CN113667743A - 一种用于布地奈德代谢标志物的检测试剂盒及其检测方法和应用 - Google Patents
一种用于布地奈德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
本发明公开了一种用于布地奈德代谢标志物的检测试剂盒及其检测方法和应用,其中,所述试剂盒针对GLCCI1A‑1106G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:样本处理液、磁珠、扩增试剂1、扩增试剂2、GLCCI1A‑1106G测序引物和阳性对照。本发明主要从两方面进行了优化,一方面采用提取扩增同管的方式,免去提取多次移管、核酸丢失风险;另一方面通过加入抗抑制剂进行快速恒温扩增;本发明的目的是以快速DNA制备、恒温PCR扩增和焦磷酸测序技术为组合对布地奈德疗效的基因多态性进行检测,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布地奈德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
布地奈德,是一具有高效局部抗炎作用的糖皮质激素,能增强内皮细胞、平滑肌细胞和溶酶体膜的稳定性,抑制免疫反应和降低抗体合成,从而使组胺等过敏活性介质的释放减少和活性降低,并能减轻抗原抗体结合时激发的酶促过程,抑制支气管收缩物质的合成和释放而减轻平滑肌的收缩反应。临床上用于糖皮质激素依赖性或非依赖性的支气管哮喘和哮喘性慢性支气管炎患者。难治性哮喘是指采用包括吸入中高剂量糖皮质激素和长效β2受体激动药两种或更多种的控制药物规范治疗至少3~6个月仍不能达到良好控制的哮喘。在门诊随诊患儿中,难治性哮喘病例比较多见。哮喘是一种由遗传因素和环境因素相互作用而形成的多基因遗传疾病,发病有明显的家族聚集现象,遗传度达70%~80%。而给予及时的正规治疗和给予合适的给药剂量,对于哮喘的控制至关重要。
糖皮质激素诱导转录因子1(glucocorticoid-induced transcript 1gene,GLCCI1)是一种蛋白质编码基因,在肺细胞和免疫细胞中表达,在哮喘条件下使用糖皮质激素,GLCCI1的表达会增加。有研究表明,GLCCI1可能是糖皮质激素诱导凋亡的早期标志物。GLCCI1(rs37973)与糖皮质激素应答息息相关,有研究报道,同AA基因型相比,携带GG基因型的哮喘患者对糖皮质激素应答较弱,而携带GA基因型的哮喘患者对糖皮质激素应答居中;G等位基因和GLCCI1的表达有关,在糖皮质激素刺激的人细胞中,同AA基因型相比,GG基因型的表达更低。因此,在糖皮质激素应答方面,GLCCI1扮演重要的角色,其机制可能是通过调节GLCCI1基因表达完成的。故开展抗哮喘药物相关基因检测可为儿童哮喘个体化治疗提供有力的依据。
目前,基因多态性检测的方法有很多种,主要采用荧光PCR。主要包括高分辨率熔解曲线法、taqman荧光探针法、等位基因特异性扩增法。高分辨率熔解曲线法步骤简单,但特异性偏低,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,但检测条件要求严格,实际操作中容易出现引物错配而产生假阳。taqman荧光探针法试验成本较高。
目前对血液DNA的获得方式主要有传统的柱式提取、磁珠法提取,此两种方法均耗时较久,操作较为繁琐。CN201610022581.8提出了一种在一管中进行磁珠提取核酸与扩增的实时荧光定量PCR方法,将混合有磁珠的裂解液和待检测样品加入到PCR扩增管中,混匀,静置,进行磁吸后吸出混合液,将得到的磁珠洗涤一次;在上述PCR扩增管中加入配制好的PCR反应液,对目标核酸进行实时荧光定量PCR反应。该发明也同样需要对磁珠进行洗涤。因此,急需建立一种简单、快速有效、价格低廉、特异性高的检测IRS-1基因多态性的焦磷酸测序方法。
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布地奈德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,本发明采用的用于布地奈德代谢标志物的检测试剂盒的技术方案如下:
所述布地奈德疗效预测的基因多态性检测试剂盒针对GLCCI1(A-1106G)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:样本处理液、磁珠、扩增试剂1、扩增试剂2、GLCCI1(A-1106G)测序引物、阳性对照。
具体的,所述特异性引物序列如下表1所示:
优选的,GLCCI1(A-1106G)扩增引物如序列表SEQ ID NO:1~2所示。
优选的,GLCCI1(A-1106G)测序引物如序列表SEQ ID NO:3所示。
优选的,GLCCI1(A-1106G)测序引物对应的测序区域为GLCCI1(A-1106G)待检序列,如序列表SEQ ID NO:4所示;优选的,GLCCI1(A-1106G)分配指令如序列表SEQ ID NO:5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,GLCCI1(A-1106G)测序引物对应的测序区域为GLCCI1(A-1106G)待检序列,如序列表SEQ ID NO:4所示;GLCCI1(A-1106G)分配指令如序列表SEQ ID NO:5所示。
优选的,所述样本处理液,不包含胍盐,通过碱性条件和表面活性剂对样本进行裂解。包括0.1%-0.6%十二烷基硫酸锂、0.1-0.5%曲拉通X-100、2-50mg/mL氢氧化钠、5-15%海藻糖、3~7mmol/L BSA、20-80mM Tris-HCl、100mM NaCl,PH=8.5-9.5。
更优选的,所述样本处理液,包括0.3%-0.5%十二烷基硫酸锂、0.2-0.4%曲拉通X-100、10-20mg/mL氢氧化钠、8-12mM甜菜碱、8-12%海藻糖、4~6mM BSA、50mM Tris-HCl、100mM NaCl,PH=9。
优选的,所述的磁珠为羧基磁珠,粒径为600mm,悬浮性较好,磁性能较强,吸附量大。反应液中磁珠浓度为0.2mg/25μL,血液样本中DNA吸附量为30ng~260ng,完全满足扩增所需的DNA量。
优选的,所述的扩增试剂1包括:扩增缓冲液,15mM醋酸镁。
优选的,所述的扩增试剂2包括:GLCCI1(A-1106G)前引物(0.32uM),GLCCI1(A-1106G)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)。
更优选的,所述的扩增试剂2包括:海藻糖(0.2%),10mM醋酸锰、0.1M山梨糖醇、5ug/mL BSA。海藻糖对生物活性物质具有非特异性保护作用,能提高DNA聚合酶热稳定性、降低DNA模板解链温度,减少G-C丰富区由于自身互补配对所形成的二级结构,因此提高PCR反应的特异性。山梨糖醇和醋酸锰具有PCR预混液稳定效应,同时在冷冻干燥过程中具有稳定作用。牛血清白蛋白能提高PCR反应的扩增效率并减少体系中PCR抑制物对反应的影响。
优选的,所述的反应体积为25ul,反应条件为:42℃25min。
优选的,所述的阳性对照,包括浓度为20ng/ul的GLCCI1(A-1106G)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布地奈德疗效预测的基因多态性检测方法,所述检测方法包括以下步骤:
a.样本处理液100ul、4ul磁珠和30ulEDTA抗凝全血样本混合,室温静置5min;
b.将所述PCR扩增管放置在磁力架上,待磁珠全部吸附至一侧后从磁珠对侧将混合液吸出;
c.将配制好的PCR反应液加入到步骤b)得到的PCR扩增管中,使磁珠与所述PCR反应液充分混匀,离心,进行恒温反应;
d.结合液(含微珠)与扩增产物进行结合;
e.变性液处理得到单链产物;
f.加入洗涤缓冲液漂洗;
g.向每个测序管中加入测序酶和测序底物;
h.取一个8排管,自圆滑一端向平端依次加入dATP、dTTP、dGTP、dCTP、GLCCI1(A-1106G)测序引物;将排管底部轻轻磕碰桌面,使得碱基平铺在排管底部;
i.焦磷酸测序。
本发明还公开了一种布地奈德疗效预测的基因多态性检测试剂盒的应用,所述检测试剂盒对GLCCI1(A-1106G)进行检测,以从基因层面反应布地奈德疗效,进一步为指导布地奈德使用给出基因层面的建议。
与现有技术相比,本发明采用样本处理液快速样本释放出DNA,样本量为30ul全血,相比一步裂解法和直接扩增(约2-10ul),可以得到足量的基因组DNA吸附在磁珠上供后续分析。通过将样本和裂解混合液移除可以去除大部分抑制物。DNA模板通过恒温PCR扩增GLCCI1(A-1106G)基因位点,产生大量的生物素标记的单链DNA。生物素标记单链DNA与链霉亲和素结合,洗涤后加入测序引物和测序原料,进行焦磷酸测序,简化了测序流程和时间。本发明以快速DNA制备、恒温PCR扩增和焦磷酸测序技术为组合对布地奈德疗效的基因多态性进行检测,为临床个性化用药给出基因角度的建议。
本发明的快速扩增方法主要从两方面进行了优化,一方面采用提取扩增同管的方式,免去提取多次移管、核酸丢失风险;另一方面通过加入抗抑制剂进行快速恒温扩增;本发明的目的是以恒温PCR和焦磷酸检测为基础,获得一种布地奈德疗效预测的基因多态性检测试剂盒及其检测方法和应用。
附图说明
图1是本发明提供的GLCCI1(A-1106G)位点GG型焦磷酸检测结果示例图;
图2是本发明提供的GLCCI1(A-1106G)位点GA型焦磷酸检测结果示例图;
图3是本发明提供的GLCCI1(A-1106G)位点AA型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用于布地奈德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、试剂盒的制备及使用
