CN113249463A - 一种用于血管紧张素ii受体抑制剂用药的基因检测试剂盒及其检测方法和应用 - Google Patents
一种用于血管紧张素ii受体抑制剂用药的基因检测试剂盒及其检测方法和应用 Download PDFInfo
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Abstract
本发明公开了一种用于血管紧张素II受体抑制剂用药的基因检测试剂盒,其中,所述试剂盒针对CYP2C9*3、AGTR1(A1166C)两个基因的多态性血管紧张素II受体抑制剂设计特异性扩增引物和测序引物,所述试剂盒包括如下组分:扩增反应液、CYP2C9*3测序引物、AGTR1(A1166C)测序引物和阳性对照。本发明采用采用非对称多重PCR扩增和优化焦磷酸测序技术为组合对血管紧张素II受体抑制剂用药相关的基因多态性进行检测,试剂盒可同时检测CYP2C9*3、AGTR1(A1166C)基因多态性,测序引物为羧基修饰素琼脂糖凝胶与氨基标记的DNA序列的复合物,既充当测序引物又作为捕获探针,实现基因多态性的快速、准确的检测,结果判读便捷、明确,能从基因层面指导血管紧张素II受体抑制剂的用药,为临床个性化用药给出基因角度的建议。
Description
技术领域
本发明涉及一种用于血管紧张素II受体抑制剂用药的基因检测试剂盒及其检测方法和应用,属于基因检测领域。
背景技术
血管紧张素II受体拮抗剂(AngiotensinIIreceptorantagonists,ARB)是一类新型抗高血压药物,降压作用显著,尤其对心血管疾病的独特疗效和保护作用,引起临床普遍关注,世界卫生组织(WHO)已将它列为一线降压药物。ARB是一类作用于肾素-血管紧张素系统的药物,通过对血管紧张素II(ANGⅡ)受体的阻滞,主要是阻断存在于许多组织中如血管和肾上腺中的血管紧张素II的I型受体(AT1),可较血管紧张素转换酶抑制剂(ACE抑制剂)更充分有效地阻断血管紧张素对血管收缩、水钠潴留及细胞增生等不利作用。血管紧张素II受体拮抗剂目前已上市的血管紧张素II受体阻滞剂有氯沙坦、伊贝沙坦、缬沙坦、芦沙坦、罗沙坦、依普罗沙坦、依贝沙坦、坎地沙坦等。
血管紧张素Ⅱ型受体基因存在1166A/C多态性,1166C突变在亚洲人的发生频率为:9%。此多态性与ACEI的降压效果有关,含有C突变基因型的血压下降幅度更大,该基因型的受体对药物敏感程度更高。CYP2C9参与抗高血压药、抗凝血药、抗惊厥药、降糖药、非甾体类解热镇痛抗炎药以及利尿药等多种药物的羟化代谢。中国人群中CYP2C9*3的频率为3%。CYP2C9遗传多态性导致其酶活性变化,从而导致药物代谢种族和个体差异现象。CYP2C9活性变化可导致这些药物体内浓度出现较大变化,甚至导致严重药物不良反应的发生。洛沙坦是一种常用的抗高血压药物,在体内主要经CYP2C9代谢活化为具有降压作用的代谢产物E-3174。携带CYP2C9*3等位基因的个体服用洛沙坦后E-3174的生成减少,洛沙坦的代谢率降低。口服单剂量洛沙坦后1h~6h后,CYP2C9*1/*3基因型个体中洛沙坦的降压作用下降,需适当增加用药剂量以增强降压疗效。
目前,对于基因多态性检测的方法有很多种,如直接测序法、芯片法、高分辨率熔解曲线法、等位基因特异性扩增法、taqman荧光探针法等。其中,测序法能够直接检测突变位点的位置和类型,但该方法操作步骤繁琐,检测周期长,且扩增产物容易产生污染;芯片法涉及基因特异扩增、杂交、检测等多个步骤,可进行高通量分析,但其成本较高,检测步骤复杂且对样本数量有一定的要求;高分辨率熔解曲线法步骤简单,不需要做扩增后处理,但其不含特异性荧光探针,特异性偏低,且对仪器设备的要求较高;等位基因特异性扩增法采用ARMS引物进行特异扩增,操作方法简单,无需扩增后处理,但其引物设计难以最优化,检测条件要求严格,实际操作中容易出现引物错配而产生假阳。taqman荧光探针法操作方法简单,无需扩增后处理,但其试验成本高,对于多个基因的扩增通量不高。因此,需要建立一种简单、快速有效、价格低廉、特异性高的检测基因多态性的方法。
不对称PCR(asymmetric PCR)是用不等量的一对引物,PCR扩增后产生大量的单链DNA(SSDNA)。这对引物分别称为非限制引物与限制性引物,其比例一般为50~100∶1。在PCR反应的最初10~15个循环中,其扩增产物主要是双链DNA,但当限制性引物(低浓度引物)消耗完后,非限制性引物(高浓度引物)引导的PCR就会产生大量的单链DNA。
