CN111187830A - 一种用于检测ApoE和SLCO1B1基因多态性的试剂盒和方法 - Google Patents
一种用于检测ApoE和SLCO1B1基因多态性的试剂盒和方法 Download PDFInfo
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Abstract
本发明公开了一种用于检测ApoE和SLCO1B1基因多态性检测的试剂盒和方法。所述试剂盒包括ApoE 526C>T与ApoE A388T>C联合检测反应液和SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液。实验证明,本发明所述联合检测反应液中的引物和探针特异性强,能实现直接采用唾液/血样进行PCR扩增分析,且可在一个反应管中实现两个SNP的分型检测,不仅明显提高了检测效率,同时也明显缩短了检测周期,并且具有敏感性好、特异性高、准确可靠、操作方便快捷等优点。
Description
技术领域
本发明是涉及一种用于检测ApoE和SLCO1B1基因多态性检测的试剂盒和方法,属于体外基因检测技术领域。
背景技术
高血脂症是指血清中的胆固醇和甘油三酯升高的一种疾病。血脂主要由四种物质组成:总胆固醇、甘油三酯、低密度脂蛋白和高密度脂蛋白。总胆固醇、甘油三酯和低密度脂蛋白这三项中只要任何一项超标就属于高血脂。血中的胆固醇又与动脉弹性硬蛋白结合,血管变硬,变脆,失去原本的弹力活性,导致重要器官动脉供血不足,从而引发冠心病、脑中风、肢体坏死等。不仅如此,高血脂还可引发诸如高血压、糖尿病、老年痴呆、肝硬化等多种疾病,甚至诱发癌症。
现有研究表明:ApoE和SLCO1B1基因均可通过多种途径参与机体的脂质代谢调节,是影响机体血脂水平的重要内在因素;其中,人类ApoE基因定位于19号染色体上,而第19号染色体是载脂蛋白E编码基因,该段编码基因的第4外显子参与编码第112位氨基酸和第158位氨基酸的密码子,而这段密码子具有多态性,可编码出3种常见的等位基因,即:ε2,ε3,ε4,又可分为6种常见的基因型,它们分别是ε2/2、ε2/3、ε3/3、ε2/4、ε3/4、ε4/4,因此,人类ApoE基因具有多态性,主要有两种单核苷酸多态性526C>T和388T>C,可以形成3种单倍型ApoE3(388T-526C)、ApoE2(388T-526T)、ApoE4(388C-526C);ApoE多态性对血脂在预防、诊断及治疗上具有重要的临床应用价值;但ApoE基因发生突变,可以形成不同的基因多态性,以致影响血脂的代谢和利用,从而影响高脂血症、动脉粥样硬化和心脑血管疾病的发病率,例如:ApoE4携带者患冠心病的风险要高40%,并且他汀类药物对ApoE4携带者疗效往往不佳或无疗效,而对ApoE2携带者的降脂作用最强;人类SLCO1B1基因定位于12号染色体,编码有机阴离子转运多肽OATP1B1。OATP1B1为跨膜转运蛋白,具有介导肝细胞膜转运内、外源性物质并对其进行代谢和消除的生理功能。研究表明:SLCO1B1基因具有遗传多态性,其中388A>G、521T>C是两种常见的单核苷酸多态性,可以形成4种单倍型:SLCO1B1*1a(388A-521T)、SLCO1B1*1b(388G-521T)、SLCO1B1*5(388A-521C)和SLCO1B1*15(388G-521C);突变型SLCO1B1基因引起编码的OATP1B1转运蛋白活力减弱,表现为肝脏摄取药物能力降低,引起血药浓度上升,会增加横纹肌溶解症或肌病的发生风险。
另外,大量研究已经证实,调脂药物不仅降低血脂,同时也可明显减少冠心病、心肌梗塞、脑中风的发生率、致残率和死亡率。在降血脂药物中,当属他汀类的临床研究和使用最为广泛。他汀类药物是通过竞争性抑制HMG-CoA(三羟基三甲基戊二酰辅酶A)还原酶从而减少胆固醇的生物合成,进而达到降低血脂的作用,但大剂量长期使用他汀类药物除了经济上的负担,还有非常严重的安全隐患,下表是SLCO1B1*1b(388A、388G)、SLCO1B1*5(521T、521C)和载脂蛋白ApoE2(526C、526T)、ApoE4(388T、388C)基因型检测对应风险提示和药效提示:
