CN113755576A - 一种用于静脉血栓风险评估的检测试剂盒及其检测方法和应用 - Google Patents
一种用于静脉血栓风险评估的检测试剂盒及其检测方法和应用 Download PDFInfo
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Abstract
本发明公开了一种用于静脉血栓风险评估的检测试剂盒及其检测方法和应用,其中,所述用于检测静脉血栓代谢的标志物为PAI‑1(4G/5G)、F5(G1691A)两个基因,试剂盒针对PAI‑1(4G/5G)、F5(G1691A)两个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、PAI‑1(4G/5G)测序引物、F5(G1691A)测序引物和阳性对照。本发明以血液直扩、快速扩增和优化焦磷酸测序技术为组合对静脉血栓风险评估相关的基因多态性进行检测,为静脉血栓风险评估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静脉血栓风险评估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
静脉血栓栓塞症(venousthromboembolism,VTE)是由遗传、环境、行为等多种因素共同作用的疾病,VTE的发生50%~60%可归因于遗传因素现已知的遗传因素在东西方人群中存在较大差异,蛋白C缺陷、蛋白S缺陷以及抗凝血酶缺陷是亚洲人群主要的遗传缺陷类型,主要表现为深静脉血栓形成(DVT)和肺血栓栓塞(PE)。随着临床工作对该疾病的关注及医疗卫生事业的发展,静脉血栓在亚洲发病率逐年上升。突变及基因多态性在疾病发生中发挥重要作用。遗传因素表现出明显的地域和种族差异性。
纤溶酶原激活物抑制剂(PAI-1)基因启动子区域4G/5G多态性:作为组织和尿纤溶酶原激活物的主要抑制物,PA1-1能抑制活性纤溶酶的产生。PAI-1的过度表达可导致纤溶系统受损进而增加血栓栓塞事件的风险单鸟瞟吟核昔插入/删除(4G/5G)变异位于PAI-1基因的启动子区域,可影响PAI-1的表达量进而对纤溶系统产生影响。等位基因4G与PA1-1水平升高有关,可增加VTE的发生风险在亚洲人群中该多态性与血栓栓塞风险也存在相关关系,且风险高于高加索人群。
凝血因子V基因Leiden突变(factorVLeiden,FVL)是遗传性易栓症中最常见的类型,属常染色体显性遗传,分为纯合型及杂合型,杂合子形成血栓的风险较正常人高5~10倍,而纯合子可高达80倍。凝血因子VLeiden是指凝血因子V基因的一个位点发生突变,使编码蛋白的密码子发生改变,致使所编码的凝血因子V虽可正常激活,但对APC产生抵抗,导致血液中凝血酶含量增高,患有静脉血栓栓塞(VTE)的风险增大。F5编码凝血因子V(FV)突变已明确为静脉血栓栓塞症(venousthromboembolism,VTE)的原发性危险因素。F 5突变纯合子发生静脉血栓的风险大大增加,这种血栓形成风险与活化蛋白C(ActivatedProteinC,APC)抵抗有关,APC可以水解活化因子V(activatedfactorV,FVa)成为具有抗凝作用的FVac,还可以间接降低凝血酶原的活性,刺激纤溶酶原激活物的释放,增加纤溶活性。F5基因突变产生的APC抵抗与FVac生产障碍有关。
目前,对于基因多态性检测的方法主要有直接测序法、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,本发明的目的是获得一种用于静脉血栓风险评估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,本发明采用的用于静脉血栓风险评估的检测试剂盒的技术方案如下:
本发明的快速反应试剂盒针对PAI-1(4G/5G)、F5(G1691A)两个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、PAI-1(4G/5G)测序引物、F5(G1691A)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PAI-1(4G/5G)的特异性引物组序列如序列表SEQ ID NO:1~SEQ IDNO:2所示;所述F5(G1691A)的特异性引物组序列如序列表SEQ ID NO:3~SEQ ID NO:4所示。
优选的,所述的PAI-1(4G/5G)测序引物、F5(G1691A)测序引物分别如序列表SEQID NO:5~SEQ ID NO:6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。与DNA结合较DNA/DNA的结合更为稳定。
