CN107034274B - 一种水貂自咬行为诊断基因及其诊断方法 - Google Patents
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Abstract
本发明提供了一种水貂自咬行为诊断基因,采用尾分析法对健康水貂和自咬行为水貂基因组DNA池进行RAPD扩增筛选随机引物,获得健康水貂和自咬行为水貂基因池中差异标记基因;回收、克隆随机引物A10的RAPD特异标记片段,经测序后获得该片段的全序列;所测的SA10‑1000基因序列的长度为959bp,将所测序列通过GenBank中的Blast序列比较分析,从比较结果得知,所测的SA10‑959扩增序列与犬属布氏杆菌的同源性达73%。通过GenBank中的Blast序列比较分析,从比较结果得知,所测的SA10‑959扩增序列与犬属布氏杆菌的同源性达73%。具有特异性强、灵敏度高、快速准确且自动化程度高等特点,使其适合于临床的快速诊断和牧区、村镇等流行病学调查大规模应用。
Description
技术领域
本发明提供了一种水貂自咬行为诊断基因,进一步本发明还涉及利用该诊断基因设计自咬行为个体特异性的环介导恒温核酸扩增引物,可用于快速诊断水貂群体中的自咬行为个体的检测方法,属于鼬科动物疾病检测技术领域。
背景技术
自咬行为(self-biting behavior,SB)是一种发生机理未明的异常行为。行为学家将其定义为有意、直接伤害自体组织的行为模式,多见于笼养条件下饲养的动物和患有精神性疾病或智力障碍、痴呆的病人。毛皮动物的自咬症是指水貂和狐狸等肉食性毛皮动物的一种以反复咬伤身体后躯和尾部为主要症状的慢性疾病,是毛皮动物养殖业中最为严重的疾病之一。目前,世界上主要的毛皮动物饲养大国均有该病发生,发病率约为5~20%。丹麦报道的发病率为20%。全世界每年仅水貂皮的生产量约为3 000~3 500万张,由于自咬症导致的等级下降的皮张在200~300万张,每年给世界毛皮动物饲养业带来严重的经济损失。我国1967~1970年已有水貂自咬症发病的报道。国内每年水貂自咬症的发病率约为5~10%。因此对水貂自咬行为的动态监测、诊断并及时治疗对预防和彻底根除该行为具有重大的意义。目前,对于水貂自咬行为的检测方法多为PCR扩增、RAPD和SCAR检测方法。但以上方法存在重复性和稳定性差,以及操作复杂不利于现场推广等问题。因此,选择高特异性的抗原,建立具有高敏感性、高特异性的水貂自咬行为诊断方法十分必要。
发明内容:
本发明公开了一种水貂自咬行为诊断基因,采用尾分析法对健康水貂和自咬行为水貂基因组DNA池进行RAPD扩增筛选随机引物,获得健康水貂和自咬行为水貂基因池中差异标记基因。回收、克隆随机引物A10的RAPD特异标记片段,经测序后获得该片段的全序列。所测的SA10-1000基因序列的长度为959bp,将所测序列通过GenBank中的Blast序列比较分析,从比较结果得知,所测的SA10-959扩增序列与犬属布氏杆菌的同源性达73%。
本发明所述的一种水貂自咬行为诊断基因,如SQY NO:1所示。
本发明所述一种水貂自咬行为诊断基因的制备方法,包括以下步骤:
用基因组 DNA 提取试剂盒从采取健康水貂和自咬症水貂肌肉样本中提取基因组DNA,分别组成基因池用于RAPD扩增;
随机引物序列如下:
A10引物序列GTGATCGCAG;
将随机引物A10对健康和自咬水貂基因组DNA进行扩增,发现该引物在自咬水貂基因组中扩增到1条特异性条带 ;回收、克隆随机引物的RAPD特异标记片段,经测序后获得该片段的全序列;
所测基因序列的长度为959bp(图1),并命名为SA10-959,将所测序列通过GenBank中的Blast序列比较分析,从比较结果得知,所测的基因序列与犬属布氏杆菌的同源性达73%;SA10-959特异标记片段的全序列如 SQY NO:1 所示。
本发明水貂自咬行为诊断基因诊断水貂群体中的自咬行为个体的检测方法,包括以下步骤:
采用水貂自咬行为诊断基因序列做为候选靶基因序列,设计了一套特异性的LMAP引物。LMAP的引物设计是采用官方软件Primer Explorer V3.0;首先通过浏览器登录LAMP官方网站 http://primerexplorer.jp,进入Primer Explorer V3.0软件使用界面,导入文本格式的靶序列;
LMAP的引物序列如下:
F3引物序列 CGGGTGATTTCCGGTTTGA
B3引物序列 ACGCAGCCATGATCTTCTTG
