Spezies- und gewebespezifischer Nachweis von bovinem ZNS ...

Summary
7 Summary
Alexandra Küfen
Species- and Tissue-Specific Detection of Bovine CNS-Tissue in Meat Products Using RT-
PCR
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°C 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
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