也就是说,在服用他汀类药物前针对ApoE和SLCO1B1基因多态性进行检测可科学、有效评估用药后疗效。
目前针对基因多态性的检测方法主要有:PCR-RFLP法、PCR-芯片杂交法、PCR-测序法和Taqman-qPCR法,其中:PCR-RFLP法主要依赖于靶标的限制性酶切位点,实验难度增大,同时后续的电泳分析容易引起实验室污染,无法大规模临床应用;PCR-芯片杂交法是将PCR技术与基因芯片技术结合使用的,同样有步骤复杂和实验室交叉污染的风险,检测灵敏度低并且特异性差,容易出现假阳性结果;PCR-测序法是基因检测的金标准,但是其操作流程过长,成本过高,同时仪器昂贵也难以适合在医院大规模应用;Taqman-qPCR法检测灵敏度高,但往往由于位点序列原因,导致检测特异性不高,并且其通过Genotyping方法进行分型,对样本数量和多态性分布有一定要求,不适合检测突变频率过低的位点和少量样本。因此,本领域急需一种能快速、准确、敏感性好、特异性高、操作方便的检测ApoE和SLCO1B1基因多态性的技术。
发明内容
针对现有技术存在的上述问题和需求,本发明的目的是提供一种能快速、准确、敏感性好、特异性高、操作方便的用于检测ApoE和SLCO1B1基因多态性的试剂盒和方法。
为实现上述目的,本发明采用的技术方案如下:
一种用于检测ApoE和SLCO1B1基因多态性的试剂盒,包括ApoE 526C>T与ApoEA388T>C联合检测反应液和SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液;所述ApoE526C>T与ApoE A388T>C联合检测反应液包括针对ApoE 526C>T位点的SEQ ID NO.1和SEQID NO.2所示序列的引物组及SEQ ID NO.3和SEQ ID NO.4所示序列的探针组,针对ApoEA388T>C位点的SEQ ID NO.5和SEQ ID NO.6所示序列的引物组及SEQ ID NO.7和SEQ IDNO.8所示序列的探针组;所述SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液包括针对SLCO1B1 388A>G位点的SEQ ID NO.9和SEQ ID NO.10所示序列的引物组及SEQ ID NO.11和SEQ ID NO.12所示序列的探针组,针对SLCO1B1 521T>C位点的SEQ ID NO.13和SEQ IDNO.14所示序列的引物组及SEQ ID NO.15和SEQ ID NO.16所示序列的探针组。
进一步,所有探针的5'端标记有报告荧光染料及3'端标记有淬灭荧光染料。
进一步,所有探针的5'端标记有报告荧光染料FAM、VIC、ROX、CY5中的任意一种,所有探针的3'端均标记有淬灭荧光染料BHQ。
进一步,针对ApoE 526C>T位点的野生型探针的5’端标记有报告荧光染料FAM,针对ApoE 526C>T位点的突变型探针的5’端标记有报告荧光染料VIC。
进一步,针对ApoE A388T>C位点的野生型探针的5’端标记有报告荧光染料ROX,针对ApoE A388T>C位点的突变型探针的5’端标记有报告荧光染料CY5。
进一步,针对SLCO1B1 388A>G位点的野生型探针的5’端标记有报告荧光染料FAM,针对SLCO1B1 388A>G位点的突变型探针的5’端标记有报告荧光染料VIC。
进一步,针对SLCO1B1 521T>C位点的野生型探针的5’端标记有报告荧光染料ROX,针对SLCO1B1 521T>C位点的突变型探针的5’端标记有报告荧光染料CY5。
