Development of an assay to determine single nucleotide polymorphisms in the prion gene for the genetic diagnosis of relative susceptibility to classical scrapie in sheep

Authors

Mary Lynn Johnson, North Dakota State University - Main Campus
Jessica M. Evoniuk, North Dakota State University - Main Campus
Charles L. Stoltenow, North Dakota State University - Main Campus
Katherine I. O'Rourke, U.S. Department of AgricultureFollow
Dale A. Redmer, North Dakota State University - Main Campus

Document Type

Article

Date of this Version

2007

Comments

Published in J Vet Diagn Invest 19:73–77 (2007)

Abstract

The objective of this study was to develop a reliable Taqman® 5' Nuclease Assay for genotyping sheep for scrapie susceptibility. The sheep prion gene contains 2 single nucleotide polymorphisms (SNPs) that may mediate resistance to classical scrapie, one at codon 136, alanine (A) or valine (V), and another at codon 171, arginine (R) or glutamine (Q). The R allele appears to confer resistance to classical scrapie, with the AA₁₃₆ RR₁₇₁ genotype the most resistant to scrapie and QR₁₇₁ only rarely infected in the US sheep population. The Assays by Design^SM protocol was used for development of probes and primers for codon 136 and Primer Express® for codon 171. Commercially available kits were used to isolate genomic DNA from blood or muscle. For validation, 70 SNP determinations for each codon were compared to commercial testing with an error rate of less than 1%. Then, 935 samples from blood (n = 818) and muscle (n = 117) were tested for both codons with 928 successful determinations and only 7 samples (<1% of total samples) that needed repeating. Genotypes were AA QQ (n = 102; 11.0%), AV QQ (n = 28; 3.0%), AA QR (n = 396; 42.7%), AV QR (n = 54; 5.8%), and AA RR (n = 348; 37.5%). Thus, 86% of the sheep tested (n = 798) contained R at codon 171 and were expected to be scrapie-resistant. This new Taqman® 59 Nuclease SNP genotyping assay is accurate, easy to perform, and useful in the study of classical scrapie in sheep and its prevention through selective breeding programs to eliminate highly susceptible animals.