CN107419024B - 犬髋关节发育不良相关snp标志物组 - Google Patents
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Abstract
本发明提供了检测SNP位点基因型的试剂在制备预测拉布拉多犬或金毛巡回猎犬犬髋关节发育不良(CHD)的试剂盒中的应用,其中SNP位点选自下列位点中的一个或多个:BICF2P772455、BICF2P548082、BICF2G630339806、BICF2G630558239、BICF2G630227898、BICF2S230609、BICF2S2452559、TIGRP2P265674、BICF2P1086886、BICF2P355865、BICF2P281364、BICF2S2367279。
Description
技术领域
本发明属于兽医分子生物学诊断领域,具体地说,本申请涉及检测SNP位点组基因型的试剂在制备预测犬髋关节发育不良(CHD)的试剂盒中的应用以及相应的试剂盒产品。
背景技术
犬髋关节发育不良(Canine hip dysplasia,CHD)是一种几乎存在于所有犬种中的常见遗传病。所有品种的犬均可发生髋关节发育不良,其发病率在品种间有差异。据动物矫形外科基金会(OFA)的资料报道,不同品种的发病率从1%-75%不等(Janutta & Distl,2006),且大型犬发病率更高,其中尤以拉布拉多犬、金毛寻回猎犬、伯恩山犬和德国牧羊犬中最常见。该病是由于在犬发育过程中肌肉与骨骼以不同速度发育成熟,致使主要依赖肌肉组织固定的髋关节变松弛,从而引发变性性骨关节病以及髋关节半脱位、甚至脱位,而最终导致髋关节发育异常。
犬髋关节发育不良是一种多基因控制的遗传性疾病,此外这种疾病的发生和发展还受到环境、营养状况等因素的影响。该病属于多基因遗传病,不同个体间病症差异较大,病理变化是一个从非常轻微的变化到几乎没有髋臼的完全脱位的连续过程。目前针对CHD的诊断和预防都是建立在通过X光拍摄进行诊断,目前被普遍认可的是美国宾西法利亚大学推广的Pennyhip方法,即通过髋关节与股骨头的位置关系和关节的炎症程度对犬进行诊断(Vezzoni & Tavola, 2015) 。但是X光片诊断法虽然最早在四月龄时就可以进行,但是繁育期之前的幼犬由于年龄较轻,犬肌肉和骨骼尚未发育完全,导致诊断的准确率很低。因此,很难通过选育的方法有效地杜绝该病的发生。
基因检测在幼犬出生后就可以进行,对于携带致病基因的犬可以在其性成熟前就得到确诊,从而避免其参与繁育,这样就可以有效提高犬质量,降低下一代的发病率(据统计,正常双亲的后代,CHD的发生率是37.5%;双亲之一患CHD,后代的发生率为44.7%;双亲都患CHD,后代患病率则高达84.1%。)。目前对于CHD致病基因的研究已经取得了一定进展,尤其是当狗的全基因组序列完成测序并且获得了超过250万个SNP位点后(Lindblad-Toh etal., 2005),更是加快了该项研究的速度。目前已经在多个犬种中定位了几十个致病位点(Bartolomé et al., 2015; Fels, Marschall, Philipp, & Distl, 2014; Pfahler &Distl, 2012)。例如Bartolomé等(Bartolomé, N., Segarra, S., Artieda, M.,Francino, O., Sánchez, E., Szczypiorska, M., Martínez, A. (2015). A geneticpredictive model for canine hip dysplasia: Integration of genome wideassociation study (gwas) and candidate gene approaches. PloS one, 10(4),e0122558)通过将患有CHD的拉布拉多犬和正常拉布拉多犬进行全基因组关联分析(GWAS),在17万个SNP位点中定位到了7个与CHD相关的突变位点。这7个SNP位点分布于犬的七条不同的染色体上,涵盖了拉布拉多犬中85%的CHD遗传力因素,文献中诊断灵敏度达到80%,而诊断特异性达到78%。
申请人阅读Bartolomé文献后,在拉布拉多犬中使用文献中的7个位点选育,并尝试将这七个位点用于尚缺乏研究的金毛巡回猎犬中。在拉布拉多犬中取得了一定的效果。但实践结果表明,该7个位点在拉布拉多犬中效果不佳,特别是特异性不足(可能与繁育基地中犬的品种和亲缘关系有关,文献中使用的是较纯正的欧洲和美国血统,国内拉布拉多犬血统纯正度一般不高),而且该7个位点在金毛巡回猎犬金毛巡回猎犬中效果更差。此外。检测SNP位点时,直接测序法和荧光探针法有反应次数多和探针标记复杂,费用高的问题。
发明内容