FIP引物序列 TCCAGCGTCACCGTCACGCGTCCGAAGAGCCGTGAGG
BIP引物序列 AAGGCAGTCGGCATGGGCGGCGGGATCAACCGCAAT
LF引物序列 GTTGGGAAGACCGATAAGAGC
LB引物序列 GGGGATCGCGGCTGTTTCT。
所述的LAMP反应体系为:2.5μL 10×buffer、1.2 mM dNTP、F3和B3引物0.2μM、FIP和BIP引物1.6μM、LF和LB引物0.8μM、1.0M甜菜碱、8mM Mg2+、1μL Bst DNA聚合酶、2μL模板;62.1°C 反应1小时,80°C加热2分钟;分别以健康水貂和自咬水貂DNA为模板,同时进行LAMP扩增,发现只有自咬水貂个体能扩增出LAMP反应特有的梯形条带。
本发明所述的诊断基因可以制备水貂群体中的自咬行为个体试剂盒。
本发明的积极效果在于:
提供了一种水貂自咬行为诊断基因,通过GenBank中的Blast序列比较分析,从比较结果得知,所测的SA10-959扩增序列与犬属布氏杆菌的同源性达73%。具有特异性强、灵敏度高、快速准确且自动化程度高等特点,使其适合于临床的快速诊断和牧区、村镇等流行病学调查大规模应用。
附图说明
图1为本发明A10随机引物对健康和自咬水貂DNA的扩增结果1~5为健康水貂 6~10为自咬水貂;
图2为本发明LAMP反应对水貂自咬症的的特异性检测电泳图:M, DL2000;泳道1,2是自咬水貂DNA;泳道3,4为健康水貂DNA;N,阴性对照。
具体实施方式:
下列实施例旨在进一步举例说明,而不是限制本发明。本领域技术人员可以理解到,在不背离本发明的精神和原则的前提下,对本发明的任何平行改变和改动都将落入本发明的待批权利要求范围内。
实施例1:
本发明一种水貂自咬行为诊断基因的制备方法,包括以下步骤:
用基因组 DNA 提取试剂盒从采取健康水貂和自咬症水貂肌肉样本中提取基因组DNA,分别组成基因池用于RAPD扩增;
随机引物序列如下:
A10引物序列GTGATCGCAG;
将随机引物A10对健康和自咬水貂基因组DNA进行扩增,发现该引物在自咬水貂基因组中扩增到1条特异性条带;
回收、克隆随机引物的RAPD特异标记片段,经测序后获得该片段的全序列;
所测基因序列的长度为959bp(图1),并命名为SA10-959,将所测序列通过GenBank中的Blast序列比较分析,从比较结果得知,所测的基因序列与犬属布氏杆菌的同源性达73%;SA10-959特异标记片段的全序列如 SQY NO:1 所示。
实施例2:
本发明水貂自咬行为诊断基因诊断水貂群体中的自咬行为个体的检测方法,包括以下步骤:
采用水貂自咬行为诊断基因序列做为候选靶基因序列,设计了一套特异性的LMAP引物。LMAP的引物设计是采用官方软件Primer Explorer V3.0;首先通过浏览器登录LAMP官方网站 http://primerexplorer.jp,进入Primer Explorer V3.0软件使用界面,导入文本格式的靶序列;
LMAP的引物序列如下:
F3引物序列 CGGGTGATTTCCGGTTTGA
B3引物序列 ACGCAGCCATGATCTTCTTG
FIP引物序列 TCCAGCGTCACCGTCACGCGTCCGAAGAGCCGTGAGG
BIP引物序列 AAGGCAGTCGGCATGGGCGGCGGGATCAACCGCAAT
LF引物序列 GTTGGGAAGACCGATAAGAGC
LB引物序列 GGGGATCGCGGCTGTTTCT。
LAMP反应体系为:2.5μL 10×buffer、1.2 mM dNTP、F3和B3引物0.2μM、FIP和BIP引物1.6μM、LF和LB引物0.8μM、1.0M甜菜碱、8mM Mg2+、1μL Bst DNA聚合酶、2μL模板;62.1°C反应1小时,80°C加热2分钟;分别以健康水貂和自咬水貂DNA为模板,同时进行LAMP扩增,发现只有自咬水貂个体能扩增出LAMP反应特有的梯形条带(图2)。
为了验证LMAP标记的特异性和适用性。选水貂养殖厂取皮期的自咬水貂和健康水貂各30只,分别提取DNA,并分别采用RAPD和LMAP标记扩增并进行验证。卡方检验结果如表1所示,特异LMAP标记在健康和自咬水貂群体之间的比例差异极显著(P<0.001)。与RAPD标记相比较,LMAP标记方法更准确的区分健康与自咬水貂个体,并且更加稳定、可靠。LAMP反应可合成109数量级的DNA,同时产生大量的焦磷酸根离子,它们能与镁离子结合生成白色的焦磷酸镁沉淀,可根据反应体系中是否形成白色沉淀来快速定性判断LAMP反应。因此,在实际生产中,可以用此简化方法对水貂群体进行大规模鉴定,既快速简便,又大大降低成本。
