Development of a real-time polymerase chain reaction assay for single nucleotide polymorphism genotyping codons 136, 154, and 171 of the prnp gene and application to Brazilian sheep herds

Abstract

Scrapie is a transmissible spongiform encephalopathy of sheep and goats and is associated with the deposition of an abnormal isoform of prion protein (PrP(sc)). This isoform presents an altered conformation that leads to its aggregation in the host's central nervous and lymphoreticular systems. A predisposition to the prion-agent infection can be influenced by specific genotypes that are related to polymorphisms in the ovine prnp gene. The most characterized polymorphisms occur at codons 136, 154, and 171, with genotype VRQ being the most susceptible and ARR the most resistant. In the current study, a real-time quantitative polymerase chain reaction (qPCR) technique based on allele-specific TaqMan probes was developed to identify single nucleotide polymorphisms in the prnp gene from Brazilian herds. Specific primers and TaqMan probes were designed for all 3 codons of interest. Samples from a total of 142 animals were analyzed by qPCR, followed by DNA sequencing of the amplicons. All of the genotypes determined by qPCR were in agreement with the data determined by DNA sequencing. In all 3 of the analyzed breeds, the majority of the animals were AA homozygous for the 136 codon. The most frequent genotype for codon 154 was RR, and genotypes QQ and QR were the most frequent for codon 171. The results are discussed in relation to establishing scrapie control measures and breeding programs for Brazilian herds.