2) washing it with a PBS solution, (3) store in ethanol 96 % at −20°C, and (4) perform DNA extraction.

They report that the pre-treated samples showed an increase of both DNA concentration and purity indicating that some of the inhibiting molecules were successfully removed. These differences between the two treatments were statistically significant (p < 0.01). At the amplification level, the pre-treatment allowed the recovery of complete fragments of the barcode region COX1 with approximately 650 base pairs.

The authors recommend their approach should be used in combination with other methodologie such as mini-barcoding.

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This research (purchase required) set out to design a rapid point-of-use test to detect cassava starch as an adulterant in higher-value starches.  The test method used Proofreading enzyme-mediated probe cleavage (Proofman) coupled with ladder-shape melting temperature isothermal amplification (LMTIA). The optimal detection temperature of this Proofman-LMTIA method was 62℃ and the reaction could be finished within 20 minutes with a detection sensitivity of 100 pg/μL of genomic cassava DNA. Nine different species were collected and verified for the specificity of cassava ITS primers and probes. The detection limit of cassava DNA derived from artificially premixed starch powders was 1 % (w/w).

As a proof-of-concept, the researchers used their Proofman-LMTIA assay to test 33 commercial products from the food and medicine sectors.  They report that – based on their assay -  16 samples contained undeclared cassava components.

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The canning process has a degrading effect on DNA, making the species verification of canned tuna more challenging than for raw fish.

In this paper (open access) the authors optimised and compared three PCR approaches: real-time PCR (RT-PCR), mitochondrial control region (CR) mini-barcode, and multiplex PCR.  They tested 24 samples labelled as either albacore, yellowfin, skipjack or light tuna.

They reported RT-PCR as having the highest identification rate (100%), followed by CR mini-barcoding (33%) and multiplex PCR (29%). They consider that the success of RT-PCR may have been due to the short (<100 bp) DNA fragments targeted. In comparison, multiplex PCR and CR mini-barcoding targeted slightly longer fragments of 127–270 and∼236 base pairs, respectively. Regarding species identification, CR mini-barcoding and multiplex PCR confirmed the presence of albacoreor yellowfin tuna in several samples; however, both methods struggled with the identification of skipjack tuna.

CR mini-barcoding enabled sequencing-based detection of a range of species in the products. The authors conclude that a combination of real-time PCR and CR mini-barcoding is the optimum approach for rapid screening of target species along with sequencing-based confirmation.

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Atlantic salmon in processed food is prone to substitution with cheaper species of similar appearance such as rainbow trout, chum salmon or masou salmon.

In this paper (open access) the authors developed a sensitive and visual PCR-based CRISPR/Cas12a detection method to identify Atlantic salmon ingredients in processed foods. A guide RNA (gRNA) was designed based on the mitochondrial genome of Atlantic salmon, with an efficient target site identified within the ATP synthase F0 subunit 6 (ATP6) gene.

Specificity was tested against 9 other species commonly associated with Atlantic salmon adulteration.  There was no evidence of cross-reactivity. 

The authors report that theirassay exhibited an absolute detection limit of 0.5 pg and a relative sensitivity of 0.1 %. The results were visually interpretable under UV light without the need for complex instrumentation. They cross-checked their results through Sanger sequencing during the authentication of commercial products.

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One of the limiting factors in DNA analyses, in terms of both the time taken and the need to send samples to a laboratory for testing.  There are a number of modern point-of-use technologies that circumvent the need for amplification (see FANs methods explainers).  Currently these cannot compete on price-per-test with “traditional” laboratory-based Polymerase Chain Reaction amplification methods.

In this paper (purchase required) the authors have developed a novel point-of-use biosensor that can detect trace levels of different species' DNA in parallel (“multiplex”).  They conducted proof of concept for low-level meat species contamination in complex food matrices.  The sensor is based on Surface Enhanced Raman Spectroscopy (SERS – a technique that has been used for sensors to detect clinical markers in biological samples).  The authors have enhanced the technique by using argonaute endonuclease coupled with guide DNA to specifically cleave the target nucleic acids and maximise the signal.  The system is programmable, and the authors report that controllable polystyrene nanoparticles encapsulating SERS probes significantly improved detection sensitivity.

