Primer extension assay for prion protein genotype determination in sheep.
Scrapie is a transmissible spongiform encephalopathy (TSE) which affects sheep and goats. TSEs are characterised by the conversion of the cellular prion protein (PrP(C)) into the pathological form PrP(Sc). The occurrence of scrapie in sheep is influenced by polymorphisms in the PrP gene; in particular, three codons (136, 154 and 171) are important in conditioning the susceptibility/resistance of sheep to the disease, with the Val/Val(136) Arg/Arg(154) Gln/Gln(171) genotype being the most susceptible and the Ala/Ala(136) Arg/Arg(154) Arg/Arg(171), the most resistant one. The latter genotype seems to confer, in sheep, resistance to the oral infection with bovine spongiform encephalopathy, as well. The selection of genetically resistant sheep populations represents the basis of the recent strategies against ovine TSE in the European Union (EU). Herein, we describe a rapid and simple method, based on the primer extension technique, for PrP genotype determination at codons 136, 154 and 171. Intra-laboratory validation of the method showed accuracy levels comparable to those of sequencing analysis. Such method could be used for both the application of the EU policies requiring PrP genotype analysis in all ovine TSE cases, and the large-scale genotyping claimed by the implementation of breeding programmes for genetic resistance to TSE in sheep.
Vaccari
G
G
Laboratory of Veterinary Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy. gvaccari@iss.it
Conte
M
M
Morelli
L
L
Di Guardo
G
G
Petraroli
R
R
Agrimi
U
U
D000818:Animals; D001483:Base Sequence; D003062:Codon; D017931:DNA Primers / Q000592:standards*; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D021141:Nucleic Acid Amplification Techniques; D015588:Observer Variation; D017096:Prion Diseases / Q000235:genetics; D011328:Prions / Q000235:genetics*; D015203:Reproducibility of Results; D012756:Sheep
D003062:Codon; D017931:DNA Primers; D011328:Prions