常规焦磷酸测序前处理操作较为复杂和耗时,因此,设计开发一款可以结合不对称PCR技术的快速检测的焦磷酸检测试剂盒是十分必要的。
发明内容
针对现有技术存在的上述问题,本发明的目的是以非对称多重PCR扩增和优化焦磷酸测序技术为基础检测基因多态性,获得一种用于血管紧张素II受体抑制剂用药的基因检测试剂盒及其检测方法和应用。
为实现上述发明目的之一,本发明采用的一种用于血管紧张素II受体抑制剂用药的基因检测试剂盒及其检测方法和应用的技术方案如下:
本发明的试剂盒针对CYP2C9*3、AGTR1(A1166C)两个基因的多态性血管紧张素II受体抑制剂设计特异性扩增引物和测序引物,所述试剂盒包括如下组分:扩增反应液、CYP2C9*3测序引物、AGTR1(A1166C)测序引物和阳性对照。
优选地,所述设计特异性引物,如下表所示:
即,所述CYP2C9*3的特异性引物组序列如序列表SEQ ID NO:1~SEQ ID NO:2所示;所述AGTR1(A1166C)的特异性引物组序列如序列表SEQ ID NO:3~SEQ ID NO:4所示。所述的CYP2C9*3测序引物、AGTR1(A1166C)测序引物分别如序列表SEQ ID NO:5~SEQ ID NO:6所示。
优选的,所述的测序引物,为琼脂糖凝胶颗粒与氨基标记的DNA序列的结合物,既充当测序引物又作为捕获探针。该引物与扩增的单链DNA结合以后,经过简单洗涤即可直接用于测序反应。
优选的,所述的阳性对照,包括浓度为20ng/ul的CYP2C9*1/*3、AGTR1(A1166C)杂合型基因组DNA。阳性对照对应所检测基因位点的杂合型,对未知样本的型别判定提供参考,同时对反应液的有效性进行质控。
优选的,所述的扩增反应液,包括CYP2C9*3、AGTR1(A1166C)特异性扩增引物,还包括PCR Buffer、dNTPS、HS-Taq、BSA、dUTP、UDG酶、海藻糖和无核酸酶水。
更优选的,反应液各组分浓度分别为:CYP2C9*3后引物(1.2uM),CYP2C9*3后引物(0.02uM),AGTR1(A1166C)前引物(1.2uM),AGTR1(A1166C)后引物(0.02uM),PCR Buffer(1.5×),dNTPS(0.3mM)、HS-Taq酶(1U),BSA(0.05mg/ml),海藻糖(0.2%),dUTP(0.5mM)、UDG酶(1U)和无核酸酶水(将体系补充至20μL)。
本发明的另一个目的是公开一种采用上述试剂盒的血管紧张素II受体抑制剂用药相关的基因多态性检测方法,所述基因检测方法对待测CYP2C9*3、AGTR1(A1166C)基因进行焦磷酸测序。所述焦磷酸测序的待测基因采用非对称多重PCR方式扩增获得。
优选的,所述检测方法包括以下步骤:
a.将所述扩增反应液与5ul待测基因组DNA,采用非对称多重PCR扩增进行扩增;
b.将10ul反应产物与3ul测序引物分别进行结合;
c.焦磷酸测序;
d.确定CYP2C9*3位点、AGTR1(A1166C)位点血管紧张素II受体抑制剂位点的基因型。
优选的,所述的反应体积为25ul,扩增条件为:酶处理37℃3min;预变性95℃,5min;40个循环,95℃15s,60℃25s,72℃25s;最后延伸72℃4min。
本发明还公开了一种用于血管紧张素II受体抑制剂用药的基因检测试剂盒及方法的应用,所述用于血管紧张素II受体抑制剂用药的基因检测试剂盒用于同时检测CYP2C9*3和AGTR1(A1166C)基因型,以判断样本源的代谢类型。以从基因层面指导血管紧张素II受体抑制剂的用药。
与现有技术相比,本发明采用非对称多重PCR扩增和优化焦磷酸测序技术为组合对血管紧张素II受体抑制剂用药相关的基因多态性进行检测,通过非对称多重PCR一管扩增CYP2C9*3、AGTR1(A1166C),产生大量的单链DNA。通过直接与羧基修饰素结合的氨基标记单链DNA特异捕获单链DNA,洗涤后加入测序引物和测序原料,进行焦磷酸测序,减少了强碱性试剂对扩增片段的损伤,简化了测序处理的流程和时间。试剂盒可同时检测CYP2C9*3、AGTR1(A1166C)基因多态性,测序引物为羧基修饰素琼脂糖凝胶与氨基标记的DNA序列的复合物,既充当测序引物又作为捕获探针,实现基因多态性的快速、准确的检测,结果判读便捷、明确,能从基因层面指导血管紧张素II受体抑制剂的用药,为临床个性化用药给出基因角度的建议。
附图说明
图1是本发明提供的CYP2C9*3焦磷酸检测结果示例图;
图2是本发明提供的AGTR1(A1166C)焦磷酸检测结果示例图。
具体实施方式
