NR ATMC
AU van Poucke,M.; Vandesompele,J.; Mattheeuws,M.; van Zeveren,A.; Peelman,L.J.
TI A dual fluorescent multiprobe assay for prion protein genotyping in in sheep
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Genetics, strains and emerging problems GEN-02
PT Konferenz-Poster
AB Scrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programs towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Here we report the development and validation of a new, dual fluorescent multiprobe assay, consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above. This fast, simple and cost-effective assay is successfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep. The assay was used to genotype more than 600 Belgian sheep within the TSE resistant breeding program framework. No discrepancies with the rules of Mendelian inheritance were observed. The assay can easily be adapted for new polymorphisms and the described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations.
AD M.van Poucke, J.Vandesompele, M.Mattheeuws, A.van Zeveren, Luc J.Peelman, Ghent University, Belgium
SP englisch
PO Deutschland