表1 健康水貂和自咬水貂中RAPD和LMAP标记分布情况
<110> 中国农业科学院特产研究所
<120>
<140>
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<211> 959
<212> DNA
<213> 水貂(Neovison vison)
<400>
1 CACTAGCGTC GCCCTGCTAA GTCGATCTTG ACGCCGCGTT GGTCGAACCC
51 TCTCTTACTA CTCGCCGCAA GTCGGGCTAA TGTGCGAAGT GTCCCTTCGT
101 GGTAGGCAAC TGCTAATAAC GGCCAACGCC CACTAAAGGC CAAACTCAGG
151 CTTCTCGGCA CTCCGCCGAG AATAGCCAGA AGGGTTGTGC CAGACGCCGC
201 ACTGCCACTG CGACCTGCCG TTCCGTCAGC CGTACCCGGC GTAGCAGCCC
251 CTAGCGCCGA CAAAGATAGT CCAGCACCTG TAACGCCAAC TAGGGCGGGT
301 AGTCCGCGCG CCCGAACCGT TCTTCTAGTA CCGACGCAAC TACCTGGTAC
351 ACCGCCGCCT TCGCGGACTA CCGCGGATAC AGTCGGAGTA GCGGCTACCG
401 GGACGGTTTG TAGAGATGCG TTTTAAGCCA AAGCTCGGCC ACTGCGGCCT
451 AAGGTAGCCG TACCGCAAGG CCCACAGCGC GTGGACTCAA GTCCGCTCGC
501 AGAAGTTGTG GTCTAGCTCG CGGCAAAGCT GCCGCAAAAC GGCCTACGAG
551 CGCGCAGGGA CTAGCCCTTT TTGTACGGCG TGTCGGACGC CGCGGACAGG
601 GAACTCCCGC TGTGGCTCGG TCTGCTCTGG TTGGCCGAAC CGGCGGCTAG
651 GCGGCGGCCC TGGACGTTGT GGGCAGTGGC GGTGCCGTTA CGGCCGCGCG
701 CTCACAACGT TACGCGGGCG CCGGTACCGG TCGCGGTGAA GAAGGCTATG
751 CCGCGGTACC CGTCACTAGT GCAGGTACCC TTACGGCTCG TAGCGCACGA
801 ACCGAAGCAA CCTTATCCAC TACTTCGGTG CTAGGTGGTG TAGCCTACTA
851 GGCGGCTACA GCCACTCGCT ACGGTGTAGT CGGGGGCACG TCTAAGCCGA
901 CACGTCGTCT ACGAGACGCG CGTCGAAGGT CTTGCGCTAC GAAAACAGAG
951 ACGCTAGTG
<210>2
<211>19
<212>DNA
<213>人工合成
<220>
<221>引物(primer)
<222> (1)..(19)
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CGGGT GATTT CCGGT TTGA 19
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<211>10
<212>DNA
<213>人工合成
<220>
<221>引物(primer)
<222> (1)..(10)
<223>
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ACGCA GCCAT GATCT TCTTG 20
<210>4
<211>37
<212>DNA
<213>人工合成
<220>
<221>引物(primer)
<222> (1)..(37)
<223>
<400>4
TCCAG CGTCA CCGTC ACGCG TCCGA AGAGC CGTGA GG 37
<210>5
