CN114480623A - 基于crispr检测脑卒中相关tbxas1基因的试剂盒 - Google Patents
基于crispr检测脑卒中相关tbxas1基因的试剂盒 Download PDFInfo
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Abstract
本发明属于基因检测技术领域,公开了基于CRISPR检测脑卒中相关TBXAS1基因的试剂盒,所述试剂盒包括针对TBXAS1基因rs2267682和rs10487667位点设计的突变型crDNA或突变型crRNA,其序列如SEQ ID NO.1~4所示,结合CRISPR‑cas12a系统,能确定待测样本中是否存在TBXAS1基因rs2267682和/或rs10487667位点的基因突变,具备特异性强、准确性高、检测速度快等优点,应用前景广阔。
Description
技术领域
本发明属于基因检测技术领域,具体涉及一种基于CRISPR检测脑卒中相关TBXAS1基因的试剂盒。
背景技术
脑卒中又称中风,在世界范围内,中风是致残和死亡的主要原因。在中国,缺血性中风约占所有中风病例的70%,其发病率表现出显著的地理差异,表现为中国北方的发病率最高,而中国南方的发病率较低。尽管年龄、高血压、糖尿病和环境因素是缺血性中风的众所周知的危险因素,但它是一种多因素多基因疾病。识别与缺血性卒中风险相关的基因变异可以阐明卒中的发病机制,并为预防和治疗这一复杂疾病提供新的方法。尽管一些全基因组关联研究评估了可能导致缺血性中风的各种多态性,而在中国个体中,与缺血性卒中易感性相关的遗传变异尚未明确确定。
血栓素A合酶1(TBXAS1)是花生四烯酸代谢的下游酶,是合成血栓素A2(TXA2)所必需的酶,现有研究认为TXA2与动脉粥样硬化病变的发展和血栓形成有关。rs2267682和rs10487667是TBXAS1基因的两种内含子单核苷酸多态性(SNP)变异,可能影响TBXAS1 mRNA的结构和稳定性,从而影响TXA2的代谢物生成;同时研究表明rs2267682TBXAS1多态性与缺血性卒中相关。
现阶段临床一般通过定量即时聚合酶链锁反应(Quantitative real timepolymerase chain reaction,Q-PCR)、非放射性原位杂交技术(fluorescence insituhybridization,Fish)、多重引物PCR(multiplex PCR)等方式对患者进行基因突变检测,荧光检测具有灵敏度高,选择性好等优点,在生物检测与生化分析中起着不可替代的作用。然而这些检测技术存在检测时间长、过程复杂或检测成本过高等问题,导致无法应用到大规模的临床样本研究中。因此亟需提供一种温度控制简单,且灵敏度、特异性高的特异性片段检测方法。
RNA引导的成簇规则间隔短回文重复序列-CRISPR(CRISPR-Cas)相关方法由于其高可靠性和特异性而在快速核酸检测方面显示出相当大的前景。目前已经开发了几种类型的Cas核酸酶用于CRISPR-Cas系统,例如Cas9、Cas12等,已显示出快速、灵敏和特异性检测核酸的强大实用潜力。
发明内容
有鉴于此,本发明的目的在于解决现有技术中检测时间长、成本过高和准确性不高以致无法大规模应用的问题,提供了一种基于CRISPR技术检测与脑卒中相关的TBXAS1基因是否存在rs2267682和/或rs10487667目标突变位点和检测该靶核酸在目标区域是否存在突变的方法和试剂盒。
为了实现上述目的,本发明具体采用以下技术方案:
一种检测试剂盒,所述检测试剂盒包括突变型crDNA或突变型crRNA;
所述突变型crDNA为如下任一:
A1).rs2267682突变型crDNA,其序列如SEQ ID NO.1所示;
A2).rs10487667突变型crDNA,其序列如SEQ ID NO.2所示;
A3).A1)和A2);
所述突变型crRNA为如下任一:
B1).rs2267682突变型crRNA,其序列如SEQ ID NO.3所示;
B2).rs10487667突变型crRNA,其序列如SEQ ID NO.4所示;
B3).B1)和B2)。
优选地,所述检测试剂盒还包括以下任一:
C1).用于特异性扩增TBXAS1基因上rs2267682多态性位点的引物对,具体为:
rs2267682-F,其序列如SEQ ID NO.5所示;