进一步,所述的ApoE 526C>T与ApoE A388T>C联合检测反应液及SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液中均还包括改良型HotStar Taq酶、直扩PCR扩增缓冲液、直扩试剂和稳定剂。
进一步,所述直扩PCR扩增缓冲液的组成为:Tris-HCl(三羟甲基氨基甲烷盐酸盐)缓冲溶液,其中含有200mmol/L~400mmol/L KCl(氯化钾)。
进一步,所述直扩试剂的组成为:25mmol/L~100mmol/L Tris-HCl(三羟甲基氨基甲烷盐酸盐),1mmol/L~10mmol/L EDTA(乙二胺四乙酸),200mmol/L~500mmol/L NaCl(氯化钠),0.1wt%~0.8wt%SDS(十二烷基硫酸钠),0.1mol/L~2.0mol/L山梨醇,0.5mmol/L~1.0mmol/L DTT(二硫苏糖醇)。
进一步,所述稳定剂的组成为:0.5wt%~1.5wt%甘油,0.2wt%~0.8wt%NP40(乙基苯基聚乙二醇),0.2wt%~0.8wt%tween20(吐温20),0.1mg/mL~2mg/mL BSA(牛血清白蛋白)。
进一步,所述试剂盒还包括阳性对照品和阴性对照品,所述阳性对照品采用人基因组,所述阴性对照品采用DEPC H2O。
一种用于检测ApoE和SLCO1B1基因多态性的方法,包括本发明上述的试剂盒,所述方法包括如下步骤:
a)分别取1份待测样本直接加入含有ApoE 526C>T与ApoE A388T>C联合检测反应液的PCR反应管Ⅰ和含有SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液的PCR反应管Ⅱ中,待混匀离心后,再放入荧光定量PCR仪器中同时进行PCR扩增反应;
b)对扩增产物进行荧光检测,并根据荧光曲线和Ct值直接对测试样本的多态性进行判读。
进一步,所述待测样本为唾液或血样。
进一步,每个PCR反应管中的待测样本量为1μL,联合检测反应液为20μL,且联合检测反应液中的引物和探针的浓度均为200nmol/L。
进一步,步骤a)进行PCR扩增反应的条件为:95℃预变性5分钟;95℃变性10秒钟;60℃退火30秒钟;40个循环。
相较于现有技术,本发明具有如下有益技术效果:
1、实验证明,本发明所设计的引物和探针特异性强,能实现直接采用唾液/血样进行PCR扩增分析,省略了繁杂的核酸提取和纯化步骤,明显节约了实验耗材和人力成本;
2、本发明可在一个反应管中实现两个SNP的分型检测,不仅明显提高了检测效率,同时也明显缩短了检测周期,而且可直接根据荧光曲线进行结果判读,判读结果简便客观;
3、采用本发明所述的ApoE 526C>T与ApoE A388T>C联合检测反应液及SLCO1B1388A>G与SLCO1B1 521T>C联合检测反应液,可确保实验的准确性和灵敏性,检测结果和金标准一代测序法相比,一致性为100%。
附图说明
图1为ApoE 526C>T野生型样品的荧光PCR曲线图;
图2为ApoE 526C>T突变型样品的荧光PCR曲线图;
图3为ApoE 526C>T杂合型样品的荧光PCR曲线图;
图4为ApoE A388T>C野生型样品的荧光PCR曲线图;
图5为ApoE A388T>C突变型样品的荧光PCR曲线图;
图6为ApoE A388T>C杂合型样品的荧光PCR曲线图;
图7为SLCO1B1 388A>G野生型样品的荧光PCR曲线图;
图8为SLCO1B1 388A>G突变型样品的荧光PCR曲线图;
图9为SLCO1B1 388A>G杂合型样品的荧光PCR曲线图;
图10为SLCO1B1 521T>C野生型样品的荧光PCR曲线图;
图11为SLCO1B1 521T>C突变型样品的荧光PCR曲线图;
图12为SLCO1B1 521T>C杂合型样品的荧光PCR曲线图。
具体实施方式
以下结合附图和具体实施例对本发明的技术方案和效果做进一步详细描述。以下采用的试剂和生物材料如未特别说明,均为商业化产品。
实施例1:制备本发明所述的用于检测ApoE和SLCO1B1基因多态性的试剂盒