优选的,PAI-1(4G/5G)测序引物对应的测序区域为PAI-1(4G/5G)待检序列,如序列表SEQ ID NO:7所示;F5(G1691A)测序引物对应的测序区域为F5(G1691A)待检序列,如序列表SEQ ID NO:8所示。
优选的,PAI-1(4G/5G)和F5(G1691A)共用一个分配指令如序列表SEQ ID NO:9所示。
优选的,所述的扩增反应液,包括,PAI-1(4G/5G)和F5(G1691A)特异性扩增引物,还包括血液样品直接PCR预混液(2×)、海藻糖。
更优选的,反应液各组分浓度分别为PAI-1(4G/5G)前引物(0.2uM),PAI-1(4G/5G)后引物(0.2uM),F5(G1691A)前引物(0.25uM),F5(G1691A)后引物(0.25uM),PCR预混液(1×),海藻糖(0.2%)。
优选的,所述的PCR预混液为Blood Direct PCR Master Mix(2X),含有耐血的HemoTaqTMDNA聚合酶,对全血中血红素等各种PCR抑制剂表现出超强的抗性。
优选的,为获得最高的检测灵敏度,20μl的PCR扩增体系中最大加入的血液量可以达到4μl,即20%体积。
优选的,所述的阳性对照,包括浓度为20ng/ul的PAI-1(4G/5G)-F5(1691GA)型基因组DNA,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,所述的反应体积为20ul,反应条件为:预变性温度设为95℃,预变性时间设为5min,变性温度设为95℃,变性时间设为0s,退火延伸温度设为58℃,退火延伸时间设为0s,扩增35个循环。
优选的,扩增所采用的PCR管密封薄膜,在PCR反应孔处具有与加热柱匹配的凹陷设计,其厚度为85μm,贴合度高,热传递快。更优选的,PCR管密封薄膜具穿透性,可用移液器枪头或探针穿透进行产物回收。
本发明还公开了一种采用上述试剂盒的静脉血栓风险评估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4ul待测EDTA抗凝全血混合均匀进行PCR扩增;
b.将含链霉亲和素标记微珠的结合液与扩增产物进行混合;
c.加入洗涤缓冲液漂洗;
d.变性液处理得到单链产物;
e.加入洗涤缓冲液漂洗;
f.向每个测序管中加入测序酶和测序底物;
g.取一个8排管,自圆滑一端向平端依次加入dATP、dTTP、dGTP、dCTP、PAI-1(4G/5G)测序引物、F5(G1691A)测序引物、ddGTP;将排管底部轻轻磕碰桌面,使得碱基平铺在排管底部;
h.焦磷酸测序;
i.确定PAI-1(4G/5G)、F5(G1691A)的基因型。
本发明还公开了一种用于静脉血栓风险评估试剂盒及方法的应用,所述检测试剂盒对PAI-1(4G/5G)、F5(G1691A)进行检测,以从基因层面指导静脉血栓风险评估。
与现有技术相比,本发明以血液直扩、快速扩增和优化焦磷酸测序技术为组合对静脉血栓风险评估相关的基因多态性进行检测,为静脉血栓风险评估临床个性化用药给出基因角度的建议。
针对现有技术存在的上述问题,本发明的快速扩增方法主要从三方面进行了优化,一方面采用血液直扩的方式,免去核酸提取的步骤,只需将样品与其他PCR必须的组分加入反应管中混匀即可。另一方面通过将PCR仪加热模块设计由加热底座和加热柱两部分组成,加热柱四周与底座相连中间与反应孔对应的,该加热模块在PCR扩增时伸入PCR反应管中,使反应液分散在加热柱和PCR管壁之间,从中间和四周同时变化温度,可以显著地提高热传递效率,降低了反应液各部分的温度变化差异,提高了反应液整体的温度一致性和变温速度,为PCR的快速扩增提供了另一关键要素。第三方面采用双重PCR扩增PAI-1(4G/5G)、F5(G1691A)两个位点,同时一次反应进行两个位点的焦磷酸测序。该测序首先加PAI-1(4G/5G)测序引物与测序原料进行焦磷酸测序,最后一个碱基加入ddGTP终止该测序反应。再加入F5(G1691A)测序引物和相应的dNTP进行测序。一次处理先后进行两个位点的测序,减少了操作的时间和提高了测序的通量。
附图说明
图1是本发明提供的PCR反应管的结构示意图;
图2是本发明提供的PAI-1(5G/5G)、F5(1691AA)型测序结果示例图;
图3是本发明提供的PAI-1(4G/5G)、F5(1691GA)型测序结果示例图;
图4是本发明提供的PAI-1(4G/4G)、F5(1691GG)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用于静脉血栓风险评估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
