荷斯坦牛瓜氨酸血症焦磷酸测序检测方法的建立与应用

为建立一种快速高通量的荷斯坦牛瓜氨酸血症(Citrullinemia,CN)分子检测方法,依据CN是由于精氨酸琥珀酸(ASS)合成酶基因编码第86位精氨酸的密码子突变为终止密码子的遗传学基础,利用Assay Design SW软件设计了1对引物和1条测序引物对三种不同基因型的基因材料进行测序分析。用建立的方法对55份进境荷斯坦牛血样进行检测,发现有1例为隐性基因携带者,将PCR产物进行常规测序,结果与焦磷酸测序检测结果一致。研究表明该方法可用于荷斯坦牛瓜氨酸血症的检测,是一种有效的新方法。

The Development and Application of Pyrosequencing for Detecting Citrullinemia in Holstein Cows

Citrullinemia （CN） is caused by a transition of C→T at codon 86 within exon 5 in the gene coding for argininosuccinate synthetase enzyme（ASS）. To develop a rapid and high-lfux molecular detection method for CN, one pair of PCR primers and one sequencing primer were designed by the Assay Design SW software. The PCR products of three different genotypes were detected via pyrosequencing method. 55 blood samples from imported Holstein cows were detected by the established protocol. There was one CN carrier in 55 samples. The result of sequencing for the PCR product of the CN carrier was consistent with the pyrosequencing. This study provided a more reliable and useful method for detecting CN and also offered a theoretical basis for molecular diagnosis in Holstein cows.