Johnson Mary Lynn, Evoniuk Jessica M, Stoltenow Charles L, O'rourke Katherine I, Redmer Dale A
Department Animal and Range Sciences, 172 Hultz Hall, North Dakota State University, Fargo, ND 58105-5727, USA.
J Vet Diagn Invest. 2007 Jan;19(1):73-7. doi: 10.1177/104063870701900111.
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 AA(136) RR(171) genotype the most resistant to scrapie and QR(171) only rarely infected in the US sheep population. The Assays by Design 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 5' 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.
本研究的目的是开发一种可靠的Taqman 5'核酸酶检测法，用于对绵羊进行痒病易感性基因分型。绵羊朊病毒基因包含2个单核苷酸多态性（SNP），可能介导对经典痒病的抗性，一个位于密码子136，为丙氨酸（A）或缬氨酸（V），另一个位于密码子171，为精氨酸（R）或谷氨酰胺（Q）。R等位基因似乎赋予对经典痒病的抗性，AA(136) RR(171)基因型对痒病最具抗性，而QR(171)在美国绵羊群体中很少被感染。通过“设计检测法”方案开发针对密码子136的探针和引物，针对密码子171则使用Primer Express软件。使用市售试剂盒从血液或肌肉中分离基因组DNA。为进行验证，将每个密码子的70次SNP测定结果与商业检测结果进行比较，错误率低于1%。然后，对来自血液（n = 818）和肌肉（n = 117）的935个样本进行两个密码子的检测，成功测定928次，仅7个样本（占总样本的<1%）需要重复检测。基因型为AA QQ（n = 102；11.0%）、AV QQ（n = 28；3.0%）、AA QR（n = 396；42.7%）、AV QR（n = 54；5.8%）和AA RR（n = 348；37.5%）。因此，所检测绵羊中的86%（n = 798）在密码子171处含有R，预计对痒病具有抗性。这种新的Taqman 5'核酸酶SNP基因分型检测法准确、易于操作，在绵羊经典痒病研究以及通过选择性育种计划消除高度易感动物以预防痒病方面很有用。