一、按下表所示分别合成针对ApoE 526C>T、ApoE A388T>C、SLCO1B1 388A>G和SLCO1B1 521T>C四个位点的引物和探针:
二、按下表所示配制ApoE 526C>T与ApoE A388T>C联合检测反应液,其中包括:
组分 | 配比 |
SEQ ID NO.1所示序列的上游引物 | 200nmol/L |
SEQ ID NO.2所示序列的下游引物 | 200nmol/L |
SEQ ID NO.3所示序列的野生型探针 | 200nmol/L |
SEQ ID NO.4所示序列的突变型探针 | 200nmol/L |
SEQ ID NO.5所示序列的上游引物 | 200nmol/L |
SEQ ID NO.6所示序列的下游引物 | 200nmol/L |
SEQ ID NO.7所示序列的野生型探针 | 200nmol/L |
SEQ ID NO.8所示序列的突变型探针 | 200nmol/L |
三、按下表所示配制SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液,其中包括:
组分 | 配比 |
SEQ ID NO.9所示序列的上游引物 | 200nmol/L |
SEQ ID NO.10所示序列的下游引物 | 200nmol/L |
SEQ ID NO.11所示序列的野生型探针 | 200nmol/L |
SEQ ID NO.12所示序列的突变型探针 | 200nmol/L |
SEQ ID NO.13所示序列的上游引物 | 200nmol/L |
SEQ ID NO.14所示序列的下游引物 | 200nmol/L |
SEQ ID NO.15所示序列的野生型探针 | 200nmol/L |
SEQ ID NO.16所示序列的突变型探针 | 200nmol/L |
另外,上述的ApoE 526C>T与ApoE A388T>C联合检测反应液及SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液中均还包括改良型HotStar Taq酶、直扩PCR扩增缓冲液、直扩试剂和稳定剂,所述直扩PCR扩增缓冲液的组成为:Tris-HCl缓冲溶液,其中含有200mmol/L~400mmol/L KCl;所述直扩试剂的组成为:25mmol/L~50mmol/L Tris-HCl(三羟甲基氨基甲烷盐酸盐),1mmol/L~5mmol/L EDTA(乙二胺四乙酸),200mmol/L~400mmol/L NaCl(氯化钠),0.1wt%~0.6wt%SDS(十二烷基硫酸钠),0.5mol/L~2.0mol/L山梨醇,0.5mmol/L~0.8mmol/L DTT(二硫苏糖醇);所述稳定剂的组成为:0.5wt%~1.0wt%甘油,0.2wt%~0.6wt%NP40(乙基苯基聚乙二醇),0.2wt%~0.6wt%tween20(吐温20),0.5mg/mL~2mg/mL BSA(牛血清白蛋白)。
四、组装试剂盒
所述试剂盒包含2个PCR反应管,其中一个PCR反应管中装有ApoE 526C>T与ApoEA388T>C联合检测反应液,另一个PCR反应管中装有SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液;
另外,所述试剂盒还包括1管阳性对照品,1管阴性对照品,所述阳性对照品采用人基因组,所述阴性对照品采用DEPC H2O。
实施例2
采用上述试剂盒检测ApoE和SLCO1B1基因多态性的方法,包括如下步骤:
a)分别取1μL待测唾液(也可以是血样)样本直接加入含有20μL ApoE 526C>T与ApoE A388T>C联合检测反应液的PCR反应管Ⅰ和含有20μL SLCO1B1 388A>G与SLCO1B1521T>C联合检测反应液的PCR反应管Ⅱ中,待混匀离心后,再放入荧光定量PCR仪器中同时进行PCR扩增反应:95℃预变性5分钟;95℃变性10秒钟;60℃退火30秒钟;40个循环;
