CN113584143A - 一种硝酸甘油代谢标志物的检测试剂盒及其检测方法和应用 - Google Patents
一种硝酸甘油代谢标志物的检测试剂盒及其检测方法和应用 Download PDFInfo
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Abstract
本发明公开了一种硝酸甘油代谢标志物的检测试剂盒及其检测方法和应用,其中,检测试剂盒用于检测硝酸甘油的代谢标志物基因位点ALDH2G1510A的基因多态性,试剂盒包括ALDH2G1510A扩增引物、ALDH2G1510A测序引物和阳性对照。本发明通过RPA扩增ALDH2(G1510A),快速、有效地在恒定温度下产生大量扩增产物,通过直接与羧基修饰素结合的氨基标记单链DNA类似物特异捕获单链DNA,洗涤后,加入测序引物与模板DNA退火后,加入测序原料进行焦磷酸测序。
Description
技术领域
本发明涉及一种硝酸甘油代谢标志物的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
硝酸甘油是心绞痛和急性心肌梗死的一线治疗药物,能够有效缓解心绞痛的症状。中国人含服硝酸甘油无效比例高达25%,硝酸甘油的临床有效性常因人而异,有些患者出现心绞痛时服用硝酸甘油会出现用药无效现象,或者是过半小时以上才有效。研究表明ALDH2基因突变是影响硝酸甘油疗效的决定性因素。ALDH2基因,位于人类第12号染色体,ALDH2同时具有乙醛脱氢酶和酯酶活性,参与乙醇、硝酸甘油等药物的代谢。ALDH2 代谢活化硝酸甘油成其活性代谢产物一氧化氮,硝酸甘油的舒血管作用是通过释放一氧化氮所介导,而ALDH2是一氧化氮形成的关键。ALDH2*2(Glu504Lys,rs671)多态导致所编码蛋白质504位谷氨酸被赖氨酸所取代,携带突变等位基因(ALDH2*2)的个体ALDH2 酶活性下降,杂合子个体酶活性仅为野生型个体的10%,突变纯合子个体酶活性缺失。因此,携带ALDH2*2等位基因的个体酒精代谢能力下降,少量饮酒即出现脸红、心跳加速等不适;代谢硝酸甘油的能力下降,硝酸甘油抗心肌缺血的效应减弱。如果病人基因中携带 ALDH2突变,硝酸酯酶活性会降低10倍以上,使硝酸甘油难以发挥药效。因此,携带 ALDH2*2等位基因的个体酒精代谢能力下降,少量饮酒即出现脸红、心跳加速等不适;代谢硝酸甘油的能力下降,硝酸甘油抗心肌缺血的效应减弱。亚洲人群中ALDH2*2等位基因的携带率为30~50%。携带ALDH2*2等位基因的心绞痛患者应尽可能改用其他急救药物,避免硝酸甘油含服无效。
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,测序法和芯片法,操作步骤繁琐,检测周期长,且扩增产物容易产生污染;高分辨率熔解曲线法步骤简单,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,其引物设计难以最优化,检测条件要求严格。Taqman荧光探针法其试验成本高,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。
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RPA扩增和焦磷酸测序技术为基础,获得一种硝酸甘油代谢标志物的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,本发明采用的硝酸甘油代谢标志物的检测试剂盒的技术方案如下:
本发明的检测试剂盒用于检测硝酸甘油的代谢标志物基因位点ALDH2 G1510A的基因多态性,试剂盒包括ALDH2 G1510A扩增引物、ALDH2 G1510A测序引物和阳性对照,针试剂盒对ALDH2(G1510A)基因的多态性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、ALDH2(G1510A)测序引物、阳性对照。
优选地,所述设计特异性引物,如下表所示:
优选的,所述ALDH2(G1510A)的特异性引物组序列如序列表SEQ ID NO:1~SEQ IDNO:2所示。
优选的,所述的ALDH2(G1510A)测序引物如序列表SEQ ID NO:3所示。