本发明的快速反应试剂盒针对PAI-1(4G/5G)、F5(G1691A)设计了特异性扩增引物和测序引物,用于扩增和焦磷酸测序检测。基于快速扩增技术设计引物是本发明的关键之一,为保证扩增速度及检测灵敏度,扩增长度应控制在60-120bp。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PCR反应液单人份配置体系如下:
PCR反应液各组分浓度分别为:PAI-1(4G/5G)前引物(0.2uM),PAI-1(4G/5G)后引物(0.2uM),F5(G1691A)前引物(0.25uM),F5(G1691A)后引物(0.25uM),PCR预混液(1×),海藻糖(0.2%);其中Easy-LoadTMBlood Direct PCR Master Mix(2X)购自上海钰博生物科技有限公司。
成分 | 体积(ul) |
Blood Direct PCR Master Mix(2×) | 10 |
Nuclease-Free Water | 4 |
PAI-1(4G/5G)前引物(10μM) | 0.4 |
PAI-1(4G/5G)后引物(10μM) | 0.4 |
F5(G1691A)前引物(10μM) | 0.5 |
F5(G1691A)后引物(10μM) | 0.5 |
海藻糖(20%) | 0.2 |
配置完成后200ul/管进行分装。
实施例2、焦磷酸检测
本发明中采用的仪器如下:扩增仪、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PCR反应液涡旋振荡15秒,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16μL/管分装至PCR反应管中。
(2)加样检测(样本制备间)
将EDTA抗凝全血、阳性对照和空白对照按4μL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20μL,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;扩增所采用的PCR管密封薄膜,在PCR反应孔处具有与加热柱匹配的凹陷设计,其厚度为85μm,PCR管密封薄膜具穿透性,可用移液器枪头或探针穿透进行产物回收;上述PCR管结构如图1所示;
2)7,000×g离心1min,弃上清;
3)加入22uL稀释后的变性液工作液,静置5min,7,000×g离心1min,EP管收集得到单链产物;
4)向EP管中加入150uL洗涤缓冲液,7,000×g离心1min;
5)将EP管中的单链产物转移至测序管中,向每个测序管中加入3uL测序酶和3uL测序底物;
6)取一个8排管,自圆滑一端向平端依次加入dATP、dTTP、dGTP、dCTP、PAI-1(4G/5G)测序引物、F5(G1691A)测序引物、ddGTP;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2~4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PAI-1(4G/5G)-F5(G1691A)型。
2)结果判定标准
a.PAI-1(4G/5G)的DNA测序峰值图中,
C的高度为A的2倍,即为型4G/4G;
C的高度为A的2.5倍,即为型4G/5G;
C的高度为A的3倍,即为型5G/5G;
b.F5(G1691A)的DNA测序峰值图中,
G的频率≧90%,A的频率≦10%,即为GG型;
40%≦G的频率≦60%,40%≦A的频率≦60%,即为GA型;
A的频率≧90%,G的频率≦10%,即为AA型。
(6)风险评估
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> 菲思特(上海)生物科技有限公司
<120> 一种用于静脉血栓风险评估的检测试剂盒及其检测方法和应用
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<220>
<221> unsure
<222> (1)..(22)
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<210> 4
<211> 24
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tgttctagcc agaagaaatt ctca 24
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<211> 15
<212> DNA