(一)特异性引物设计
本发明的试剂盒针对GLCCI1(A-1106G)基因多态性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,引物序列如下表1所示。
表1
(二)试剂盒组成
检测试剂盒包括如下表2所示的组分:
表2试剂盒组分表
(三)样本处理液配置体系如下:
样本处理液,包括0.4%十二烷基硫酸锂、0.3%曲拉通X-100、15mg/mL氢氧化钠、10mM甜菜碱、10%海藻糖、5mM BSA、50mM Tris-HCl、100mM NaCl,PH=9。
(四)本实施例的检测试剂盒扩增试剂1单人份配置体系如下:
成分 | 体积(ul) |
扩增缓冲液 | 24 |
200mM醋酸镁 | 1 |
(五)本实施例的检测试剂盒扩增试剂2单人份配置体系如下:
GLCCI1(A-1106G)前引物(0.32uM),GLCCI1(A-1106G)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%),10mM醋酸锰、0.1M山梨糖醇、5ug/mL BSA。PCR体系如表3所示:
表3 PCR体系表
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(25mM) | 0.3 |
GLCCI1(A-1106G)前引物(20μM) | 0.4 |
GLCCI1(A-1106G)后引物(20μM) | 0.4 |
海藻糖(20%) | 0.25 |
1M醋酸锰 | 0.25 |
10M山梨糖醇 | 0.25 |
5mg/mL BSA | 0.25 |
配置完成后96.8ul/管进行分装、冻干。
实施例2、试剂盒检测步骤
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)取30μL EDTA抗凝全血样品于PCR扩增管中;
2)加入100μL样本处理液和4ul磁珠,静置5min;
3)将所述PCR扩增管放置在磁力架上,待磁珠全部吸附至一侧后从磁珠对侧将混合液吸出;
4)将扩增试剂1加入扩增试剂2干粉中,充分溶解混匀;
5)将配制好的PCR反应液加入25ul到步骤4)得到的PCR扩增管中,使磁珠与所述PCR反应液充分混匀,离心,进行恒温反应;
6)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℃ | 25min | 1 |
7)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20μL,置于台式振荡器上,1100rpm振荡10min,使微珠和PCR产物充分结合;
8)7,000×g离心1min,弃上清;
9)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;
10)向EP管中加入150uL洗涤缓冲液,7,000×g离心1min。(重复3次);
11)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;
12)测序管中分别加入3uL测序酶和3uL测序底物;
13)取一个8排管,自圆滑一端向平端依次加入dATP、dTTP、dGTP、dCTP、GLCCI1(A-1106G)测序引物;将排管底部轻轻磕碰桌面,使得碱基平铺在排管底部;
14)焦磷酸测序;如图1~3所示;
15)结果判读。
i.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GLCCI1(A-1106G)杂合突变型。
ii.结果判定标准
GLCCI1(A-1106G)的DNA测序峰值图中,
A的频率≧90%,G的频率≦10%,即为AA型;
40%≦A的频率≦60%,40%≦G的频率≦60%,即为GA型;
G的频率≧90%,A的频率≦10%,即为GG型;
16)基因检测结果与布地奈德疗效预测抵抗的相关性表
五、试剂盒的性能测试结果
5.1.特异性
对特异性样本(包括非人类DNA模板和人类同一基因不同位点或同源性位点扩增产物稀释物)进行检测,结果为阴性。
5.2.准确性
对试剂盒范围内的不同基因型的参考品(包括GLCCI1(A-1106G)野生、杂合、突变三种基因型)进行检测,应均能检出相应基因型。
5.3.最低检出限
最低检出限应不高于2ng/ul。
5.4.重复性
检测试剂盒范围内的不同基因型的重复性参考品,每个参考品进行10次检测,结果应均为相应突变类型,且相应检测通道的Ct值的变异系数(CV)应≤5.0%。
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> 一种用于布地奈德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