更优选的,所述的测序引物,为核酸类似物,其骨架为肽键而非磷酸二酯键,肽键骨架连有相应的碱基。该结构具有生物学性质稳定,无论蛋白酶还是核酸酶都不易将其降解。
更优选的,所述的测序引物,为琼脂糖凝胶颗粒与氨基标记的PNA序列的结合物,该结合物与DNA结合较DNA/DNA的结合更为稳定且特异性高于DNA/DNA的捕获,且不受盐离子浓度的影响。可既充当测序引物又作为捕获探针,与扩增的产物结合以后,经过简单洗涤即可直接用于测序反应。
优选的,ALDH2(G1510A)测序引物对应的测序区域为ALDH2(G1510A)待检序列,如序列表SEQ ID NO:4所示。ALDH2(G1510A)待检序列对应ALDH2(G1510A)分配指令如序列表SEQ ID NO:5所示。
优选的,所述的试剂1包括:扩增缓冲液,18mM醋酸镁;
优选的,所述的试剂2包括:ALDH2(G1510A)前引物,ALDH2(G1510A)后引物,,dNTPS、链置换DNA聚合酶,单链DNA结合蛋白,结合单链核酸的重组酶,海藻糖;可在恒温条件下进行ALDH2(G1510A)扩增。
更优选的,试剂2各组分浓度分别为:ALDH2(G1510A)后引物(0.32uM),ALDH2(G1510A)后引物(0.32uM),dNTPS(0.3mM)、链置换DNA聚合酶(1.2ng/μL),单链DNA结合蛋白(3.2ng/μL),结合单链核酸的重组酶(4.8ng/μL),海藻糖(0.2%);
优选的,所述的阳性对照,包括浓度为20ng/ul的ALDH2(G1510A)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。
本发明还公开了一种采用上述试剂盒的昂丹司琼用药相关的基因多态性检测方法,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RPA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.向每个测序管中加入测序酶和测序底物;
d.取一个dNTP排管,自圆滑一端向平端依次加入dATPαS、dTTP、dGTP、dCTP;
将排管底部轻轻磕碰桌面,使得碱基平铺在排管底部;
e.焦磷酸测序;
f.确定ALDH2(G1510A)位点硝酸甘油位点的基因型;
优选的,所述的反应体积为25ul,反应条件为:42℃20min。
本发明还公开了一种硝酸甘油代谢标志物的检测试剂盒及方法的应用,所述检测试剂盒对ALDH2(G1510A)进行检测,以从基因层面指导硝酸甘油疗效预测。
重组酶聚合酶扩增(RPA),被称为是可以替代PCR的核酸检测技术。RPA技术主要依赖于三种酶:能结合单链核酸(寡核苷酸引物)的重组酶、单链DNA结合蛋白(SSB)和链置换DNA聚合酶。这三种酶的混合物在常温下也有活性,最佳反应温度在37℃左右。重组酶与引物结合形成的蛋白-DNA复合物,能在双链DNA中寻找同源序列。一旦引物定位了同源序列,就会发生链交换反应形成并启动DNA合成,对模板上的目标区域进行指数式扩增。被替换的DNA链与SSB结合,防止进一步替换。在这个体系中,由两个相对的引物起始一个合成事件。整个过程进行得非常快,一般可在二十分钟之内获得可检出水平的扩增产物。
与现有技术相比,本发明以RPA扩增和优化焦磷酸测序技术为组合对硝酸甘油用药相关的基因多态性进行检测,试剂盒可同时检测ALDH2(G1510A)基因多态性硝酸甘油疗效,为临床个性化用药给出基因角度的建议。本发明通过RPA扩增ALDH2(G1510A),快速、有效地在恒定温度下产生大量扩增产物。通过直接与羧基修饰素结合的氨基标记单链DNA类似物特异捕获单链DNA,洗涤后,加入测序引物与模板DNA退火后,加入测序原料进行焦磷酸测序。
附图说明
图1是本发明提供的ALDH2(G1510A)GG型测序结果示例图;
图2是本发明提供的ALDH2(G1510A)GA型测序结果示例图;
图3是本发明提供的ALDH2(G1510A)AA型测序结果示例图。
具体实施方式
下面结合实施例对本发明提供的硝酸甘油代谢标志物的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。
实施例1、试剂盒的制备