下面结合实施例对本发明提供的一种用于血管紧张素II受体抑制剂用药的基因检测试剂盒及其检测方法和应用作进一步详细、完整地说明。下面描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的实验材料如无特殊说明,均为市场购买得到。
1、试剂盒的制备
本发明的试剂盒针对CYP2C9*3、AGTR1(A1166C)设计了特异性扩增引物和测序引物,用于焦磷酸PCR检测。基因多态性序列以Genebank内的公开序列为准。设计特异性引物,如下表所示:
测序引物,为琼脂糖凝胶颗粒与氨基标记的DNA序列的结合物,既充当测序引物又作为捕获探针。该引物与扩增的单链DNA结合以后,经过简单洗涤即可直接用于测序反应。测序引物的制备过程包括将合成氨基标记的测序引物在结合液的情况下与羧基修饰素琼脂糖凝胶微颗粒混合,洗涤去除游离的测序引物,在保存液中2-8℃保存。
本实施例的检测试剂盒包括如下组分:
序号 | 组份 | 标示装量 |
1 | 扩增反应液 | 430μL×1管 |
2 | CYP2C9*3测序引物 | 70ul×1管 |
3 | AGTR1(A1166C)测序引物 | 70ul×1管 |
5 | 阳性对照 | 50μL×1管 |
本实施例的检测试剂盒扩增反应液单人份配置体系如下:
以上引物探针购自生工、dNTP(25mM)购自诺唯赞、10×PCR buffer、HS Taq(5U/μL)购自TAKARA、UNG酶(5U/μL)Thermo Fisher。
本实施例的检测试剂盒测序引物制备过程如下:
(1)MES溶液配置
①100mM MES,pH 4.8(100mL):
②2.13g MES(2-[N-morpholino]ethane sulfonic acid,MW 213.25).
③pH 4.8
(2)TT Buffer(50mL)
①12.5mL of 1M Tris buffer pH 8
(3)封闭液
(4)捕获磁珠制备
①取500ul磁珠,吸磁1min,去上清;
②取1000ml 100MES溶液洗涤吸磁1min,去上清,重复洗涤2次;
③取10mg/ml EDS和10mg/ml NHS各500ul,加入捕获引物10ul;
④25℃杂交30min;
⑤取1000ml封闭液混合均匀,吸磁1min,去上清;
⑥取1000ml TT Buffer混合均匀,吸磁1min,去上清;重复洗涤3次;
⑦加入1ml TE缓冲液溶解。
2、焦磷酸检测
本发明中采用的仪器如下:PCR扩增仪:ABI 2720PCR仪;
焦磷酸测序仪:武汉菲思特生物科技有限公司。
(1)试剂准备
提前将试剂取出,室温融化,并将各组分涡旋振荡15秒,将试剂盒各组分低速离心待用。确定反应数N,N=待检样本数(n)+质控品数(1)+空白对照。建议每次PCR实验同时进行阳性对照、空白对照分析。然后将反应液按20μL/管分装至PCR反应管中。
(2)加样检测
将样本DNA、阳性对照和空白对照按5μL加样量加入到PCR反应管中,盖紧管盖,低速离心15秒将管壁上的液体全部甩至管底,然后立即进行PCR扩增反应。所加入的待测样本DNA应大于20copies。样本一般为EDTA抗凝全血样本,样本处理参见常规处理方式。
(3)PCR扩增
采用PCR仪进行PCR扩增,PCR反应体系为25μL,扩增条件:
(4)焦磷酸测序
1)在PCR反应管中加入结合液40μL和琼脂糖凝胶颗粒与DNA序列的结合物3ul,再向其中加入PCR产物10μL,置于台式振荡器上,1100rpm振荡10min,使测序引物和单链PCR产物充分结合;
2)7,000×g离心1min,弃上清;
3)向EP管中加入150uL洗涤缓冲液,7000g离心1min,弃上清;
4)测序管中分别加入3uL测序酶和3uL测序底物;
5)取一个dNTP排管,自圆滑一端向平端依次加入20μldATPαS、20μl dTTP、20μldGTP、20μl dCTP。将排管底部轻轻磕碰桌面,使得碱基平铺在排管底部;
6)根据仪器使用说明进行测序。测序结果如图1和图2所示。
(5)结果判读
1)有效性判定:
本试剂盒空白对照品的不通过,阳性对照品的检出结果为CYP2C9*1/*3、AGTR11166CA型。
2)结果判定标准
a.CYP2C9*3的DNA测序峰值图中,
C的频率≧90%,A的频率≦10%,即为CC型;
40%≦C的频率≦60%,40%≦A的频率≦60%,即为CA型;
A的频率≧90%,C的频率≦10%,即为AA型;
b.AGTR1(A1166C)的DNA测序峰值图中,
A的频率≧90%,C的频率≦10%,即为ADRB1 1165AA型;
40%≦A的频率≦60%,40%≦C的频率≦60%,即为ADRB1 1165AC型;
