New methodology for the species determination of animal products in feeds : combination of micro-spectroscopy techniques and Real Time PCR
|Type of the activity: research project
Duration of the project: 2/2006-1/2008
Since the outbreak of Bovine Spongiform Encephalopathy (BSE), the European legislation on animal by-products in feed drastically limits the use of Processed Animal Proteins (PAPs). Moreover, it recommends the development of alternative methods able to detect and identify animal meals at species or group of species level such as Ruminants (European Regulations 999/2001, 1774/2002 et 1234/2003). Even if classical microscopy remains the only official method to detect animal ingredients in compound feeds, the technique shows its limitations for a complete taxonomic classification of animal particles. Moreover, classical microscopy is mainly based on bone detection and identification while evidencing muscles or any other animal tissue may also be essential to an efficient control.
Results already obtained by the CRA-W in previous national (Project RCS N° S-6112 – « Detection and quantification of animal meals in feedingstuffs ») and European projects (http://stratfeed.cra.wallonie.be) show the potential of both micro-spectroscopic techniques (Near infrared microscope – NIRM and NIR camera) and Real Time PCR. The project aims to develop an original methodology by combining the advantadges of the two techniques and by answering to the legal requirements, namely the detection and the determination of the animal origin up to species level even in the case of low contamination (down to 0.1% in weight) with animal particles from different tissues like bones, muscles,…
Micro-spectroscopic techniques (NIR microscope and NIR camera) are able to detect specifically meat and bone meal particles. They are non-destructive and therefore allow another type of analysis on the studied object. PCR is able to detect any DNA source coming from an animal origin even at very low level but some authorised ingredients such as milk powder, egg products, blood or fats can interfere. However, at the present time, PCR is the only technique able to determine which animal species is present in the sample. So the strategy to develop is :
- to locate meat and bone particles by means of micro-spectroscopy and to isolate them
- to transfer each particle in a well of PCR plate
- to perform a PCR analysis on it in order to
- to determine its species origin.
This project will also aim to evaluate the potential of Raman spectroscopy and of Fluorescence.
Expected results :
Thanks to this new methodology, it should be possible to detect the presence of animal proteins coming from meat and bone meals and to determine their genetic origin up to the species level without any interference with any other authorised products (fats and milk powders).
Achieved results :
A new DNA extraction protocol using a special buffer has been developped. It allows to perform 5 PCR with the DNA extracted from a single particle. A protocol for an efficient cleaning of the particle – assuring that the amplification is only due to the DNA from the inside of the particle – has still to be optimised. Specific spectral databases have been built. They cover the main species used for the production of meat and bone meals. Discriminant models able to recognize particles from these different species have been developped. Specific spectral markers (specific spectral peaks) for fish, cattle, pig and poultry species have been identified. The potential of Raman spectroscopy for the analysis of sediment has also been studied and discriminant wavelenghts have been identified at 960 cm-1 and 1087 cm-1. A study on a limited sample set was also conducted and demonstra tes the usefullness of Fluorescence for the detection of meat and bone meal in feedingstuffs. These different works will be continued within the framework of the European SAFEED-PAP project.
JRC-IRMM: Dr Christoph von Holst and Dr Ana Boix
AFSCA: Ir Jeroen Van Cutsem
UCL: Prof. Marc Meurens
Dr Gilbert Berben
CRA-W – Quality Department for Agricultural Products
Chaussée de Namur, 24
B 5030 Gembloux
Tel : +32 (0)81 62 03 50
Fax : +32 (0)81 62 03 88
Email : firstname.lastname@example.org
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