Page 6

eFOOD-Lab_International_02_2014

Figure 2: A representative Terminal-Restriction Fragment Length Polymorphism (T-RFLP) chromatogram. 6 2/14 eFOOD-Lab international Figure 3: Analyzable and identifiable animal species using T-RFLP analysis, developed by Fraunhofer Institute - IME (Schmallenberg - DE). differentiates microbial populations based on terminal restriction fragment (T-RF) size. Mixed DNA samples are amplified with fluorescence-labeled universal primers, digested with select restriction enzymes, and separated by capillary electrophoresis with molecular standards for estimating fragment size. Only the labeled 5’ terminal fragments are detected and compared to a database to determine which species (grouped as operational taxonomic units, OTUs) are represented by these fingerprint-like markers. Fluorescence intensity is integrated to pseudoquantitatively determine OTU abundance relative to total fluorescence. Results The T-RFLP method for animal species differentiation, developed by the IME in 2002, completely avoids the disadvantages of conventional methods. By introducing an intermediate step – a so-called nested PCR – it is possible to analyze not only food, but also feed – depending on the degree of processing. Initial results indicate that the method can also be used successfully for the analysis of animal leather or skins. Moreover, this method could be used for forensic analysis, and to monitor the illegal trade of exotic and protected animals. This improved version of the detection system now allows all the animal species in a given sample to be detected simultaneously, considerably shortening the overall analysis time. The method is powerful enough to determine the species present in highly processed feed, within which the DNA tends to be extensively degraded, and to detect components which represent only 0.5 % of the total. It is possible to distinguish between very closely related animal species. Even the presence of exotic animal species such as crocodile, can be verified easily. Currently, Fraunhofer-Institute - IME (Schmallenberg - DE) is able to identify more than 50 animal species using T-RFLP analysis (Figure 3). Conclusions T-RFLP analysis has many applications and contributes an important and efficient method to guarantee quality of the finished product. The suggested approach allows a more accurate assessment on the food supply chain, through the identification of the most common animal species composing processed food and feed. The objective is to improve or complement existing detection methods, consenting the generation of a wider library of terminal restriction fragments. Beyond the crucial role represented by a such faster and cheaper species differentiation technique, its real innovation resides in the possibility to verify the presence of a particular specie and, at the same time, identify the composition of the entire sample, thus ensuring high quality standards and safety levels for the consumer. Qual ity Mana gement


eFOOD-Lab_International_02_2014
To see the actual publication please follow the link above