C的频率≧90%,A的频率≦10%,即为ADRB1 1165CC型;
3、基因检测结果与代谢活性的相关性
通过检测结果可以判断样本源的代谢类型,从而进一步指导相应代谢途径的药物的用药剂量。
最后有必要在此说明的是:以上实施例只用于对本发明的技术方案作进一步详细地说明,不能理解为对本发明保护范围的限制,本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。
序列表
<110> 湖南菲思特精准医疗科技有限公司
<120> 一种用于血管紧张素II受体抑制剂用药的基因检测试剂盒及其检测方法和应用
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Claims (10)
1.一种用于血管紧张素II受体抑制剂用药的基因检测试剂盒,其特征在于,所述试剂盒针对CYP2C9*3、AGTR1A1166C两个基因的多态性血管紧张素II受体抑制剂设计特异性扩增引物和测序引物,所述试剂盒包括如下组分:扩增反应液、CYP2C9*3测序引物、AGTR1A1166C测序引物和阳性对照。
2.根据权利要求1所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒,其特征在于,所述扩增反应液包括CYP2C9*3、AGTR1A1166C二个基因的多态性血管紧张素II受体抑制剂设计特异性扩增引物,所述CYP2C9*3的特异性引物组序列如序列表SEQ ID NO:1~SEQ ID NO:2所示;所述AGTR1A1166C的特异性引物组序列如序列表SEQ ID NO:3~SEQ IDNO:4所示。
3.根据权利要求1所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒,其特征在于,所述阳性对照包括浓浓度为20ng/ul的CYP2C9*3/*1、AGTR1A1166C杂合型基因组DNA。
4.根据权利要求1所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒,其特征在于,所述CYP2C9*3测序引物、AGTR1A1166C测序引物核酸序列分别如序列表SEQ ID NO:5~SEQ ID NO:6所示,所述测序引物为琼脂糖凝胶颗粒与氨基标记的DNA序列的结合物。
5.根据权利要求1所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒,其特征在于,所述扩增反应液还包括PCR Buffer、dNTPS、HS-Taq、BSA、dUTP、UDG酶和海藻糖。
6.根据权利要求2或5所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒,其特征在于,所述反应液体系各组分浓度分别为:CYP2C9*3前引物1.2uM,CYP2C9*3后引物0.02uMAGTR1A1166C前引物1.2uM,AGTR1A1166C后引物0.02uM,PCR Buffer1.5×,dNTPS0.3mM、HS-Taq酶1U,BSA0.05mg/ml,海藻糖0.2%,dUTP0.5mM、UDG酶1U。
7.一种采用根据权利要求1~6任一所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒的基因检测方法,其特征在于,所述基因检测方法对待测CYP2C9*3、AGTR1A1166C基因进行焦磷酸测序。
8.根据权利要求7所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒的检测方法,其特征在于,所述焦磷酸测序的待测基因采用非对称多重PCR方式扩增获得。
9.根据权利要求8所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒的检测方法,其特征在于,所述非对称多重PCR反应体积为25ul,扩增条件为:酶处理37℃3min;预变性95℃,5min;40个循环,95℃15s,60℃25s,72℃25s;最后延伸72℃4min。
10.一种根据权利要求1~6任一所述的用于血管紧张素II受体抑制剂用药的基因检测试剂盒的应用,其特征在于,所述用于血管紧张素II受体抑制剂用药的基因检测试剂盒用于同时检测CYP2C9*3和AGTR1A1166C基因型,以判断样本源的代谢类型。
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