Van Poucke Mario, Vandesompele Jo, Mattheeuws Marc, Van Zeveren Alex, Peelman Luc J
Department of Animal Genetics and Breeding, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium.
BMC Infect Dis. 2005 Mar 15;5:13. doi: 10.1186/1471-2334-5-13.
Scrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programmes towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Therefore, a reliable, fast and cost-effective method for routine PRNP genotyping in sheep, applicable in standard equipped molecular genetic laboratories, will be a vital instrument to fulfill the need of genotyping hundreds or thousands of sheep.
A dual fluorescent multiprobe assay consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above.
The assay is successfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep.
We report the development and validation of a robust, reliable and reproducible method for PRNP genotyping of a few to many sheep samples in a fast, simple and cost-effective way, applicable in standard equipped molecular genetic laboratories. The described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations.
羊瘙痒病和牛海绵状脑病属于一类称为传染性海绵状脑病（TSE）的致命性、传染性神经退行性疾病。为了将绵羊自然感染羊瘙痒病以及疑似自然感染牛海绵状脑病的风险降至最低，许多国家开展了针对TSE抗性的育种计划，该计划基于密码子136（A/V）、154（R/H）和171（R/H/Q）处的抗性赋予PRNP多态性。因此，一种可靠、快速且经济高效的绵羊PRNP基因分型常规方法，适用于配备标准设备的分子遗传实验室，将成为满足对数百或数千只绵羊进行基因分型需求的重要工具。
一种双荧光多重探针检测法，由2个封闭管PCR反应组成，分别包含4个和3个双标记荧光ASO探针，用于实时检测上述绵羊PRNP的7个等位基因变体。
该检测法以未纯化的DNA作为PCR模板成功进行，无需任何PCR后处理，且可半自动确定PRNP基因型。在对50只绵羊进行的交叉验证研究中，通过PCR-RFLP和测序证实了该检测法的性能。
我们报告了一种稳健、可靠且可重复的方法的开发与验证，该方法可快速、简单且经济高效地对几只到许多只绵羊样本进行PRNP基因分型，适用于配备标准设备的分子遗传实验室。所描述的引物/探针设计策略也可用于检测其他多态性或致病突变。
