奶牛白细胞黏附缺陷症焦磷酸测序检测方法的建立及应用

奶牛白细胞黏附缺陷症（bovine leukocyte adhesion deficiency，BLAD）是一种常染色体隐性、致死性、遗传性疾病，严重影响奶牛生产性能和奶牛场的经济效益。为建立快速可靠的BLAD检测方法，本试验根据GenBank中BLAD CD18序列设计引物，PCR扩增后纯化产物，构建A/A、A/G和G/G 3种基因型标准质粒。用标准质粒建立焦磷酸测序检测方法。用建立的方法检测奶牛血液样品，其结果与Sanger测序结果进行比较，分析和验证检测结果的准确性。用建立的方法对300份奶牛血液样品进行检测，结果显示样品中A/A基因型检出294例，占总体比例的98%；A/G基因型检出6例，占总体比例的2%。随机抽取30份已检测样品进行Sanger测序,结果显示与焦磷酸测序法结果一致。本试验结果表明焦磷酸测序法检测BLAD，具有特异、灵敏、快速的特点，其检测结果准确可靠，适合于试剂盒的研发及应用于BLAD的临床诊断。

Development and Application of Pyrosequencing Method for Detection of BLAD

Bovine leukocyte adhesion deficiency (BLAD) was an autosomal recessive lethal genetic disease, the caused BLAD had a bad effect on the bovine production trait and dairy economic returns. In order to establish a rapid and reliable method for BLAD detection, in this experiment, according to BLAD CD18 gene sequences published in GenBank, PCR primers were designed. After PCR amplification and purification, three standard plasmids including genotypes A/A, A/G and G/G were constructed. Pyrosequencing method were established by using these constructed standard plasmids. Then, the bovine blood samples were detected by the method. The results were compared with Sanger sequencing results to analyze and verify the accuracy of the test results. 300 bovine blood samples of BLAD were detected by pyrosequencing method，the results showed A/A genotype were 294 cases, accounting for 98% of the overall proportion; A/G genotype were 6 cases, accounting for 2%; and the results of two detection methods were consistent. It showed that pyrosequencing method to detect BLAD were specific, sensitive and rapid, and the test results were accurate and reliable. It was suitable for kit development and applied in clinical diagnosis of BLAD.