奶牛白细胞黏附缺陷症HRM检测方法的建立及应用  

Development and application of high resolution melting analysis for detection of BLAD alleles

作　　者：贾广乐[1] 周莉[1,2] 林祥梅[1] 廖娟红[1] 

机构地区：[1]中国检验检疫科学研究院,北京100121 [2]河南省商业科学研究所有限责任公司,河南郑州450002

出　　处：《中国兽医科学》2013年第12期1274-1279,共6页Chinese Veterinary Science

基　　金：质检公益项目(201210122)

摘　　要：根据GenBank中奶牛白细胞黏附缺陷症(BLAD)CD18序列设计引物,进行PCR扩增后纯化产物,构建A/A型、A/G型和G/G型3种标准质粒,用标准质粒建立HRM检测方法。用建立的方法对300份奶牛血液样品进行检测,将其结果与Sanger测序结果进行对比,分析和验证检测结果的准确性。结果显示,样品中A/A基因型检出294例,占总体的98%;A/G基因型检出6例,占总体的2%,且两种检测方法的结果一致。结果表明,HRM检测BLAD具有简便快速、特异和灵敏等优点,可作为BLAD携带者突变筛查的优选方法。Bovine leukocyte adhesion deficiency（BLAD） is an autosomal recessive lethal genetic dis- ease,caused by single nucleotide mutation（A-＊G） of CD18 gene,resulting in the significantly reduction or deficiency of β2 integrin expression on leukocyte surface. Thus this disease has a bad effect on bovine productivity and dairy economic benefit. 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 genotype A/A, A/G and G/G were constructed. High resolution melting（HRM） analy- sis was established using these constructed standard plasmids. Then, bovine blood samples were detected by this method. 300 bovine blood samples were detected by the method of high resolution melting analysis, and the results showed that A/A genotype were 294 cases, accounting for 98% of the overall samples. A/G genotype were 6 cases,accounting for 2 %. The results were consistent with those by Sanger sequencing. It showed that high resolution melting curve analysis was a simple,accurate and sensitive approach for rapid detecting of BLAD and could he used as an optimized method for genetic test.

关 键 词：奶牛白细胞黏附缺陷症 CD18基因 高分辨熔解曲线分析 

分 类 号：S856.1[农业科学—临床兽医学]