rs2267682-R,其序列如SEQ ID NO.6所示;
C2).用于特异性扩增TBXAS1基因上rs10487667多态性位点的引物对,具体为:
rs10487667-F,其序列如SEQ ID NO.7所示;
rs10487667-R,其序列如SEQ ID NO.8所示;
C3).C1)和C2)。
可以理解的是,引物对的选择依据试剂盒中突变型crDNA或突变型crRNA的序列而定。
优选地,所述试剂盒包括荧光探针和cas12a蛋白;更加优选地,所述cas12a蛋白为Fncas12a蛋白。
优选地,所述试剂盒包括荧光探针,所述荧光探针的5’端标记有荧光报告基团,所述荧光探针的3’端标记有荧光猝灭基团;更加优选地,所述荧光报告基团选自FAM、VIC、HEX、TRT、Cy3、Cy5、ROX、JOE、Texas Red中的任意一种,所述荧光猝灭基团选自TAMRA、DABCYL、MGB、BHQ1、BHQ2、BHQ3中的任意一种。
优选地,所述试剂盒包括DNA酶抑制剂和无酶水。
优选地,所述试剂盒中包括阴性对照物,所述阴性对照物为野生型crDNA或野生型crRNA;
所述野生型crDNA为如下任一:
D1).rs2267682野生型crDNA,其序列如SEQ ID NO.9所示;
D2).rs10487667野生型crDNA,其序列如SEQ ID NO.10所示;
D3).D1)和D2);
所述野生型crRNA为如下任一:
E1).rs2267682野生型crRNA,其序列如SEQ ID NO.11所示;
E2).rs10487667野生型crRNA,其序列如SEQ ID NO.12所示;
E3).E1)和E2)。
可以理解的是,阴性对照物的选择依据试剂盒中突变型crDNA或突变型crRNA的序列而定。
本发明还提供了上述检测试剂盒在检测与脑卒中相关的TBXAS1基因突变中的应用。
优选地,所述应用的过程方法具体为:对待测DNA样品进行PCR扩增,扩增完成后加入检测混合液反应,并采集荧光变化;所述检测混合液含有荧光探针和所述突变型crRNA;所述突变型crDNA在T7 RNA聚合酶作用下生成RNA,经回收纯化得到对应的crRNA。
更加优选地,所述PCR扩增的程序为:95℃3min进行预变性;95℃10s,60℃20s,循环30次;最后降温至25℃,反应结束。
更加优选地,所述检测混合液共50ul,具体为:250~500nM纯化的cas12a蛋白,250~500nM突变型crRNA,1~5μL荧光探针,2μL DNA酶抑制剂,无酶水余量。
本发明的有益效果:针对TBXAS1基因的rs2267682和rs10487667位点设计突变型crDNA和突变型crRNA,筛选得到的突变型crRNA的荧光值相较于野生型变化显著,灵敏度高;使用所述突变型crRNA判断样本是否存在待检基因突变时,检测结果与临床诊断结果基本一致,准确率高。另外,本发明基于CRISPR-Cas12a系统设计crRNA,cas12a核酸酶与crRNA配合,对DNA切割的效率更高、更精确,且构建方式简单、转化效率高。
附图说明
图1为TBXAS1基因中rs2267682位点的crRNA荧光检测结果图;
图2为TBXAS1基因中rs10487667位点的crRNA荧光检测结果图;
图3为TBXAS1基因中rs2267682(图3左)和rs10487667(图3右)位点野生型(上)、纯合突变(中)、杂合突变(下)的测序图谱。
具体实施方式
为了更好的理解本发明,下面结合具体实施例进一步阐明本发明的内容,但以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1靶向基因突变位点crRNA的设计及获得
(1)靶向基因突变位点crRNA设计原则
由于CRISPR-Cas12a系统是一种新型的靶向DNA基因编辑系统,其中Cas12a与crRNA结合形成监测复合物,crRNA的向导区域用互补序列识别目标DNA,并且Cas12a降解目标DNA链。其中所述crRNA设计要求:crRNA包括蛋白锚定序列和向导序列,序列格式为:5’-与Cas12a蛋白结合的锚定序列-crRNA向导序列-3’,蛋白锚定序列需要根据Cas12a蛋白来确定,使其能够与选定的Cas12a蛋白匹配并结合;向导序列则与靶向DNA中的片段匹配。
(2)crDNA序列的选择
