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

中国畜牧兽医

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

周莉¹^，2，贾广乐¹，林祥梅¹，廖娟红¹

（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）

收稿日期:2013-08-19 出版日期:2014-05-20 发布日期:2014-06-25

Development and Application of Pyrosequencing Method for Detection of BLAD

ZHOU Li¹^，2，JIA Guang-le¹，LIN Xiang-mei¹，LIAO Juan-hong¹

（1.Chinese Academy of Inspection and Quarantine，Beijing 100121，China；2.Institute of Commercial Sciences Co.,Ltd. of Henan Provice，Zhengzhou 450002，China）

Received:2013-08-19 Online:2014-05-20 Published:2014-06-25

摘要/Abstract

摘要： 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。

关键词:

奶牛白细胞黏附缺陷症; CD18基因; 焦磷酸测序

Abstract: 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.

Key words: font-size: 10.5pt; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA" lang="EN-US">bovine leukocyte adhesion deficiency; CD18 gene; pyrosequencing method

周莉，贾广乐，林祥梅，廖娟红. 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[J]. 中国畜牧兽医.

ZHOU Li，JIA Guang-le，LIN Xiang-mei，LIAO Juan-hong. Development and Application of Pyrosequencing Method for Detection of BLAD[J]. .

参考文献

1 马金柱，宋佰芬，王北艳，等.黑龙江省某牛场种公牛牛白细胞粘附缺陷病（BLAD）的调查［J］.黑龙江八一农垦大学学报，2011，23(5):35～37.
2 王洪梅，李建斌，许尚忠，等.中国荷斯坦牛白细胞黏附缺陷症检测方法的研究进展［J］.生物技术通报，2007，3:155～158.
3 张晓丹，武海萍，周国华.焦测序技术及其在遗传分析中的应用［J］.中国分析化学，2006，34(4):582～586.
4 李艳华，张胜利，韩广文，等.利用单链构象多态性检测牛白细胞黏附缺陷症方法的建立［J］.中国兽医学报，2007，29(12):1471～1474.
5 李艳华，韩广文，张胜利，等.牛白细胞黏附缺陷症(BLAD)的检测与单倍型分析［J］.畜牧兽医学报，2008，39(9):1285～1288.
6 周莉，贾广乐，张映，等.奶牛白细胞黏附缺陷症(BLAD)检测方法的研究进展［J］.中国畜牧兽医，2012，39(2):137～140.
7 范学华，张毅，孙东晓，等.中国荷斯坦种公牛BLAD遗传缺陷的分子检测及系谱分析［J］.中国奶牛，2011，8(2):1～3.
8 倪红兵，王惠民.焦磷酸测序技术及其应用进展［J］.医学检验与临床，2006，17(2):1～3.
9 袁群英，金庆生.焦磷酸测序技术及其在水稻单核苷酸多态性研究中的应用［J］.浙江农业学报，2007，19(4):325～328.
10 崔玉东，朴范泽.牛白细胞黏附缺陷病的研究进展［J］.中国兽医学报，2001，21(2):204～207.
11 曹宏伟.牛白细胞黏附缺陷病（BLAD）诊断及白细胞功能研究［D］.大庆：黑龙江八一农垦大学，2005.
12 葛剑徽. 焦磷酸测序技术的原理及应用［J］.科技资讯，2006(4):216～217.
13 Alifrangis M,Enosse S,Pearce R,et al.A simple,high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase,dihydropteroate synthase,and P.falciparum chloroquine resistance transporter genes using polymerase chain reaction and enzyme-linked immunosorbent assay-based technology［J］.Am J Trop Med Hyg,2005,72(2):155～162.
14 Chessa S,Chiatti F,Ceriotti G,et al.Development of a single nucleotide polymorphism genotyping microarray platform for the identification of bovine milk protein genetic polymorphisms［J］.J Dairy Sci,2007,90(1):451～464.
15 Gerardi A S.Bovine leukocyte adhesion deficiency:A review of a modern disease and its implications［J］.Res Vet Sci,1996,61(3):183～186.
16 Gharizadeh B,Akhras M,Nourizad N,et al.Methodological improvements of pyrosequencing technology［J］.J Biotechnol,2006,124(3):504～511.
17 Kehrli M E Jr,Schmalstieg F C,Anderson D C,et al.Molecular definition of the bovine granulocytopathy syndrome: Identification of deficiency of the Mac-1(CD11b/CD18) glycoprotein［J］.Am J Vet Res,1990,51(11):1826～1836.
18 Nagahata H,Higuchi H,Teraoka H,et al.Decreased apoptosis of β2 integrin deficient bovine neutrophils［J］.Immunol Cell Biol,2004,82(1):32～37.
19 Nyren P. The history of pyrosequencing［J］.Methods Mol Biol,2006,373:1～14.
20 Ronagi M，Uhlen M，Nylen P.A sequencing method based on real time pyrophosphate［J］.Science,1998,281(5375):363～365.
21 Schutz E,Scharfenstein,Brenig B.Implication of complex vertebral malformation and bovine leukocyte adhesion deficiency DNA-based testing on disease frequency in the Holstein population［J］.Journal of Dairy Science,2008,91(12):4854～4859.