为解决上述问题,一方面,申请人查阅大量相关文献总结出数十个可能与CHD相关的位点,对拉布拉多犬和金毛巡回猎犬中这些SNP与疾病的关联进行了重新统计分析,确定了适合中国常见拉布拉多犬和金毛巡回猎犬种犬髋关节发育不良预测的SNP组合;另一方面,申请人采用等位基因特异性PCR技术(allele-specific PCR)和后标记荧光技术(post-labeling method),从而达到在同一反应中同时检测多个SNP位点的目的,并且只需要两条标记荧光的通用引物即可,节省了费用。
一方面,本发明提供了检测SNP位点基因型的试剂在制备预测犬髋关节发育不良(CHD)的试剂盒中的应用,其中SNP位点选自下列位点中的一个或多个:BICF2P772455、BICF2P548082、BICF2G630339806、BICF2G630558239、BICF2G630227898、BICF2S230609、BICF2S2452559、TIGRP2P265674、BICF2P1086886、BICF2P355865、BICF2P281364、BICF2S2367279。
进一步的方面,其中犬为拉布拉多犬或金毛巡回猎犬。
进一步的方面,其中犬为金毛巡回猎犬。
进一步的方面,其中检测SNP位点基因型的试剂为引物,引物序列和标记为:
通用引物:SEQ ID NO.1和SEQ ID NO.2,SEQ ID NO.1和SEQ ID NO.2的5’端分别标记6-FAM和ROX;
BICF2P772455:SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5;
BICF2P548082:SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8;
BICF2G630339806:SEQ ID NO.9、SEQ ID NO.10和SEQ ID NO.11;
BICF2G630558239:SEQ ID NO.12、SEQ ID NO.13和SEQ ID NO.14;
BICF2G630227898:SEQ ID NO.15、SEQ ID NO.16和SEQ ID NO.17;
BICF2S230609:SEQ ID NO.18、SEQ ID NO.19和SEQ ID NO.20;
BICF2S2452559:SEQ ID NO.21、SEQ ID NO.22和SEQ ID NO.23;
TIGRP2P265674:SEQ ID NO.24、SEQ ID NO.25和SEQ ID NO.26;
BICF2P1086886:SEQ ID NO.27、SEQ ID NO.28和SEQ ID NO.29;
BICF2P355865:SEQ ID NO.30、SEQ ID NO.31和SEQ ID NO.32;
BICF2P281364:SEQ ID NO.33、SEQ ID NO.34和SEQ ID NO.35;
BICF2S2367279:SEQ ID NO.36、SEQ ID NO.37和SEQ ID NO.38。
进一步的方面,其中SNP位点为:
BICF2G630227898,BICF2G630339806,BICF2S230609,BICF2G630558239,BICF2P355865,和BICF2P281364;或者BICF2S2367279,BICF2P281364,BICF2P1086886,BICF2P355865,BICF2G630227898,BICF2S230609,和BICF2P548082。
另一方面,本发明提供了预测犬髋关节发育不良(CHD)的试剂盒,其中包含检测SNP位点基因型的试剂,其中SNP位点选自下列位点中的一个或多个:BICF2P772455、BICF2P548082、BICF2G630339806、BICF2G630558239、BICF2G630227898、BICF2S230609、BICF2S2452559、TIGRP2P265674、BICF2P1086886、BICF2P355865、BICF2P281364、BICF2S2367279。
进一步的方面,其中检测SNP位点基因型的试剂为引物,引物序列和标记为:
通用引物:SEQ ID NO.1和SEQ ID NO.2,SEQ ID NO.1和SEQ ID NO.2的5’端分别标记6-FAM和ROX;
BICF2P772455:SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5;
BICF2P548082:SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8;
BICF2G630339806:SEQ ID NO.9、SEQ ID NO.10和SEQ ID NO.11;
BICF2G630558239:SEQ ID NO.12、SEQ ID NO.13和SEQ ID NO.14;
BICF2G630227898:SEQ ID NO.15、SEQ ID NO.16和SEQ ID NO.17;
