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Final optimisation and evaluation of DNA based methods for the authentication and quantification of meat species - FA0210

The Food Labelling and Standards Division of the FSA (formally of MAFF) funded 4 laboratories to design and optimise DNA based species-specific assays for turkey, chicken, pork, beef and lamb.

Another laboratory was funded to develop methods to identify meat adulteration in vegetarian foods. The application of the developed assays and their technological transfer to enforcement and other analytical laboratories, has yet to be fully realised.

The objective of this study was therefore to validate these assays and produce a set of protocols which would provide comprehensive guidelines for the analysis of meat species in commercial meat products, and be suitable for enforcement of product labelling. These protocols were to encompass the DNA extraction technique, the species-specific assay conditions and quality control criteria for each assay.

The project was divided into two strands which ran concurrently:
· The optimisation and comparison of qualitative species-specific real-time PCR assays
· The comparison of different approaches to quantify species using real-time PCR assays.

The first strand of the project identified the most appropriate DNA extraction protocol suitable for the purification of high quality DNA, (in the absence of inhibitors), from raw and canned meats, including samples made from mixed species that also contained fat or rusk. It was found that a single DNA
extraction protocol was suitable for all meat matrices tested: the CTAB, proteinase K, wizard protocol and additionally, sensitive real-time PCR assays for chicken and porcine meats were also identified.

The final work for this strand of the project involved a proficiency trial of the CTAB, proteinase K, Wizard DNA extraction protocol, coupled to the porcine and chicken real-time PCR assay, used by each of the contractors to analyse a set of sausage samples. Additionally, contractors used in-house real-time PCR assays for chicken, pork, beef, lamb and turkey on the DNA extracted from the samples. Upon comparison of the results it was found that all the real-time PCR assays using both mitochondrial and nuclear DNA targets, previously developed by the contractors, were able to correctly identify the species present within a sample, with no false positive results. This strand of the project was completed with the provision of a comprehensive set of Standard Operating Procedures for the identification of meat species in complex food products using real-time PCR-based methods.

The second strand of the project involved an investigation into different approaches to quantitate the amount of meat of a given species present in a commercial sample. This work focused on the identification of the most appropriate calibrants for quantification, and investigated the use of single
and mixed meat samples, dilutions of DNA and plasmid standards. Real-time PCR assays detecting both multi-copy mitochondrial targets and single copy nuclear targets were also compared. The results of this work showed promising trends which could be used for meat quantification, however
none of the calibration methods proved robust enough to be considered suitable for enforcement of product labelling.

In conclusion, the project allowed the successful identification of a DNA extraction protocol and realtime PCR assays for the qualitative determination of common species present in complex foods, and which are suitable to assist public analysts and enforcement laboratories. Also, a set of standard
operating procedures for these methods has been provided to the Food Standards Agency.
01: DNA extraction from raw tissue, raw tissue admixtures (different species), autoclaved, autoclaved admixtures (different species)
and canned tissue Expected achievement – DNA extractions available for all participants

02: Methodology validation
Expected achievement – direct comparison between the assay

03: Comparison of approaches for quantification. Expected achievement Evaluation of CCFRA and VLA method for application to raw and processed meat products. Identification of appropriate method for standard curve construction

04 Proficiency trial of the optimised methods. Expected achievement –– identification of the most appropriate
methods for the identification and quantification of meat species in food products

05 Technology Transfer: Expected achievement Preparation of protocols and SOPs
Project Documents
• FRP - Final Report : species id final report   (1131k)
• SUM - Summary Report : species id rcua5   (196k)
Time-Scale and Cost
From: 2003

To: 2005

Contractor / Funded Organisations