A real-time polymerase chain reaction assay to detect single nucleotide polymorphisms at codon 171 in the prion gene for the genotyping of scrapie susceptibility in sheep

Abstract

The objective of this study was to report a reliable real-time polymerase chain reaction assay compatible with the Roche LightCycler 2.0 capable of genotyping sheep for scrapie susceptibility at codon 171. The single nucleotide polymorphisms (SNPs) in the prion protein gene in sheep that may govern resistance to scrapie at codon 171 encode for lysine (K), histidine (H), glutamine (Q), and arginine (R). A modified proteinase K method for leukocytes or whole blood was used to isolate genomic DNA from sheep blood. Fluoresentric developed and optimized primers and probes for the codon 171 SNP assay. The assay was initially validated using 218 determinations from whole blood of known genotypes with 100% correct identity. The assay was further validated through a whole-blood check test provided annually by the National Veterinary Services Laboratory with a correct identification rate of 100%. From January 2005 to December 2006, 3,672 samples from blood were genotyped at codon 171. The genotypes were QR(171) (n = 1,838, 50.05%), RR(171) (n = 1,423, 38.75%), QQ(171) (n = 407, 11.08%), HR(171) (n = 2, 0.05%), and HQ(171) (n = 2, 0.05%). The combination of this simple extraction method and the novel Fluoresentric assay is very accurate, is capable of identifying all 4 SNPs at codon 171 in one reaction, and has proven to be a useful tool for producers in their selective breeding programs.