BICF2S230609:SEQ ID NO.18、SEQ ID NO.19和SEQ ID NO.20;
BICF2S2452559:SEQ ID NO.21、SEQ ID NO.22和SEQ ID NO.23;
TIGRP2P265674:SEQ ID NO.24、SEQ ID NO.25和SEQ ID NO.26;
BICF2P1086886:SEQ ID NO.27、SEQ ID NO.28和SEQ ID NO.29;
BICF2P355865:SEQ ID NO.30、SEQ ID NO.31和SEQ ID NO.32;
BICF2P281364:SEQ ID NO.33、SEQ ID NO.34和SEQ ID NO.35;
BICF2S2367279:SEQ ID NO.36、SEQ ID NO.37和SEQ ID NO.38。
进一步的方面,其中SNP位点为:
BICF2G630227898,BICF2G630339806,BICF2S230609,BICF2G630558239,BICF2P355865,和BICF2P281364;或者BICF2S2367279,BICF2P281364,BICF2P1086886,BICF2P355865,BICF2G630227898,BICF2S230609,和BICF2P548082。
进一步的方面,该试剂盒用于预测拉布拉多犬或金毛巡回猎犬的犬髋关节发育不良(CHD)。
进一步的方面,该试剂盒用于预测金毛巡回猎犬的犬髋关节发育不良(CHD)。
附图说明
图1为BICF2G630558239基因型判定结果示意图。
图2为BICF2G630227898,BICF2G630339806,BICF2S230609,BICF2G630558239,BICF2P355865,BICF2P281364组合在拉布拉多犬中的ROC曲线。
图3为BICF2S2367279,BICF2P281364,BICF2P1086886,BICF2P355865,BICF2G630227898,BICF2S230609,BICF2P548082在金毛巡回猎犬中的ROC曲线。
具体实施方式
实施例1试剂盒的设计和制备
位点选择参考Bartolomé, N., Segarra, S., Artieda, M., Francino, O., Sánchez, E., Szczypiorska, M., Martínez, A. (2015). A genetic predictive modelfor canine hip dysplasia: Integration of genome wide association study (gwas)and candidate gene approaches. PloS one, 10(4), e0122558等文献。从broadinstitute网站的犬基因组信息库Canfam 2.0调取BICF2P772455、BICF2P548082、BICF2G630339806、BICF2G630558239、BICF2G630227898、BICF2S230609、BICF2S2452559、TIGRP2P265674、BICF2P1086886、BICF2P355865、BICF2P281364、BICF2S2367279。的序列信息,以oligo 7.57设计引物,引物由英骏生物合成。
表1:引物信息
引物名称 | 引物序列(5’—3’) | 5’端标记荧光类型 | PCR产物大小(bp) |
FAM-tag | CTAGTATCAGGACTAC(SEQ ID No.1) | 6-FAM | |
ROX-tag | CTAGTATTAGGATGTG(SEQ ID No.2) | ROX | |
CHD-1F | gtttcttTGGCTGGTCGTCTTCAGGTG(SEQ ID No.3) | ||
CHD-1RW | CTAGTATCAGGACTACACAGGGGTGTCCTGACaACT(SEQ ID No.4) | 无 | 88 |
CHD-1RM | CTAGTATTAGGATGTGAGACAGGGGTGTCCTGACaACC(SEQ ID No.5) | 无 | 92 |
CHD-2F | gtttcttAGATAAGCATTTTCATTTTTACTGTCA(SEQ ID No.6) | 无 | |
CHD-2RW | CTAGTATCAGGACTACCTCAGGGATGGTAGGTACATTcTA(SEQ ID No.7) | 无 | 96 |
CHD-2RM | CTAGTATTAGGATGTGCACTCAGGGATGGTAGGTACATTcTG(SEQ ID No.8) | 无 | 100 |
CHD-3F | gtttcttAATCTGGATAGTTGTGAGGCTTTC(SEQ ID No.9) | 无 | |
CHD-3RW | CTAGTATCAGGACTACTCCAGAAGAGATGAGTATCTACAAGTgAC(SEQ ID No.10) | 无 | 105 |