This paper (open access) reports the results of an authenticity testing survey of insect-containing food and feed products, purchased both within and outside the EU.

119 commercial products were tested for the declared insect species, using two DNA-based methods, real-time PCR and metabarcoding,. All samples (test portions of 100 mg) were extracted following the method recommended by the European Union Reference Laboratory for Animal Proteins in Feedingstuffs

The authors report that 50% of the products contained insect species not listed on the label, or lacked the species that were declared. In particular, cross-contamination was observed when manufacturers worked with more than one type of insect.  Some products contained insects that are not currently allowed for use in the European Union. Some insect meals also contained traces of animal DNA, which may come from the substrates the insects were raised on. The authors point out that this could cause legal problems if these meals are used in certain types of animal feed.

The authors conclude that their study highlights the need for better quality control in the insect production chain. It also shows that DNA tests are useful tools for authenticating the declared insect species in food and feed products.

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This work (open access) provides genetic marker information and proposes a standard method to support the regulatory classification of premium rice varieties including Basmati, Jasmine, Sushi and Risotto.  It builds on previous reference data sets provided by the same research group (including the University of Bangor, one of FAN’s Centre of Expertise laboratories).

Updated DNA fingerprinting was done for reference samples of 158 commercial rice varieties from 14 countries, collected since 2004. Most samples were obtained directly from the appropriate regulatory body in each jurisdiction, with provenance further substantiated by genetic cluster analysis.

DNA fingerprinting based on 10 SSR (Simple Sequence Repeat) markers was introduced in the early 2000s for authenticity testing of Basmati rice. Subsequently the addition of 5 SSRs and the fragrance gene fgr have refined the method for routine use.

This new study evaluated the applicability of the 15-SSR method for authenticity testing of more diverse types of commercially relevant rice that are traded on an international scale. The extensive range of reference samples covered this commercial scope. Most varieties were found to have distinct marker profiles except for eight near isogenic lines and eight closely related traditional varieties. The fgr marker detected several non-fragrant varieties that were incorrectly labelled as Jasmine fragrant rice, one of which was listed as fragrant and tariff-exempt in the EU Viet Nam Free Trade Agreement.

To assess the authenticity of samples obtained from unofficial sources in the trade, UPGMA algorithm and Principal Coordinate Analysis (PCoA) were used for marker-based clustering of samples. Most of the unofficially sourced samples clustered according to their expected geographical and genetic origin, supporting their authenticity. The study supports the broader utility of this 15-SSR test, supplemented by the fgr marker, for global rice variety authentication.

The authors conclude that their proposed markers are ideal to underpin ond enforce industrial, legal and free trade agreement standards.

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This study (purchase required) reports development of a Loop-Mediated Isothermal Amplification (LAMP) assay to detect several common avian meat species as adulterants in raw and heat- and pressure-treated meat products. This is an on-site test, taking about 1 hour, with the results visualised by colour changes in the SYTO 24 nucleic acid marker dye.

Conserved regions of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene were targeted to design a LAMP primer set specific to avian species. To assess the assay’s performance, six common avian species (chicken, turkey, goose, duck, ostrich, quail) and four non-avian species (sheep, cattle, goat, camel) were tested. DNA was extracted using a salt-based method, and the assay’s specificity and sensitivity were evaluated on raw, cooked, and autoclaved samples.

The authors report that the LAMP assay successfully detected chicken, turkey, goose, and duck DNA. They report detection limits of 110 femtograms chicken DNA In chicken–beef mixtures, 0.1 % chicken in raw and cooked samples and 1 % in autoclaved samples.

For the principle of LAMP, see FAN’s method explainer pages.

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This study (purchase required) reports the development of a novel recombinase aided amplification (RAA) assisted Cas12a assay to authenticate the commercially important Pacific oyster.

The COI gene was selected as a genetic marker for primer design. The authors report that the developed species-specific RAA assay was optimal at 40 °C for 25 min. The Cas12a assay successfully detected the target Pacific oyster DNA sequence in RAA products using 0.05 μM gRNA and 0.05 μM Cas12a enzyme within 40 min at 37 °C. The developed RAA primers and gRNA for CRISPR-Cas12a assay showed no cross-amplification and high specificity for C. gigas compared with C. belcheri and C. iridalei. The sensitivity test showed the ability of the assay to detect DNA concentrations as low as 10 fg/reaction. In addition, the developed assay successfully authenticated oyster samples in all processed forms, including boiled, steamed, fried, and canned samples.

