Chang Hye-Sook, Arai Toshiro, Yabuki Akira, Hossain Mohammad A, Rahman Mohammad M, Mizukami Keijiro, Yamato Osamu
Laboratory of Clinical Pathology, Department of Veterinary Clinical Sciences, Faculty of Agriculture, Kagoshima University, 1-21-24 Kohrimoto, Kagoshima 890-0065, Japan.
J Vet Diagn Invest. 2010 Mar;22(2):234-7. doi: 10.1177/104063871002200210.
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 (FTA cards). The amplification of the targeted sequence in all the samples was sufficient to determine the genotypes of GM1 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.
研究了使用TaqMan小沟结合剂（MGB）探针的实时聚合酶链反应（PCR），以建立一种快速可靠的柴犬GM1神经节苷脂贮积症基因分型技术。该技术应用于从血液、脐带或死后肝脏组织标本中提取的DNA样本，以及应用于弗林德斯技术协会滤纸（FTA卡）的血液和唾液制备的含DNA溶液。所有样本中目标序列的扩增足以确定GM1神经节苷脂贮积症的基因型。使用这种实时PCR技术对之前从柴犬的血液或组织标本中获得的47个DNA样本进行了检测，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，将这项新技术与FTA卡结合用于采样可以显著缩短基因分型所需的时间，并简化操作程序。此外，在本研究中，使用直接将储存的含粗颊细胞DNA的溶液作为DNA模板的这种实时PCR技术，对捷克共和国96只柴犬先前的流行病学筛查结果进行了重新检查。尽管早期的PCR-RFLP分析不能在所有情况下确定基因型，但结果在所有样本中都提供了明确的基因分型。总之，这种新的实时PCR技术是大规模筛查检测柴犬中指示GM1神经节苷脂贮积症的异常等位基因的简单、快速且可靠的选择。
