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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  

Photo by Florencia Viadana on Unsplash

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

An introduction to DNA melting curve analysis

This e-seminar, entitled “An introduction to DNA melting curve analysis”, describes the principles behind, as well as best practice guidelines for the application of the post-PCR analytical method of DNA melting curve analysis. The information presented will provide the viewer with a general introduction to PCR-based DNA melting analysis as a method for food authenticity testing, and provide guidance on how to design, implement and analyse PCR DNA melting assay data. Topics covered will include the principles underpinning DNA melting analysis, designing PCR DNA melting assays, examples of PCR instruments compatible with DNA melting analysis, and guidance on troubleshooting. 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.

View e-seminar here.

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.