本实施例中选用的cas12a蛋白为FnCas12a,因此选定与Cas12a蛋白结合的锚定序列为AAUUUCUACUGUUGUAGAU,所述crRNA序列根据现有技术已知的TBXAS1基因rs2267682和rs10487667两个突变位点区域设计,每个突变位点设计了一个相应的crDNA,其序列具体为:
TBXAS1基因rs2267682突变位点1个crDNA:其序列如SEQ ID NO.1所示;
TBXAS1基因rs10487667突变位点1个crDNA:其序列如SEQ ID NO.2所示。
(3)crRNA的获得
使用T7高产量转录试剂盒(NEB)将crDNA用作crRNA生物合成的模板,转录反应体系如表1所示。在37℃下孵育12h后,将DNase I添加到反应溶液中,并在37℃下孵育30min以消化剩余的DNA片段。使用RNA纯化试剂盒纯化转录的crRNA,并储存在-80℃。
表1转录反应体系
所得crRNA为:
TBXAS1基因rs2267682突变位点1个crRNA:其序列如SEQ ID NO.3所示;
TBXAS1基因rs10487667突变位点1个crRNA:其序列如SEQ ID NO.4所示。
实施例2脑卒中相关TBXAS1基因突变位点的检测试剂盒及检测方法
1、用于TBXAS1基因与脑卒中相关突变位点检测的试剂盒的组成
1)2个突变型crRNA(如实施例1所示获得),其序列如SEQ ID NO.3~4所示;
或2个突变型crDNA(当试剂盒中为crDNA时,需要使用者先将crDNA片段分别在T7RNA聚合酶作用下生成RNA,回收纯化得到crRNA,具体见实施例1),其序列如SEQ ID NO.1~2所示。
2)一条特异性荧光探针,在本实施例中,荧光探针具体为表2中任一:
表2荧光探针序列
3)cas12a蛋白,在本实施例中,采用Fncas12a。
4)无酶水和DNA酶抑制剂。
5)用于特异性扩增TBXAS1基因上rs2267682和rs10487667多态性位点的引物对,所述引物对序列如下所示:
rs2267682-F,其序列如SEQ ID NO.5所示;
rs2267682-R,其序列如SEQ ID NO.6所示;
rs10487667-F,其序列如SEQ ID NO.7所示;
rs10487667-F,其序列如SEQ ID NO.8所示。
6)阴性对照物,具体为野生型crDNA或野生型crRNA;
所述野生型crDNA包括:
rs2267682野生型crDNA,其序列如SEQ ID NO.9所示;
rs10487667野生型crDNA,其序列如SEQ ID NO.10所示;
所述野生型crRNA包括:
rs2267682野生型crRNA,其序列如SEQ ID NO.11所示;
rs10487667野生型crRNA,其序列如SEQ ID NO.12所示。
实施例3与脑卒中相关的TBXAS1基因突变位点的检测
1)取10~50ng待测突变型模板DNA,加入如表3所示的扩增体系中,所述引物混合物为如SEQ ID NO.5~8所示的引物对。
表3 PCR扩增体系
2)将所述得到的扩增产物加入检测混合液(混合液中包含100~200nM纯化的FnCas12a,100~200nM crRNA,1~5μL合成的荧光探针,2μL DNA酶抑制剂和无酶水),在检测缓冲液(NEBuffer 2.1)37℃孵育0.5~1h,检测体系如表4所示:
表4检测体系
反应全部结束后待统计分析检测反应(37℃)内反应前后荧光值的变化情况,同时采用野生型crRNA设立阴性对照组,根据阴性阈值判断待测样本DNA中是否存在相应的脑卒中风险筛查相关分子标志物基因突变。
rs2267682位点和rs10487667位点合成野生株(WT)和突变株(MUT)的靶序列,分别利用实施例1设计的crRNA构建检测体系进行检测筛选,其中rs2267682突变型crRNA,其序列如SEQ ID NO.3所示;rs10487667突变型crRNA,其序列如SEQ ID NO.4所示。检测结果如图1、2所示,根据检测结果,突变株中荧光值相较于野生株变化显著,灵敏度高,验证了所使用的crRNA效果优异。
实施例4试剂盒在临床样本中的检测
根据实施例3的方法和结果,选择多例临床样本检测rs2267682和rs10487667突变位点,并对结果进行汇总比对。
一、检测方法
1、利用DNA提取试剂盒提取患者血液样本的DNA,操作步骤严格按照说明书进行;
2、利用实施例1设计的crRNA构建检测体系,按实施例3所述的检测方法进行扩增及检测反应,最后通过荧光值判断检测结果。
