STRATFEED PCR Method

The bovine spongiform encephalopathy (BSE) has led the European Union (EU) to take several drastic decisions in order to ensure exclusion of its causal agent from the food chain with the aim to guarantee the safety of the food and feed supply. These decisions include specific treatment conditions (133°C for 20 min at a pressure of 3 bar) for the manufacturing of meat and bone meal (MBM) and a temporary ban of these products as well as their derived proteins in compound feed production.

The STRATFEED project aims to provide innovative protocols by development of new methods. One of those methods is the PCR (Polymerase Chain Reaction), able to detect specific DNA segments through genetic amplification.

The qualitative search of taxon specific animal DNA sequence as markers of MBM in animal nutrition is carried out in 5 steps:

- the sample reception/storage

- the sample preparation

- the DNA extraction-purification

- the preparation of the reaction mixture

- the DNA amplification by PCR

The results produced by PCR are showed through the STRATFEED explorer

The STRATFEED project aims also at editing publications

1^st Step : the sample reception/storage (Figure 1)

The first step is the sample reception.

As soon as the laboratory receives the sample, this one is identified by a code used during the subsequent steps of the analysis.

Figure 1. : The sample reception/storage.

2^nd Step : the sample preparation (Figure 2)

Each solid sample is ground in order to :

- firstly, have a good homogeneity of the sample,
- secondly, have very fine particles and higher contact surface with the extraction reagents

Figure 2. : The sample preparation.

3^rd Step : the DNA extraction-purification (Figure 3)

The basic principle of nucleic acid extraction consists in releasing the DNA present in a given matrix by lysis of remaining cell structures. The DNA in solution is then purified by removing all the other components particularly the PCR inhibitors. Different extraction methods can be used depending of the sample analysed.

Figure 3. : The DNA extraction-purification.

4^th Step : the preparation of the reaction mixture (Figure 4)

A sub sample of the extract is used for the DNA amplification by PCR.

The PCR consists in an enzymatic reaction that multiplies a specific DNA target sequence present in the MBM.

The reaction mixture contains:

- Primers: oligonucleotides used to delimit the segment of DNA target.
- dNTP: deoxynucleotides triphosphates used by the DNA polymerase to synthesize the DNA target sequence.

- MgCl₂: cofactor required for the polymerase activity.

- Thermostable DNA polymerase: heat resistant enzymatic preparation, purified from the thermophilic bacterium Thermus aquaticus (Taq DNA polymerase), allowing the polymerization of DNA under the selected reaction conditions.

These components are added as a mix in vials and the DNA extract to be analysed is added afterwards (together with appropriate controls)

Figure 4. : The preparation of the reaction mixture.

5^th Step : the DNA amplification by PCR (Figure 5)

The reaction mixture is put in a thermocycler. This device supports the continuous enzymatic production of DNA through thermal cycles consisting of a succession of well- defined heat steps that allow a synchronised reaction on the available substrates. As each newly produced DNA molecule is also a new substrate in subsequent cycles, the reaction accumulates the targets in an exponential fashion at least during the first cycles.

Figure 5. : The PCR amplification.

This amplification can be performed by 2 types of system:

- the classical PCR system
- the real time PCR system

The classical PCR system (Figure 6)

This system involves an electrophoresis step to visualize the amplified DNA products.

Figure 6. : The classical PCR System.

The real time PCR system (Figure 7)

This system is quicker then the classical PCR system and makes it possible to follow the amplification reaction during its achievement.

Figure 7. : The real time PCR System.

Publications

Journal article

Bellorini, S., Strathmann, S., Baeten, V., Fumière, O., Berben, G., Tirendi, S. & von Holst, C. (2005). Discriminating animal fats and their origins : assessing the potentials of Fourrier transform infrared spectroscopy, gas chromatography, immunoassay and polymerase chain reaction. Anal. Bioanal. Chem., 382, 1073-1083.

