New developments in classical microscopy; what can be expected for the official control?

L2-7

For the monitoring of the presence of prohibited animal proteins several methods can be applied. The strengths of the microscopic detection method are, among others, the low level of detection as low as 0.02% and the precise indication of the detected materials. The different detection and identification methods such as PCR (DNA detection), immunoassays (protein detection) and microscopy together provide the possibility to identify detected animal proteins in terms of avian or mammalian origin, or ruminant vs. non-ruminant. This information, additional to the mere presence or absence, is vital for support of the species-to-species ban.

In Work package 5, aimed at support and enhancement of the official control, microscopy related methods and markers are further developed for detection and identification of animal proteins.

Bone fragment analyses . Values for ten characteristics of the lacunae enclosed in bone fragments have been extracted from a range of different samples. Multivariate analysis revealed that a good discrimination could be achieved between mammalian and avian materials. Within the group of mammalian samples the two rabbit samples are the most deviating from the average appearance of mammalian bone fragments. Deviating results were also found in a sample of cattle material, which is used in several previous studies. The main discriminating characters are the diameter and the area of the lacunae, and the smoothness of the border of the lacunae.

Mammalian hairs can provide information on the presence of mammalian animal proteins because of their mere presence. Furthermore some specific problems can be solved, e.g. unintentional contamination with rodents, by specific characteristics of the structure of the hairs.

Combination of methods can be achieved in order to combine the strengths of several methods, whereas the disadvantages can be minimalised. In situ detection or hybridisation is known for years as a powerful method for detection and identification of small quantities and small particles. The combination of either PCR or immunoassay analysis and microscopy adds the possibility of identifying individual particles to the low level of detection and the specificity of the microscopic method.

In the framework of SA FE ED-PAP muscles identification by means of antibodies has been chosen. The chosen target, present at low frequencies if any is found, has to be concentra­ted and selected from the feed sample. A second step is to immobilise the particles from the concentrate on a microscopic slide. Optimal circumstances have to be established for hybridisation of the first and second antibody, and for the staining procedure. Optimal discrimination between the targeted and other muscle fibres has to be established. It appears that several enzyme-substrate combinations connected to a goat-anti-mouse antibody can be used effectively, either with alkaline phosphatise (blue staining) or with horse-radish peroxidise (red staining). These systems can be used simultaneously, allowing a discrimination system for muscle fibres from different target animal species.

1 RIKILT - Institute of Food Safety, The Netherlands

2 University of Milan , Italy

3 Walloon Agricultural Research Centre , Belgium

4 Danish Plant Directorate, Denmark