Genotyping of ovine prion protein gene (PRNP) variants by PCR with melting curve analysis.

BACKGROUND Scrapie is the transmissible spongiform encephalopathy in sheep. Because genetic variants of the ovine PrP gene (PRNP) can be associated with disease risk, the European Union initiated programs to eradicate high-risk PRNP genotypes from sheep livestock. For this purpose, reliable and cost-effective genotyping is needed. METHODS We amplified DNA to cover the 3 risk codons in exon 3 encoding amino acids 136, 154, and 171. Amplicons were mixed with dye-labeled probe sets, and melting curves were recorded in a LightCycler by use of color and temperature multiplexing. Probe design was based on thermodynamic calculations to ensure unequivocal results for the 3 codons of interest, taking the additional F141 and T137 sequence variants into account. RESULTS The fluorescence resonance energy transfer (FRET) method, when compared with sequencing, gave exactly the predicted melting temperatures for all possible genotypes. When we validated the method with samples from official certification programs, it showed completely matching results. Turnaround time was approximately 5 h after receipt of a whole-blood sample. The method detected the rare sequence variants T137 and F141, which were clearly distinguishable from the other known genotypes by melting curve analysis. One scrapie sheep was ARR/ARR, which is considered the haplotype with the lowest risk. CONCLUSIONS The FRET-based PRNP genotyping method for sheep is rapid and can differentiate all genotypes at each locus in 1 capillary. The assay is fast and has lower costs than restriction fragment length polymorphism analysis or sequencing.