Chiappini, B., Brambilla, G., Agrimi, U., Vaccari, G., Aarts, H., Berben, G., Giambra, V.& Frezza, D. (2005). A real-time PCR approach for ruminant-specific DNA quantification indicates a correlation between DNA amount and MBM heat treatments. Journal of AOAC International, 88(5), 1399-1403.

Gizzi, G., von Holst, C., Baeten, V., Berben, G. & van Raamsdonk, L. (2004). Determination of processed animal proteins, including meat and bone meal, in animal feed. Journal of AOAC International, 87 (6), 1334-1341.

Gizzi, G., van Raamsdonk, L., Baeten, V., Murray, I., Berben, G., Brambilla, G. & von Holst, C. (2003). Risk analysis of prion diseases in animal. An overview of tests for animal tissues in feeds applied in response to public health concerns regarding BSE. Revue Scientifique et Technique, Office International des Epizooties, 22 (1), 311-331.

Book

Berben, G., Fumière, O., Brambilla, G., Vaccari, G., Aarts, H., von Holst, C. & Portetelle, D. (2005). PCR Detection of animal by-products in feeds (WP4). In: Strategies and methods to detect and quantify mammalian tissues in feedingstuffs, Brussels, European Commission, 16p.

Posters

Chiappini, B., Aarts, H., Agrimi, U., Berben, G., Brambilla, G., Frezza, D. & Vaccari, G. (2004). Relationship between rendering process temperatures and DNA degradation in Meat and Bone Meals by real time PCR assay. Poster in: International symposium on Food and Feed safety in the context of prion diseases, Namur - Belgium, 16-18 June 2004.

Fumière, O., André, C., Berben, G., Dardenne, P. & Baeten, V. (2004). An original strategy coupling NIRM and PCR for detection and species identification of MBM particles. Poster in: International symposium on Food and Feed safety in the context of prion diseases, Namur - Belgium, 16-18 June 2004.

Fumière, O., Osmanaj, I., Chiappini, B., Brambilla, G., Aarts, H., Vaccari, G. & Baeten, V. (2004). PCR analysis can help microscopist to identify animal species present in the sediment fraction of compound feed. Poster in: International symposium on Food and Feed safety in the context of prion diseases, Namur - Belgium, 16-18 June 2004.

Rwagasore, F., Fumière, O., Baeten, V., Berben, G., Dardenne, P., Bellorini, S., Strat????????E???d?d??hmann, S., Tirendi, S. & von Holst, C. (2004). Possibilities of FT-IR and PCR to discriminate species by animal fats. Poster in: International symposium on Food and Feed safety in the context of prion diseases, Namur - Belgium, 16-18 June 2004.

Fumière, O., Théwis, A. & Dardenne, P. (2003). Detection of frauds of certified chicken cuts by NIR analysis. Poster in: 11th International Conference on Near Infrared Spectroscopy, Cordoba - Spain, 6-11 April 2003.

Contributions

This work was coordinated by CRA-W in collaboration with JRC-IRMM, AFSCA, FUSAGx, RIKILT, NUTRECO, SAC, ISS, UCO, LAGC, ALP, LUFA and PDIR

	Walloon Agricultural Research Centre CRA-W (B) Dr Pierre Dardenne, Dr Vincent Baeten, Dr Gilbert Berben, Dr R. Oger, Ir Philippe Vermeulen
	Institute for Reference Materials and Measurements European Commission - DG - Joint Research Centre JRC-IRMM (B) Dr Christoph von Holst
	Gembloux Agricultural University FUSAGx (B) Prof Daniel Portetelle
	Institute of Food Safety RIKILT (NL) Dr Jacob de Jong, Dr Leo van Raamsdonk, Dr Henk Aarts, Mr Rob Frankhuizen
	Italian National Institute of Health ISS (I) Dr Gianfranco Brambilla, Dr Gabriele Vaccari