b)对扩增产物进行荧光检测,并根据荧光曲线和Ct值直接对测试样本的多态性进行判读:
b1)对PCR反应管Ⅰ的PCR反应产物进行荧光检测,并根据FAM、VIC、ROX和CY5四个通道的荧光曲线和Ct值直接对该待测样本的ApoE基因多态性进行判读:若FAM荧光信号起线但VIC荧光信号未起线(如图1所示),则判定为ApoE 526C>T位点为野生型;若FAM荧光信号未起线但VIC荧光信号起线(如图2所示),则判定为ApoE 526C>T位点为突变型;若FAM荧光信号和VIC荧光信号均起线(如图3所示),则判定为ApoE 526C>T位点为杂合型;若ROX荧光信号起线但CY5荧光信号未起线(如图4所示),则判定为ApoE A388T>C位点为野生型;若ROX荧光信号未起线但CY5荧光信号起线(如图5所示),则判定为ApoE A388T>C位点为突变型;若ROX荧光信号和CY5荧光信号均起线(如图6所示),则判定为ApoE A388T>C位点为杂合型;
b2)对PCR反应管Ⅱ的PCR反应产物进行荧光检测,并根据FAM、VIC、ROX和CY5四个通道的荧光曲线和Ct值直接对该待测样本的SLCO1B1基因多态性进行判读:若FAM荧光信号起线但VIC荧光信号未起线(如图7所示),则判定为SLCO1B1 388A>G位点为野生型;若FAM荧光信号未起线但VIC荧光信号起线(如图8所示),则判定为SLCO1B1388A>G位点为突变型;若FAM荧光信号和VIC荧光信号均起线(如图9所示),则判定为SLCO1B1 388A>G位点为杂合型;若ROX荧光信号起线但CY5荧光信号未起线(如图10所示),则判定为SLCO1B1 521T>C位点为野生型;若ROX荧光信号未起线但CY5荧光信号起线(如图11所示),则判定为SLCO1B1 521T>C位点为突变型;若ROX荧光信号和CY5荧光信号均起线(如图12所示),则判定为SLCO1B1521T>C位点为杂合型。
并且,结合图1至图12所示还可见:采用本发明所述的ApoE 526C>T与ApoE A388T>C联合检测反应液及SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液,可得到扩增良好的基因型别,没有非特异性扩增。
另外,本发明人还实验证明得到:
依据本发明上述试剂盒和方法对ApoE 526C>T、ApoE A388T>C、SLCO1B1 388A>G和SLCO1B1 521T>C基因检测位点以外其它区段的基因序列合成的阳性质粒,以及金黄色葡萄球菌和大肠杆菌的基因组DNA均不会发生交叉,所设计的引物和探针特异性强;
依据本发明上述试剂盒和方法对质粒样本的各个型别的最低检测限均为1×102copies/μL,对人基因组DNA样本的各个型别的最低检测为0.1ng/μL,灵敏度高;
采用阳性参考品检测结果均为阳性,阳性符合率100%,准确度好;
用精密度参考品(1×105copies/μL)进行检测(n=10),检测结果Ct值的变异系数(CV,%)均<5.0%,精密度非常高。
综上所述,本发明所述试剂盒和方法具有敏感性好、特异性高、准确可靠、操作方便快捷等显著进步性。
最后有必要在此指出的是:以上所述仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种用于检测ApoE和SLCO1B1基因多态性的试剂盒,其特征在于:包括ApoE 526C>T与ApoE A388T>C联合检测反应液和SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液;所述ApoE 526C>T与ApoE A388T>C联合检测反应液包括针对ApoE 526C>T位点的SEQ ID NO.1和SEQ ID NO.2所示序列的引物组及SEQ ID NO.3和SEQ ID NO.4所示序列的探针组,针对ApoE A388T>C位点的SEQ ID NO.5和SEQ ID NO.6所示序列的引物组及SEQ ID NO.7和SEQID NO.8所示序列的探针组;所述SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液包括针对SLCO1B1 388A>G位点的SEQ ID NO.9和SEQ ID NO.10所示序列的引物组及SEQ IDNO.11和SEQ ID NO.12所示序列的探针组,针对SLCO1B1 521T>C位点的SEQ ID NO.13和SEQID NO.14所示序列的引物组及SEQ ID NO.15和SEQ ID NO.16所示序列的探针组。
2.根据权利要求1所述的试剂盒,其特征在于:所有探针的5'端标记有报告荧光染料FAM、VIC、ROX、CY5中的任意一种,所有探针的3'端均标记有淬灭荧光染料BHQ。
3.根据权利要求2所述的试剂盒,其特征在于:针对ApoE 526C>T位点的野生型探针的5’端标记有报告荧光染料FAM,针对ApoE 526C>T位点的突变型探针的5’端标记有报告荧光染料VIC,针对ApoE A388T>C位点的野生型探针的5’端标记有报告荧光染料ROX,针对ApoEA388T>C位点的突变型探针的5’端标记有报告荧光染料CY5。
4.根据权利要求2所述的试剂盒,其特征在于:针对SLCO1B1 388A>G位点的野生型探针的5’端标记有报告荧光染料FAM,针对SLCO1B1 388A>G位点的突变型探针的5’端标记有报告荧光染料VIC,针对SLCO1B1 521T>C位点的野生型探针的5’端标记有报告荧光染料ROX,针对SLCO1B1 521T>C位点的突变型探针的5’端标记有报告荧光染料CY5。
5.根据权利要求1所述的试剂盒,其特征在于:所述的ApoE 526C>T与ApoE A388T>C联合检测反应液及SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液中均还包括改良型HotStar Taq酶、直扩PCR扩增缓冲液、直扩试剂和稳定剂。
6.根据权利要求5所述的试剂盒,其特征在于,所述直扩PCR扩增缓冲液的组成为:Tris-HCl缓冲溶液,其中含有200mmol/L~400mmol/L KCl;
所述直扩试剂的组成为:25mmol/L~100mmol/L Tris-HCl,1mmol/L~10mmol/L EDTA,200mmol/L~500mmol/L NaCl,0.1wt%~0.8wt%SDS,0.1mol/L~2.0mol/L山梨醇,0.5mmol/L~1.0mmol/L DTT;
所述稳定剂的组成为:0.5wt%~1.5wt%甘油,0.2wt%~0.8wt%NP40,0.2wt%~0.8wt%tween20,0.1mg/mL~2mg/mL BSA。
7.根据权利要求1所述的试剂盒,其特征在于:所述试剂盒还包括阳性对照品和阴性对照品,所述阳性对照品采用人基因组,所述阴性对照品采用DEPC H2O。
8.一种用于检测ApoE和SLCO1B1基因多态性的方法,包括权利要求1至7中任一项所述的试剂盒,其特征在于,所述方法包括如下步骤:
a)分别取1份待测样本直接加入含有ApoE 526C>T与ApoE A388T>C联合检测反应液的PCR反应管Ⅰ和含有SLCO1B1 388A>G与SLCO1B1 521T>C联合检测反应液的PCR反应管Ⅱ中,待混匀离心后,再放入荧光定量PCR仪器中同时进行PCR扩增反应;
b)对扩增产物进行荧光检测,并根据荧光曲线和Ct值直接对测试样本的多态性进行判读。
9.根据权利要求8所述的方法,其特征在于:所述待测样本为唾液或血样。
10.根据权利要求8所述的方法,其特征在于,步骤a)进行PCR扩增反应的条件为:
95℃预变性5分钟;95℃变性10秒钟;60℃退火30秒钟;40个循环。
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