二、检测结果
检测结果如表5所示:
表5 20例脑卒中临床样本检测结果
由上述结果可知,本发明实施例提供的试剂盒能有效实现脑卒中相关TBXAS1基因rs2267682和rs10487667位点的突变筛查检测,判断标本是否存在待检基因突变。且检测结果与临床诊断结果基本一致,正确率高达95%。
以上所述本发明的具体实施方式,并不构成对本发明保护范围的限定;任何根据本发明的技术构思所做出的各种其他相应的改变与变形,均应包含在本发明权利要求的保护范围内。
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Claims (10)
1.一种检测试剂盒,其特征在于,所述检测试剂盒包括突变型crDNA或突变型crRNA;
所述突变型crDNA包括:
rs2267682突变型crDNA,其序列如SEQ ID NO.1所示;
和/或rs10487667突变型crDNA,其序列如SEQ ID NO.2所示;
所述突变型crRNA包括:
rs2267682突变型crRNA,其序列如SEQ ID NO.3所示;
和/或rs10487667突变型crRNA,其序列如SEQ ID NO.4所示。
2.根据权利要求1所述的检测试剂盒,其特征在于,所述检测试剂盒包括:
用于特异性扩增TBXAS1基因上rs2267682位点的引物对:
rs2267682-F,其序列如SEQ ID NO.5所示;
rs2267682-R,其序列如SEQ ID NO.6所示;
和/或用于特异性扩增TBXAS1基因上rs10487667位点的引物对:
rs10487667-F,其序列如SEQ ID NO.7所示;
rs10487667-R,其序列如SEQ ID NO.8所示。
3.根据权利要求1所述的检测试剂盒,其特征在于,所述检测试剂盒包括cas12a蛋白。
4.根据权利要求1所述的检测试剂盒,其特征在于,所述检测试剂盒包括荧光探针,所述荧光探针的5’端标记有荧光报告基团,所述荧光探针的3’端标记有荧光猝灭基团。
5.根据权利要求1所述的检测试剂盒,其特征在于,所述检测试剂盒包括DNA酶抑制剂。
6.根据权利要求1所述的检测试剂盒,其特征在于,所述检测试剂盒包括阴性对照品,所述阴性对照品为野生型crDNA或野生型crRNA;
所述野生型crDNA包括:
rs2267682野生型crDNA,其序列如SEQ ID NO.9所示;
和/或rs10487667野生型crDNA,其序列如SEQ ID NO.10所示;
所述野生型crRNA包括:
rs2267682野生型crRNA,其序列如SEQ ID NO.11所示;
和/或rs10487667野生型crRNA,其序列如SEQ ID NO.12所示。
7.如权利要求1所述检测试剂盒在检测与脑卒中相关TBXAS1基因突变中的应用。
8.根据权利要求7所述的应用,其特征在于,所述应用的过程具体为:对待测DNA样品进行PCR扩增,扩增完成后加入检测混合液反应,并采集荧光变化;所述检测混合液含有荧光探针和权利要求1所述突变型crRNA;所述突变型crDNA在T7 RNA聚合酶作用下生成RNA,经回收纯化得到对应的crRNA。
9.根据权利要求8所述的应用,其特征在于,所述PCR扩增的程序为:95℃3min进行预变性;95℃10s,60℃20s,循环30次;最后降温至25℃,反应结束。
10.根据权利要求8所述的应用,其特征在于,所述检测混合液共50ul,具体为:250~500nM纯化的cas12a蛋白,250~500nM突变型crRNA,1~5μL荧光探针,2μL DNA酶抑制剂,无酶水余量。
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CN113136429A (zh) * | 2021-04-21 | 2021-07-20 | 江苏博嘉生物医学科技有限公司 | Idh1或idh2基因突变的检测试剂盒与检测方法 |
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CN113136429A (zh) * | 2021-04-21 | 2021-07-20 | 江苏博嘉生物医学科技有限公司 | Idh1或idh2基因突变的检测试剂盒与检测方法 |
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