本发明的试剂盒针对ALDH2(G1510A)设计了特异性扩增引物和测序引物,用于恒温扩增和焦磷酸测序检测。基于重组酶聚合酶扩增技术设计引物是本发明的关键之一,该技术的引物设计无法辅助软件进行,只能依靠人工设计。为保证扩增速度及检测灵敏度,引物长度应控制在30~35bp,引物设计过短易增加非特异性扩增导致假阳性,若设计过长易导致无法进行扩增。基因多态性序列以Genebank内的公开序列为准。
(一)本实施例的引物序列如下:
(二)本实施例的检测试剂盒包括如下组分:
(三)本实施例的检测试剂盒试剂1单人份配置体系如下:
成分 | 体积(ul) |
扩增缓冲液 | 18.8 |
300mM醋酸镁 | 1.2 |
(四)本实施例的检测试剂盒试剂2单人份配置体系如下:
试剂2各组分浓度分别为:ALDH2(G1510A)前引物(0.32uM),ALDH2(G1510A) 后引物(0.32uM),,dNTPS(0.3mM)、链置换DNA聚合酶(1.2ng/μL),单链DNA结合蛋白(3.2ng/μL),结合单链核酸的重组酶(4.8ng/μL),海藻糖(0.2%);
成分 | 体积(ul) |
结合单链核酸的重组酶(100ng/μL) | 1.2 |
单链DNA结合蛋白(100ng/μL) | 0.8 |
链置换DNA聚合酶(100ng/μL) | 0.3 |
dNTPs(2.5mM) | 3 |
ALDH2(G1510A)前引物(10μM) | 0.8 |
ALDH2(G1510A)后引物(10μM) | 0.8 |
海藻糖(20%) | 0.25 |
配置完成后161.7ul/管进行分装、冻干。
实施例2、焦磷酸检测
本发明中采用的仪器如下:恒温仪;
焦磷酸测序仪:武汉菲思特生物科技有限公司。
(1)试剂准备(试剂准备室)
提前将试剂取出,并将试剂1涡旋振荡15秒,低速离心待用。直接向试剂2(冻干)中加入440ul试剂1,涡旋振荡15秒充分混匀。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(样本制备间)
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。
(3)PCR扩增(扩增间)
采用PCR仪进行扩增,反应体系为25μL,扩增条件:
扩增温度 | 时间 | 循环数 |
42℃ | 20min | 1 |
(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3ul,再向其中加入PCR产物10μL,置于台式振荡器上,1100rpm振荡10min,使微珠和PCR产物充分结合;
2)7,000×g离心1min,弃上清;
3)7,000×g离心1min,弃上清;
4)向EP管中加入150uL洗涤缓冲液,7000g离心1min,弃上清;
5)测序管中分别加入3uL测序酶和3uL测序底物;
6)取一个dNTP排管,自圆滑一端向平端依次加入20μldATPαS、20μl dTTP、20 μldGTP、20μl dCTP。将排管底部轻轻磕碰桌面,使得碱基平铺在排管底部;
7)焦磷酸测序,测序结果如图1~3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ALDH2(G1510A)型。
2)结果判定标准
ALDH2(G1510A)的DNA测序峰值图中,
a.G的频率≧90%,C的频率≦10%,即为GG型,又称为*1/*1型;
b.40%≦G的频率≦60%,40%≦A的频率≦60%,即为GA型,又称为*1/*2型;
c.A的频率≧90%,T的频率≦10%,即为AA型,又称为*2/*2型;
实施例3、基因检测结果与药物疗效的相关性
采用96名使用舌下含服硝酸甘油药物的患者的血样,对ALDH2进行基因分型,分析药物疗效与基因型的相关性。结果表示,ALDH2野生型舌下含服硝酸甘油的有效率明显比含有*2等位基因的基因型高。
硝酸甘油疗效 | ALDH2*1/*1型 | ALDH2*1/*2、*1/*2型 |
硝酸甘油有效组 | 43(86%) | 18(39.13%) |
硝酸甘油无效组 | 7(14%) | 28(60.87%) |
ALDH2对应的基因型有ALDH2*1/1,ALDH2*1/2,ALDH2*2/2。突变型对硝酸甘油的催化活性较野生型严重下降。ALDH2基因型和硝酸甘油药物疗效的相关性的总结如下:
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> 一种硝酸甘油代谢标志物的检测试剂盒及其检测方法和应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> unsure
<222> (1)..(32)
<400> 1
caactgctat gatgtgtttg gagcccagtc 30
<210> 2
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> unsure
<222> (1)..(30)
<400> 2
agcccccagc aggtcccaca ctcacagttt 30
<210> 3
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> unsure
<222> (1)..(15)
<400> 3
gggctgcagg catac 15
<210> 4
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> unsure
<222> (1)..(29)
<400> 4
actraagtga aaactgtgag tgtgggacc 29
<210> 5
<211> 10
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> unsure
<222> (1)..(10)
<400> 5
cacgtgagtg 10
Claims (10)
1.一种硝酸甘油代谢标志物的检测试剂盒,其特征在于,所述检测试剂盒用于检测硝酸甘油的代谢标志物基因位点ALDH2 G1510A的基因多态性,试剂盒包括ALDH2G1510A扩增引物、ALDH2 G1510A测序引物和阳性对照。
2.根据权利要求1所述的硝酸甘油代谢标志物的检测试剂盒,其特征在于,所述ALDH2G1510A扩增引物如序列表SEQ ID NO:1~2所示。
3.根据权利要求1所述的硝酸甘油代谢标志物的检测试剂盒,其特征在于,所述ALDH2G1510A测序引物如序列表SEQ ID NO:3所示。
4.根据权利要求1所述的硝酸甘油代谢标志物的检测试剂盒,其特征在于,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所述的硝酸甘油代谢标志物的检测试剂盒,其特征在于,所述试剂盒还包括扩增缓冲液、18mM醋酸镁、dNTPS、链置换DNA聚合酶、单链DNA结合蛋白、结合单链核酸的重组酶和海藻糖。
6.根据权利要求5所述的硝酸甘油代谢标志物的检测试剂盒,其特征在于,所述试剂盒中各组分终浓度为:扩增前后引物各0.32uM,dNTPS 0.3mM,链置换DNA聚合酶1.2ng/μL,单链DNA结合蛋白3.2ng/μL,结合单链核酸的重组酶4.8ng/μL,海藻糖0.2%。
7.根据权利要求1所述的硝酸甘油代谢标志物的检测试剂盒,其特征在于,ALDH2G1510A测序引物对应的测序区域为ALDH2 G1510A待检序列,如序列表SEQ ID NO:4所示。
8.根据权利要求1所述的硝酸甘油代谢标志物的检测试剂盒,其特征在于,ALDH2G1510A待检序列对应ALDH2 G1510A分配指令如序列表SEQ ID NO:5所示。
9.一种采用权利要求1~8任一项所述的硝酸甘油代谢标志物的检测试剂盒的检测方法,其特征在于,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RPA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.向每个测序管中加入测序酶和测序底物;
d.取一个dNTP排管,自圆滑一端向平端依次加入dATPαS、dTTP、dGTP、dCTP;
e.焦磷酸测序;
f.确定ALDH2 G1510A位点硝酸甘油位点的基因型。
10.一种采用权利要求1~8任一项所述的硝酸甘油代谢标志物的检测试剂盒的应用,其特征在于,所述检测试剂盒用于体外检测待测样本中的ALDH2 G1510A基因多态性。
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