两种用于对杜宾犬血管性血友病 I 型突变进行基因分型的新型实时 PCR 方法。
Two novel real-time PCR methods for genotyping the von Willebrand disease type I mutation in Doberman Pinscher dogs.
机构信息
Department of Veterinary Medical Sciences, Alma Mater Studiorum, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano dell'Emilia, Bologna, Italy.
出版信息
Vet J. 2013 Aug;197(2):457-60. doi: 10.1016/j.tvjl.2013.02.023. Epub 2013 Aug 2.
Two single tube real-time PCR methods were designed to genotype the mutation responsible for von Willebrand disease type I (von Willebrand factor c.7437G>A) in Doberman Pinscher dogs: (1) the Divergent PCR assay, which is a modification of the bi-directional PCR amplification of a specific allele (BI-PASA) technique, and (2) a minor groove binder (MGB) real-time PCR assay using fluorescently labelled probes. There was complete agreement between the genotypes determined using the two real-time PCR methods and the results of sequencing of PCR products generated by conventional PCR from genomic DNA purified from the blood of 27 Doberman Pinscher dogs. The Divergent PCR assay yielded reliable results with ≥ 6.4 ng genomic DNA per reaction and the MGB real-time PCR assay yielded reliable results with ≥ 150 pg genomic DNA per reaction. Both real-time PCR methods are suitable for routine genetic testing for the von Willebrand disease type I mutation using blood samples.
两种单管实时 PCR 方法被设计用于对杜宾犬的 von Willebrand 病 I 型（von Willebrand 因子 c.7437G>A）突变进行基因分型：（1）差分型 PCR 检测法，它是对特定等位基因的双向 PCR 扩增（BI-PASA）技术的改进，以及（2）使用荧光标记探针的小沟结合（MGB）实时 PCR 检测法。用两种实时 PCR 方法确定的基因型与从 27 只杜宾犬血液中提取的基因组 DNA 进行常规 PCR 生成的 PCR 产物的测序结果完全一致。差分型 PCR 检测法在每个反应中使用≥6.4ng 基因组 DNA 时可产生可靠的结果，MGB 实时 PCR 检测法在每个反应中使用≥150pg 基因组 DNA 时可产生可靠的结果。两种实时 PCR 方法都适用于使用血液样本对 von Willebrand 病 I 型突变进行常规遗传检测。
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