CHD-3RM | CTAGTATTAGGATGTGCTTCCAGAAGAGATGAGTATCTACAAGTgAT(SEQ ID No.11) | 无 | 109 |
CHD-4F | gtttcttCTCTCTCCATACTGGGGCAAG(SEQ ID No.12) | 无 | |
CHD-4RW | CTAGTATCAGGACTACGTGTAATGTGCTATTATTCATGAtCC(SEQ ID No.13) | 无 | 120 |
CHD-4RM | CTAGTATTAGGATGTGTTGTGTAATGTGCTATTATTCATGAtCT(SEQ ID No.14) | 无 | 124 |
CHD-5F | gtttcttGGAAGAGCTCAGGGCACATAG(SEQ ID No.15) | 无 | |
CHD-5RW | CTAGTATCAGGACTACCGAAAGCCAGGGAAGAtGG(SEQ ID No.16) | 无 | 130 |
CHD-5RM | CTAGTATTAGGATGTGAACGAAAGCCAGGGAAGAtGA(SEQ ID No.17) | 无 | 134 |
CHD-6F | gtttcttCGTGATTTTCCTAAGCAGATACAT(SEQ ID No.18) | 无 | |
CHD-6RW | CTAGTATCAGGACTACGCAGGTGAATCTGGGTGAcTC(SEQ ID No.19) | 无 | 141 |
CHD-6RM | CTAGTATTAGGATGTGCTGCAGGTGAATCTGGGTGAcTT(SEQ ID No.20) | 无 | 145 |
CHD-7F | gtttcttGACCTCATCTATTGGTTCCTATCC(SEQ ID No.21) | 无 | |
CHD-7RW | CTAGTATCAGGACTACCAAGCTCTGTAGAGTACATGTTCtCT(SEQ ID No.22) | 无 | 163 |
CHD-7RM | CTAGTATTAGGATGTGTGCAAGCTCTGTAGAGTACATGTTCtCC(SEQ ID No.23) | 无 | 167 |
CHD-8F | gtttcttACCTCAATGACCTTCTAACCCAG(SEQ ID No.24) | 无 |
注:小写字母书写的gtttctt为添加在5’端的“猪尾”序列;靠近3’端的小写字母书写的为人为引入的错配碱基。
实施例2试剂盒的使用
使用20μL的PCR反应体系,含有约10ng的待检测的总基因组DNA、6mM的氯化镁、1×PCR的缓冲液、0.05mM的每种dNTP、7条正向引物每条均为2 pmole、14条反向引物每条均为0.5 pmole、14 pmole的FAM-tag、14 pmole的ROX-tag、1U的HotStarTaq Plus DNA聚合酶(Qiagen)。在Applied Biosystems 9700 PCR仪上运行如下程序:(1) 94℃ 5:00分钟,(2)30次循环:98℃ 10秒、58℃ 90秒和72℃ 30秒,(3)4次循环:94℃ 30秒、49℃ 90秒和72℃30秒,(4)60℃ 30分钟。由于体系中反向引物的量只有正向引物的四分之一,因此在前30个循环中所有反向引物均已经消耗完毕。由于前30个循环的PCR产物均已经被加上反向引物上携带的通用标签序列,因此随后的4个循环以前期的PCR产物为模板,以剩余的正向引物和标记了荧光团的通用标签引物为上下游引物进行同管内的二次PCR,此时的所有PCR产物均被标记了相应颜色的荧光团。
随后将PCR产物在ABI 3100遗传分析仪上进行Genescan分析,使用GS500 LIZ(Applied Biosystems)作为内部尺寸标准。使用GeneMarker软件对测得的基因型数据进行分析。图二为一个样品所有七个位点的Genescan检测结果。蓝色峰代表存在野生型等位基因,红色峰代表存在突变型等位基因。图三为位点四的基因型判定结果示意图,当该位点为野生型等位基因纯合体时,只会在120bp位置检测到一个蓝色的峰;当该位点为突变型等位基因纯合体时,只会在124bp位置检测到一个红色的峰;当该位点为杂合体时,会同时检测到蓝色峰和红色峰。
经过已知各种基因型样本或sanger测序的验证,本申请的引物组可以特异性地区分出各位点的不同基因型,BICF2G630558239示例如图1所示。
实施例3
CHD诊断标准:使用国际上常用的OFA判断体系,将E、G、F、B等级归为正常,将M、MOD、S等级归为患病。
实验用犬:实验用犬为选自北京某拉布拉多犬繁育基地和两家宠物诊所的拉布拉多犬189只(125只患CHD,64只正常)和取自郑州某金毛巡回猎犬繁育基地和三家宠物诊所的金毛巡回猎犬167只(107只患病,60只正常),所有犬只无两代内直系亲缘关系。所有犬只年龄在24个月以上。