A small follow-up survey found that 1 of the 15 commercial samples tested was mislabeled.

The authors conclude that the developed assay was a valuable technique with high potential for food safety authorities and stakeholders in ensuring authenticity, in which substitution and adulteration of seafood products can be detected.

The  aim of this proof-of-principle study (open access) was to design a universal DNA microarray (“DNA Chip”) to distinguish all edible fish species by comparing hybridization signal patterns from samples with patterns obtained from reference specimens.

The researchers designed a universal set of 96 DNA probes that cover all fish species of food interest.  These were narrowed down by virtual modelling experiments from a long-list of 28,000 candidates which they had generated experimentally. They also included 4 control probes (sequences not present in edible fish).  All probes were based on sequences from either 16S ribosomal RNA or cytochrome b.

DNA was isolated with either a CTAB method or with commercial DNA extraction kits. The gene markers cytb (approx. 464 bp) and 16S rDNA (approx. 600 bp) and an additional pUC57 vector DNA region (542 bp) were amplified in triplex PCRs. The DNA probes were spotted contactless using piezoelectric dispensing technology as 19 × 19 arrays. For hybridization of the generated PCR amplicons on the prepared microarrays the INTER-ARRAY Hybridization Kit was used according to manufacturer's specifications. The arrays were measured directly after staining and then processed using the INTER-VISION GENOTYPING 1.2.0 software.

The authors tested 86 fish fillets sourced from verified suppliers and were able to correctly identify all species by hierarchical clustering analysis of the results.  The entire process takes a few hours.  They conclude that the method is ready for further validation and ruggedness testing. More replicates and species should be analyzed to confirm current results. Likewise, the robustness of the DNA array should be determined, e. g. by using different thermocycler or users and laboratories.

Graphical abstract from the paper

DNA analysis is rarely used for the verification of edible oil species, because of the low amount of intact DNA in the refined oil and the genetic similarities between different oil varieties.  In this study (open access pre-print, not yet peer-reviewed) the authors compared different DNA extraction kits and PCR protocols and new genetic markers to try and resolve the issue.  They reported that DNA extraction kits such as NucleoSpin Food, DNeasy mericon Food, and Olive Oil DNA Isolation as well as modified CTAB method were found to be able to isolate amplifiable genomic DNA from highly processed oils. Novel uniplex, double, and nested PCR systems targeting the sunflower-specific helianthinin gene were developed for efficient identification of sunflower. New sunflower DNA markers were revealed by uniplex PCRs.

They concluded that a combination of modified CTAB and nested PCR gave the best performance, and was demonstrated as a reliable, rapid, and cost-effective technology for detecting traces of sunflower in highly processed oil, including refined and used cooking oil.

Synthetic technologies allow companies to ‘print’ DNA and RNA.  Applications are cross-sector, and include nucleic acids that are used as the basis of selective analytical test methods.  This voluntary best-practice guidance emphasises the UK government’s intent for a pro-innovation culture in the engineering biology ecosystem through providing well-defined guardrails for customers and producers of synthetic nucleic acid.

This link has been added to FAN’s Quality page, which contains links to a range of other best-practice guidance for both laboratories and for customers looking to choose a laboratory

Highlights

Earlier this year, the Food Standards Agency (FSA) commissioned the UK National Reference Laboratory for GMOs based at LGC (Teddington), to deliver a desk-based review of the current state-of-the-art associated with methods for the potential detection of (PBOs) in the food and feed supply chains.

Precision Bred Organisms represent organisms which possess genetic variability resulting from the application of modern biotechnology, which could also have arisen through traditional processes. In March 2023 the Genetic Technology (Precision Breeding) Act was passed in the UK, which brought forward primary legislation to amend the regulatory definition of a Genetically Modified Organism (GMO), to exclude from it those organisms that have genetic changes that could also have arisen through traditional processes.