<211>36
<212>DNA
<213>人工合成
<220>
<221>引物(primer)
<222> (1)..(36)
<223>
<400>5
AAGGC AGTCG GCATG GGCGG CGGGA TCAAC CGCAA T 36
<210>6
<211>21
<212>DNA
<213>人工合成
<220>
<221>引物(primer)
<222> (1)..(21)
<223>
<400>6
GTTGG GAAGA CCGAT AAGAG C 21
<210>7
<211>19
<212>DNA
<213>人工合成
<220>
<221>引物(primer)
<222> (1)..(19)
<223>
<400>7
GGGGA TCGCG GCTGT TTCT 19
Claims (2)
1.一种水貂自咬行为诊断基因,如SEQ ID NO:1所示。
2.一种水貂自咬行为诊断基因诊断试剂盒的制备方法,包括以下步骤:
采用权利要求1所述的水貂自咬行为诊断基因做为候选靶基因序列,设计一套特异性的LMAP引物;
LMAP的引物序列如下:
F3引物序列 CGGGTGATTTCCGGTTTGA;
B3引物序列 ACGCAGCCATGATCTTCTTG;
FIP引物序列 TCCAGCGTCACCGTCACGCGTCCGAAGAGCCGTGAGG;
BIP引物序列 AAGGCAGTCGGCATGGGCGGCGGGATCAACCGCAAT;
LF引物序列 GTTGGGAAGACCGATAAGAGC;
LB引物序列 GGGGATCGCGGCTGTTTCT;
所述的LAMP反应体系为:2.5μL 10×buffer、1.2 mM dNTP、F3和B3引物0.2μM、FIP和BIP引物1.6μM、LF和LB引物0.8μM、1.0M甜菜碱、8mM Mg2+、1μL Bst DNA聚合酶、2μL模板;62.1℃反应1小时,80℃加热2分钟;分别以健康水貂和自咬水貂DNA为模板,同时进行LAMP扩增。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102154457A (zh) * | 2011-01-05 | 2011-08-17 | 吉林大学 | 一种笼养水貂自咬症的分子标记诊断方法 |
CN104928391A (zh) * | 2015-06-26 | 2015-09-23 | 山东省农业科学院生物技术研究中心 | 鉴别4种犬科动物源性成分的引物探针组合物、试剂盒及多重实时荧光pcr检测方法 |
CN105796974A (zh) * | 2016-04-29 | 2016-07-27 | 青岛海之星生物科技有限公司 | 一种治疗水貂自咬症的饲料添加剂及其制备方法 |
CN107034274A (zh) * | 2017-04-13 | 2017-08-11 | 中国农业科学院特产研究所 | 一种水貂自咬行为诊断基因及其诊断方法 |
-
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102154457A (zh) * | 2011-01-05 | 2011-08-17 | 吉林大学 | 一种笼养水貂自咬症的分子标记诊断方法 |
CN104928391A (zh) * | 2015-06-26 | 2015-09-23 | 山东省农业科学院生物技术研究中心 | 鉴别4种犬科动物源性成分的引物探针组合物、试剂盒及多重实时荧光pcr检测方法 |
CN105796974A (zh) * | 2016-04-29 | 2016-07-27 | 青岛海之星生物科技有限公司 | 一种治疗水貂自咬症的饲料添加剂及其制备方法 |
CN107034274A (zh) * | 2017-04-13 | 2017-08-11 | 中国农业科学院特产研究所 | 一种水貂自咬行为诊断基因及其诊断方法 |
Non-Patent Citations (2)
Title |
---|
水貂自咬症病因RAPD遗传分析;李玉梅等;《生物工程学报》;20080430;第24卷(第4期);摘要,第565页1材料与方法 * |
水貂自咬症遗传病因的SCAR分子标记分析;李秋芳等;《西北农业学报》;20101231;第19卷(第4期);摘要,第29页1材料与方法 * |
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