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<211> 16
<212> DNA
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<220>
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<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
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<220>
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Claims (10)
1.一种用于静脉血栓风险评估的检测试剂盒,其特征在于,所述用于检测静脉血栓代谢的标志物为PAI-1 4G/5G、F5 G1691A两个基因,试剂盒针对PAI-1 4G/5G、F5G1691A两个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、PAI-1 4G/5G测序引物、F5 G1691A测序引物和阳性对照。
2.根据权利要求1所述的用于静脉血栓风险评估的检测试剂盒,其特征在于,所述PAI-1 4G/5G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F5 G1691A的特异性引物组序列如序列表SEQ ID NO:3~SEQ ID NO:4所示。
3.根据权利要求1所述的用于静脉血栓风险评估的检测试剂盒,其特征在于,所述PAI-1 4G/5G测序引物、F5 G1691A测序引物分别如序列表SEQ ID NO:5~SEQ ID NO:6所示。
4.根据权利要求1所述的用于静脉血栓风险评估的检测试剂盒,其特征在于,所述PAI-1 4G/5G和F5 G1691A共用一个分配指令如序列表SEQ ID NO:9所示。
5.根据权利要求1所述的用于静脉血栓风险评估的检测试剂盒,其特征在于,所述扩增反应液包括PAI-1 4G/5G和F5 G1691A特异性扩增引物、血液样品直接PCR预混液和海藻糖。
6.根据权利要求1所述的用于静脉血栓风险评估的检测试剂盒,其特征在于,所述阳性对照包括浓度为20ng/ul的PAI-1 4G/5G和F5 1691GA型基因组DNA。
7.根据权利要求1所述的用于静脉血栓风险评估的检测试剂盒,其特征在于,扩增所采用的PCR管为密封薄膜且薄膜可穿透的PCR管。
8.一种采用根据权利要求1~7任一项所述的用于静脉血栓风险评估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4ul待测EDTA抗凝全血混合均匀进行PCR扩增;
b.将含链霉亲和素标记微珠的结合液与扩增产物进行混合;
c.加入洗涤缓冲液漂洗;
d.变性液处理得到单链产物;
e.加入洗涤缓冲液漂洗;
f.向每个测序管中加入测序酶和测序底物;
g.取一个8排管,自圆滑一端向平端依次加入dATP、dTTP、dGTP、dCTP、PAI-1 4G/5G测序引物、F5 G1691A测序引物、ddGTP;
h.焦磷酸测序。
9.根据权利要求8所述的用于静脉血栓风险评估的检测方法,其特征在于,所述扩增反应体积为20ul体系,体系中血液最大加入量为4ul。
10.一种根据权利要求1~9任一项所述的用于静脉血栓风险评估的检测试剂盒及其检测方法的应用,其特征在于,所述检测试剂盒及其检测方法对PAI-1 4G/5G、F5G1691A进行检测。
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Cited By (3)
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CN114317724A (zh) * | 2022-02-09 | 2022-04-12 | 湖南时代基因医学检验技术有限公司 | 一组用于中国汉族人群静脉血栓栓塞症遗传风险预测的生物标志物、试剂盒及其应用 |
CN114672548A (zh) * | 2022-03-10 | 2022-06-28 | 华捷生物科技(青岛)有限公司 | 人类静脉血栓风险基因多态性检测试剂盒及工艺和应用 |
CN114672548B (zh) * | 2022-03-10 | 2024-04-19 | 华捷生物科技(青岛)有限公司 | 一种人类静脉血栓风险基因pai-1,thbd和proc基因多态性检测试剂盒及其制备方法和应用 |
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