The report, written by Malcolm Burns and Gavin Nixon, captures use of sector specific terminology and related international developments. A focus is given on the current scope and challenges for the analytical detection of specific DNA sequences alongside supportive traceability tools inclusive of reference materials and databases. The report provides a series of recommendations towards helping develop a framework for the traceability of PBOs as well as some of the future analytical challenges this presents.

The report will be of interest to scientists and analysts involved in developing molecular biology assays for the detection of small DNA sequence changes, government departments and related stakeholders involved in assessing the efficacy of methods for the traceability of PBOs, as well as to a broader audience (e.g., academia, industry, retailers, etc.,) who are interested in some of the scope and challenges that detection of PBOs may present.

The report can be found here and a pdf version here. 

Telemeres are genetic book-ends that cap the ends of coding sequences within chromosomes.  Their length is chipped away every time DNA replicates.  Telemeric length is an indicator of an animals age but can also be suggestive of stress or environmental conditions. 

An interesting review article has been published that examines telomeric length as potential verification for fish authenticity descriptions such as organic vs conventional rearing or wild-caught vs aquaculture.

Read abstract  

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A new POSTnote on genome edited food crops has been published by the Parliamentary Office of Science and Technology with contributions from Dr Malcolm Burns (Head of LGC's GMO unit), Dr Julian Braybrook and our Executive Director, Selvarani Elahi MBE.

◼ The Government is proposing that genomeedited crop plants are exempted from Genetically Modified Organisms (GMOs) regulations, provided the genetic changes could occur naturally or via existing conventional breeding techniques.
◼ Genome editing can manipulate DNA atspecific positions in the genome to shorten timeframes for plant breeding of useful traits. This process can lead to unintended alterations of the genome, but these may be fewer than for conventional breeding.
◼ Some stakeholders believe this regulation change for genome-edited food crops could provide health and environmental benefits and make use of UK-funded research.
◼ Key issues for public acceptance and trust of genome-edited crops are tightly bound to transparency and how the public view potential risks and benefits.

Read the full POSTnote.

One of the frequently encountered types of adulteration is the adulteration of meat and animal products. In its most recent annual report [1] , the Food Fraud Network showed data that in the top ten product categories, fish and fish products take the second place, meat and meat products the third and poultry the fifth. Jointly, these three animal product categories eclipse any other product category.

There are different types of fraud that can be found in animal products. These include addition of illegal substances like melamine to milk, the treatment of tuna with carbon monoxide, and the replacement of high-quality species with lower quality ones, or even illegal ones. An example for this can be found in the publication by Fang and Zhang [2], where the addition of murine meat to substitute mutton has been reported.

Since there are many animal species that can be used for adulteration, using a species-specific PCR is often not economically viable when the adulterant species is not known. Here, the DNA barcoding approach is the better choice to cover a much wider range of species.

In the literature, numerous publications can be found that describe different primer sets to be used for barcoding. Unfortunately, not all methods have been thoroughly validated for the species they can, and, equally important, cannot detect.

The German §64 Food and Feed Law Methods Group for Animal and Plant Speciation has developed a tool that will help scientists to quickly determine which species can be detected and which cannot with a specific set of primers.

The tool, called BaTAnS – short for Barcoding Table for Animal Species – lists relevant publications, identifies the level of validation that has been performed for a specific method (and set of primers).

Read full article.

Following the previous LGC e-seminars on quantitative PCR assay design and PCR assay optimisation, this e-seminar, entitled “An introduction to quantitative PCR assay validation”, will introduce the viewer to the topic of qPCR assay validation and provide best practice guidance on how to undertake the process. The information presented will equip viewers with the necessary knowledge and skills to ensure that methods are validated and fit for purpose. Key stages in the validation process are indicated, routinely employed evaluation parameters described and critical performance criteria highlighted. Links to useful resources, additional guidance and references are also provided.

Those who should consider viewing this e-seminar include individuals currently working within the foods molecular testing area, particularly representatives from UK Official Control Laboratories, industry and members of organisations associated with the UK official control network.

The production of this e-seminar was funded by Defra, FSA, FSS and BEIS under the Joint Knowledge Transfer Framework for Food Standards and Food Safety Analysis.

This e-seminar can be be viewed on LGC's YouTube channel at  https://youtu.be/grf4tZQOArM