Rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs by real-time polymerase chain reaction with TaqMan minor groove binder probes
Rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs by real-time polymerase chain reaction with TaqMan minor groove binder probes
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DOI：
10.1177/104063871002200210
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发表时间：
2010-03-01
影响因子：
1.5
通讯作者：
Yamato, Osamu
中科院分区：
文献类型：
--
作者：
Chang, Hye-Sook;Arai, Toshiro;Yamato, Osamu
为建立一种快速、可靠的神经节苷脂代谢病基因分型方法，采用TaqMan MGB探针进行实时荧光聚合酶链反应（PCR）。该技术应用于从血液、脐带或死后肝组织标本中提取的DNA样本，以及从血液和唾液中制备的已应用于弗林德斯技术协会滤纸（ETA卡）的含DNA溶液。所有样本中靶序列的扩增足以确定GM I神经节苷脂沉积症的基因型。使用这种实时PCR技术检查了先前从Shiba犬的血液或组织标本中获得的47个DNA样本，结果与早期PCR限制性片段长度多态性（RFLP）分析获得的数据一致。此外，使用这种新技术结合FTA卡进行采样可以显着缩短基因分型所需的时间，以及简化程序。此外，在本研究中，在捷克共和国的96只柴犬先前的流行病学筛查的结果进行了重新检查，通过这种实时PCR技术，使用存储的粗颊细胞DNA含有直接作为DNA模板的解决方案。结果提供了明确的基因分型在所有的样本，虽然早期的PCR-RFLP分析不能确定在所有情况下的基因型。总之，这种新的实时PCR技术是一种简单，快速，可靠的选择，大规模筛选，以检测异常等位基因指示GM 1神经节苷脂沉积症在柴犬。
Real-time polymerase chain reaction (PCR) with TaqMan minor groove binder (MGB) probes was examined to establish a rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs. This technique was applied to DNA samples extracted from the blood, umbilical cord, or postmortem liver tissue specimens, and to DNA-containing solutions prepared from blood and saliva that had been applied to Flinders Technology Associates filter papers (ETA cards). The amplification of the targeted sequence in all the samples was sufficient to determine the genotypes of GM I gangliosidosis. Forty-seven DNA samples that had previously been obtained from blood or tissue specimens of Shiba dogs were examined using this real-time PCR technique, and the findings were consistent with the data obtained by the earlier PCR restriction fragment length polymorphism (RFLP) assay. In addition, the use of this new technique in combination with FTA cards for sampling could markedly shorten the time required for genotyping, as well as simplify the procedure. Furthermore, in the present study, the results of a previous epidemiological screening of 96 Shiba dogs in the Czech Republic were rechecked by this real-time PCR technique using stored crude buccal cell DNA-containing solutions directly as DNA templates. The results provided clear-cut genotyping in all the samples although the earlier PCR-RFLP assay could not determine the genotype in all cases. In conclusion, this new real-time PCR technique is a simple, rapid, and reliable choice for large-scale screening to detect an abnormal allele indicating GM1 gangliosidosis in Shiba dogs.