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<221> unsure
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tgacataaaa ttccttgttg acccctgcta 30
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gctgagtttt cgtgaccagt agccttatta 30
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<222> (1)..(18)
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gagaaatgtc tggaacct 18
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<212> DNA
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gcabtgaaca aagtaaaata ctcttacag 29
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<213> 人工序列(Artificial Sequence)
<220>
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<222> (1)..(11)
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cgcgacgtga c 11
Claims (10)
1.一种用于布地奈德代谢标志物的检测试剂盒,其特征在于,所述用于布地奈德代谢的标志物为GLCCI1 A-1106G的基因多态性,试剂盒针对GLCCI1 A-1106G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:样本处理液、磁珠、扩增试剂1、扩增试剂2、GLCCI1 A-1106G测序引物和阳性对照。
2.根据权利要求1所述的用于布地奈德代谢标志物的检测试剂盒,其特征在于,所述GLCCI1 A-1106G扩增引物如序列表SEQ ID NO:1~2所示。
3.根据权利要求1所述的用于布地奈德代谢标志物的检测试剂盒,其特征在于,所述GLCCI1 A-1106G测序引物如序列表SEQ ID NO:3所示。
4.根据权利要求1所述的用于布地奈德代谢标志物的检测试剂盒,其特征在于,GLCCI1A-1106G分配指令如序列表SEQ ID NO:5所示。
5.根据权利要求1所述的用于布地奈德代谢标志物的检测试剂盒,其特征在于,所述样本处理液包括0.1%-0.6%十二烷基硫酸锂、0.1-0.5%曲拉通X-100、2-50mg/mL氢氧化钠、5-15%海藻糖、3~7mmol/L BSA、20-80mM Tris-HCl、100mM NaCl,PH=8.5-9.5。
6.根据权利要求1所述的用于布地奈德代谢标志物的检测试剂盒,其特征在于,所述磁珠为羧基磁珠。
7.根据权利要求1所述的用于布地奈德代谢标志物的检测试剂盒,其特征在于,所述扩增试剂1包括:扩增缓冲液,15mM醋酸镁。
8.根据权利要求1所述的用于布地奈德代谢标志物的检测试剂盒,其特征在于,所述扩增试剂2包括:GLCCI1 A-1106G前引物0.32uM,GLCCI1 A-1106G后引物0.32uM,dNTPS0.3mM、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。
9.一种采用根据权利要求1~8任一项所述的用于布地奈德代谢标志物的检测试剂盒的检测方法,其特征在于,所述检测方法包括如下步骤:
a.样本处理液100ul、4ul磁珠和30ulEDTA抗凝全血样本混合,室温静置5min;
b.将所述PCR扩增管放置在磁力架上,待磁珠全部吸附至一侧后从磁珠对侧将混合液吸出;
c.将配制好的PCR反应液加入到步骤b)得到的PCR扩增管中,使磁珠与所述PCR反应液充分混匀,离心,进行恒温反应;
d.将含磁珠的结合液与扩增产物进行结合;
e.变性液处理得到单链产物;
f.加入洗涤缓冲液漂洗;
g.向每个测序管中加入测序酶和测序底物;
h.取一个8排管,自圆滑一端向平端依次加入dATP、dTTP、dGTP、dCTP、GLCCI1A-1106G测序引物;
i.焦磷酸测序。
10.一种根据权利要求1~9任一项所述的用于布地奈德代谢标志物的检测试剂盒及其检测方法的应用,其特征在于,所述检测试剂盒对GLCCI1 A-1106G进行检测。
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