Protein- und Nukleinsäureextraktion aus Fleisch- und Knochenmehlen und Nachweis der Tierart mittels PCR (Polymerasekettenreaktion)

Alemán. The feeding of processed animal protein to livestock (EC Regulation No. 999/2001) is prohibitedin the European Union. As a consequence, prohibition of animal protein in fodder to ruminantssignificantly reduces BSE cases. Suitable detection methods are required to monitor the legalguidelines in order to guarantee consumer protection. The validated method, which is currentlyapplied in Europe to identify animal components in fodder, is based on microscopy. The polymerasechain reaction (PCR) technique is employed as an alternative, which provides more informationabout the origin of the animal components. All PCR detection systems described in theliterature are based on multicopy target sequences. These systems are, in fact, highly sensitive,but can slightly lead to unspecific signals in the PCR.For this reason, this work reviews alternative methods for the determination to identify thermallystable animal proteins or their nucleic acids in different animal meal samples and single-copyreal-time PCR-system detection methods specific to animal species were employed.A suitable procedure to extract animal proteins was investigated in the first section of the presentwork. Hitherto, neither optimised protein extraction procedures from meat and bone meal (MBM)nor protein samples in the SDS-Page for this matrix have been published in the literature. As aresult, in this thesis, different protein isolation methods (protocol I to IV) have been investigatedin the pure bovine MBM and, conclusively, the protein content is measured by means of the BCAtechnique. When determining the protein contents, sufficient protein contents are established inthe isolation using Protocol II (according to Aije et al., 1991) and Protocol IV (according to Coll etal., 2007), in which the maximum protein contents in the extracts are established using ProtocolIV. Correspondingly, the protein content was sufficiently high to be adopted for protein separationby electrophoresis.In previous studies, the Laemmli (1970) separation of animal proteins using a glycine buffer wasunsuccessful. As a consequence, a Tricine-SDS-PAGE system - as described by Schägger(2006) - was implemented in this work, which enables the separation of particularly smaller proteinsand peptides within the low-molecular range between 2 to 20 kDa. However, no proteinseparation could be achieved by using two different staining methods, one being the Coomassiestaining and the other silver staining, or by using additional purification or precipitation methodssuch as the modified gel electrophoresis protocol. Accordingly, no protein masses of individualpeptides could be determined.In parallel, a DNA isolation method was optimised in the second part of the work. In addition tothe traditional cetyl trimethylammonium bromide (CTAB) and the Whole Genome AmplificationAbstract(WGA) procedures, different commercial kits were also employed, particularly DNA extractionkits were developed for forensic issues. The partial goal of this work was to optimise DNA isolationto apply single-copy real time PCR-systems (as described by Laube et. al., 2007) to differentanimal meals and animal meals in fodder mixtures. Specific primer and probe systems (cattle,pigs, poultry, chicken and turkey) were used to detect animal DNA.Experiments using poultry meals and DNA extraction using the Investigator Kit as well as theuse of the single-copy PCR system with a MYw primer pair to detect the myostatin gene broughtoutstanding reproducible results. Similarly, the single-copy real-time PCR system used to detectpoultry species containing the specific gallL and mellL primers (chicken- and turkey-specific)also showed reproducible results.When the Investigator Kit and the MYw primer system were applied, pig meals from LUFA(North-West, Oldenburg) produced good results. Fish meals, in contrast, exhibited low DNA yieldwith the Investigator Kit, in which the single-copy MYw primer system was most applicable in thiscase.The CTAB extraction method shows excellent extraction of DNA quantities in fodder mixturescontaining 0.1% cow meal proportion.Depending on the material used, the different results significantly show that there is no optimalprocedure for certain sample types. The DNA extraction method employed must be evaluatedfor each sample. However, there is a tendency to show that the CTAB method is particularlyvery suitable for fodder mixtures containing plant proportions, whereas the Investigator Kit is themethod of choice for certain animal meals.Both the single-copy MYw primer system and the multicopy bos-primer system show a positivesignal with these extracts and in real-time PCR, the bos primer system shows a very early fluorescenceincrease.However, in a negative sample of 100% pig DNA extract, the bos primer system showed a positivesignal for the "cattle" animal species. This system is, therefore, uncertain and may lead toerroneous results.In unknown animal meal mixtures in the fodder samples (ring experiment samples used for microscopy)No. 46/47/48, excellent extractions of DNA were obtained using the CTAB extractionmethod. The myostatin sequence was detected in all samples when the extract was diluted by1:5 for PCR use. In detection systems specific to animal species, in the detection of cattle andpig species, "pig" could not be detected and "cattle" only be shown in one sample. The discoveryof unknown samples showed that a sample was of pure plant material and two samples eachcontained 0.2 % cattle MBM, in which a sample also contained 3 % fish meal. Consequently,detection of the cattle species using the single-copy real-time system was unreliable. The obserAbstractvation of a positive myostatin signal in the pure plant sample may, by contrast, also originatefrom the presence of low DNA traces from the environment (rodents, birds) in the field material.In animal meals consisting of feather, cattle or pig meals from the Chemical Veterinary StudyOffice of Krefeld (Chemischen Veterinäruntersuchungsamtes Krefeld in North Rhine Westphalia[NRW]), no distinct detections were made by using the Investigator Kit and a subsequent WholeGenome Amplification (WGA) with the single-copy real-time PCR systems employed here.Therefore, this combination is not recommended.Overall, the PCR experiments using the extracts from animal meals and fodder, however, showthat the single-copy real-time MYw primer system is well suited to detect unequivocally animalDNA in fodder samples. Therefore, the procedure is suitable as an alternative screening procedurefor microscopy. In contrast, the detection limits of the single-copy real-time PCR systemsare reached to detect cattle and pig in well processed animal meals (with the exception of poultrymeal) or mixtures containing only trace levels of the specific material.