NR AUMN
AU Küfen,A.
TI Spezies- und gewebespezifischer Nachweis von bovinem ZNS-Gewebe in Fleischerzeugnissen mittels RT-PCR
QU Inaugural-Dissertation zur Erlangung des Grades einer Doktorin der Veterinärmedizin (Dr. med. vet.) durch die Tierärztliche Hochschule Hannover, vorgelegt von Alexandra Küfen aus Köln, 2.12.2003
PT Dissertation
AB The bovine spongiform encephalopathy (BSE), the possible routes of infection of BSE, and the new variant of the Creuzfeld-Jakob-disease (vCJD) which is closely connected with the first, have gained special attention with regard to preventive protection of consumers. The possible entry of specific risk material, particularly the entry of CNS tissue in the human food chain, is considered as a main root of infection. The illegal use of mechanically recovered meat in retail meat products and CNS-tissue as an emulgator in cooked sausages can not be excluded. Therefore appropriate control procedures for food and feeding are necessary in order to prevent CNS-tissue infiltrating the food- and feeding-chain. The method used in thesis in order to detect CNS-tissue in retail meat products is based on the detection of a168 bp sized fragment of mRNA of the glial acidic fibrillary protein (GFAP) by using reverse transcriptase-polymerase chain reaction (RT-PCR). Subsequent to the RT-PCR the classification of the species cattle by using a restriction fragment length polymorphism analysis (RFLP) had been carried out. Investigations made with regard to the tissue specificity of the GFAP-mRNA in porcine and ovine tissues revealed that the specification of CNS-tissue of these species is not possible so far, using the168 bp sized amplification product of the GFAP-mRNA as marker. Therefore only the bovine GFAP-mRNA is considered to be a save marker for the presence of CNStissue. Consequently the bovine origin of the detected GFAP-mRNA has to be proved. By the development of the RT-PCR using the primer pair bGFAPw1 and bGFAPw2 the necessary sequence information from sheep, pork, horse, fallow deer, roe deer and red deer to differentiate the respective amplification products were obtained. Using the restriction enzymes Hae III and Msc I the bovine GFAP-mRNA can be clearly identified. With beef sausage-products and liver sausages, which were spiked with bovine CNS-tissue in concentrations of 0 %, 0,25 %, 0,5 %, 1 % and 5 %, and which had a heating process up to 100° temperature to undergo, the proof of GFAP-mRNA up to a concentration of 0,25 % was possible over a period of 35 days. Over a period of 28 days, the sensitivity of this method is up to 97 % in retail meat products, which were produced by a heating temperature up to 100°. The specificity of the GFAP-mRNA RT-PCR assay for bovine CNS-tissue is 100 % in the experiments conducted here. Examination of canned food endowed with bovine CNS which were heated to FS-values over 5 revealed that the proof of GFAP-mRNA with the described method was not safely possible. Within the present thesis it can be stated that the used method is suitable to detect bovine CNS-tissue in meat products which were heated up to a temperature of 100°.
SP deutsch
PO Deutschland