结合现有文献的数据,共分析了24个4-7位点的SNP组合与CHD的关联,logistic回归模型分析,SPSS绘制ROC曲线。
发现对拉布拉多犬效果最好的组合为BICF2G630227898,BICF2G630339806,BICF2S230609,BICF2G630558239,BICF2P355865,BICF2P281364,其灵敏度为76.3%,特异性为80.9%(ROC曲线见图2),超过了Bartolomé7位点组合的实际效果(灵敏度为76.8%,特异性为68.5%)。该SNP组合检测位点更少,费用更低,而且对于国内拉布拉多犬种的检测效果优于Bartolomé7位点组合。
对金毛巡回猎犬效果最好的SNP组合为BICF2S2367279,BICF2P281364,BICF2P1086886,BICF2P355865,BICF2G630227898,BICF2S230609,BICF2P548082,其灵敏度为72.1%,特异性为65.9%(ROC曲线见图3)。超过了Bartolomé7位点组合的实际效果(灵敏度为62.8%,特异性为53.2%)虽然由于尚无条件专门全面筛选金毛寻回猎犬的SNP位点,该SNP组合效果不如上面拉布拉多犬组合,但也已经完全可以用于金毛巡回猎犬的CHD遗传筛查预测。
2016年5月起,发明人将上述位点的组合实际应用于拉布拉多犬和金毛巡回猎犬的选育中,截止目前为止准确性均在80%(拉布拉多犬)和70%以上(金毛巡回猎犬)(CHD一般在24个月骨骼发育完全后才确切诊断,因此诊断效果随时间推移可能还会有变化)。
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Claims (4)
1.检测SNP位点基因型的试剂在制备预测犬髋关节发育不良(CHD)的试剂盒中的应用,其中SNP位点为:BICF2S2367279,BICF2P281364,BICF2P1086886,BICF2P355865,BICF2G630227898,BICF2S230609,和BICF2P548082;其中的犬为金毛巡回猎犬。
2.根据权利要求1的应用,其中检测SNP位点基因型的试剂为引物,引物序列和标记为:
通用引物:SEQ ID NO.1和SEQ ID NO.2,SEQ ID NO.1和SEQ ID NO.2的5’端分别标记6-FAM和ROX;
BICF2P548082:SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8;
BICF2G630227898:SEQ ID NO.15、SEQ ID NO.16和SEQ ID NO.17;
BICF2S230609:SEQ ID NO.18、SEQ ID NO.19和SEQ ID NO.20;
BICF2P1086886:SEQ ID NO.27、SEQ ID NO.28和SEQ ID NO.29;
BICF2P355865:SEQ ID NO.30、SEQ ID NO.31和SEQ ID NO.32;
BICF2P281364:SEQ ID NO.33、SEQ ID NO.34和SEQ ID NO.35;
BICF2S2367279:SEQ ID NO.36、SEQ ID NO.37和SEQ ID NO.38。
3.预测犬髋关节发育不良(CHD)的试剂盒,其中包含检测SNP位点基因型的试剂,其中SNP位点为:BICF2S2367279,BICF2P281364,BICF2P1086886,BICF2P355865,BICF2G630227898,BICF2S230609,和BICF2P548082;其中的犬为金毛巡回猎犬。
4.权利要求3的试剂盒,其中检测SNP位点基因型的试剂为引物,引物序列和标记为:
通用引物:SEQ ID NO.1和SEQ ID NO.2,SEQ ID NO.1和SEQ ID NO.2的5’端分别标记6-FAM和ROX;
BICF2P548082:SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8;
BICF2G630227898:SEQ ID NO.15、SEQ ID NO.16和SEQ ID NO.17;
BICF2S230609:SEQ ID NO.18、SEQ ID NO.19和SEQ ID NO.20;
BICF2P1086886:SEQ ID NO.27、SEQ ID NO.28和SEQ ID NO.29;
BICF2P355865:SEQ ID NO.30、SEQ ID NO.31和SEQ ID NO.32;
BICF2P281364:SEQ ID NO.33、SEQ ID NO.34和SEQ ID NO.35;
BICF2S2367279:SEQ ID NO.36、SEQ ID NO.37和SEQ ID NO.38。
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