CN111909996A - 一种个体化用药相关基因多态性检测试剂盒 - Google Patents
一种个体化用药相关基因多态性检测试剂盒 Download PDFInfo
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Abstract
本发明涉及一种用于检测他汀类药物个体化用药相关基因多态性的特异性引物探针组合,所述引物探针组合包括针对rs7412、rs4149015、rs17244841、rs662799基因多态性的特异性引物探针组合。本发明还提供了包括所述引物对、探针对组合的试剂盒及其应用。本发明的有益效果在于:本发明提供了一种同时检测rs7412、rs4149015、rs17244841、rs662799四个基因多态性,并能准确区分杂合子与纯合子的方法,并提供了一种用于同时指导洛伐他汀、普伐他汀、阿托伐他汀、辛伐他汀的个体化用药相关基因多态性的分型的试剂盒及其应用,本发明的试剂盒检测用时短,操作简便,结果精确可靠,适合临床推广应用。
Description
技术领域
本发明涉及基因检测技术领域,具体的涉及一种用于他汀类药物个体化用药指导的基因多态性检测的引物、探针组合,试剂盒及其应用。
背景技术
他汀类药物是世界范围内应用最为广泛的降脂药物,包括洛伐他汀、普伐他汀、阿托伐他汀、辛伐他汀等。临床研究证实他汀类药物可减缓动脉粥样硬化斑块的发展,甚至使斑块消退,显著降低冠心病的发病率和死亡率。
随着他汀类药物的广泛使用,他汀类药物的不良反应,特别是肌肉毒性,如肌痛、肌病和横纹肌溶解等越来越被关注。许多临床证据表明不同个体服用他汀类药物后的疗效和不良反应存在较大差异,进而研究发现个体差异与相关基因的遗传多态性密切相关。
所有他汀类药物都是HMG-CoA还原酶抑制剂,通过降低血浆胆固醇水平有效地预防及治疗动脉粥样硬化的发生。HMGCoA还原酶(HMGCR)在合成低密度脂蛋白-胆固醇过程中期关键作用。HMG-CoA还原酶基因(HMGCRG)多态性可能通过直接影响HMG-CoA还原酶的活性影响细胞内、外胆固醇水平,对他汀类药物降脂治疗的临床效果产生影响。
阴离子转运多肽(OATP1BI)是一种重要的肝特异性转运体,由SLCO1B1基因编码。SLCO1B1基因突变引起编码的有机阴离子转运多肽活力减弱,表现为肝脏摄取药物能力降低,引起他汀类药物血药浓度上升,增加横纹肌溶解症或肌病的发生风险。通过检测SLCO1B1基因位点多态性,可评估他汀类药物的毒性反应及疗效情况,为临床合理、安全使用他汀提供科学依据,实现患者个体化用药。
载脂蛋白E(Apolipoprotein E,ApoE)在脂质代谢中发挥重要作用。编码ApoE的基因有3个等位基因,分别为E2、E3和E4。其中E3型为野生型,占人类的78%,属于常见基因型;E2型个体冠心病的风险降低而易患黄斑变性及Ⅲ类高脂血症; E4型个体增加老年痴呆症、冠心病、脑梗塞、视网膜色素变性等疾病风险。他汀类药物对E4型的疗效往往不佳,而对E2携带者的降脂作用最强。
载脂蛋白A5(ApoA5)基因特异表达与肝脏。ApoA5经肝脏合成后分泌入血,主要分布于高密度脂蛋白、极低密度脂蛋白和乳糜微粒等脂蛋白颗粒上。大量证据显示,ApoA5是体内甘油三酯代谢的负性调节因子。
因此,对正在或者考虑接受他汀降脂药物治疗的患者,进行基因多态性检测,可以有效预估他汀类药物的不良反应发生情况及降脂治疗的效果,为合理选择他汀类药物的种类及起始用量提供科学依据,实现精准治疗,提高他汀类药物治疗的有效性和安全性。
目前市场上的基因检测多采用传统基因多态性检测方法,如酶切,限制性片段长度多态性分析(restriction fragment lengt hpolymorphism,RFLP),变性高效液相色谱分析(denaturing high performance liquid chromotography,DHPLC),直接测序等。这些传统方法或不能定性,或耗时费力、所需设备和耗材昂贵,更重要的是这些方法难以同时对不同基因的多个突变位点进行检测。到目前为止,市场上还没有针对他汀类药物个体化用药基因快速检测的试剂盒,综上,开发一种快速,高效、精确的用于指导他汀类药物安全用药的基因检测试剂盒,具有重大的临床意义及社会意义。
发明内容
本发明提供了一种同时检测ApoE(rs7412)、SLCO1B1(rs4149015)、HMGCR(rs17244841)、ApoA5(rs662799)位点基因多态性的引物对和探针对,具体序列如下:
ApoE基因rs7412位点:
ApoE-F 5’-3’:CCTCCGCGATGCCGAT
ApoE-R 5’-3’:CGGCCCTGTTCCACCA
ApoE-C 5’-3’:CTGCAGAAGcGCCT
ApoE-T 5’-3’ :CTGCAGAAGtGCCTG
SLCO1B1基因rs4149015位点:
SLCO1B1-F 5’-3’: CCATATTCTCATATATGCATCCTCACA
SLCO1B1-R 5’-3’: GCTCTCTTCTGAAATAAAGTACAGACCC
SLCO1B1-G 5’-3’: TGTGTATACAgGTAAAAG
SLCO1B1-A 5’-3’: TGTGTATACAaGTAAAAGT
HMGCR基因rs17244841位点:
HMGCR-F 5’-3’: TGATAAAACTAACAGGTATTCAAGATACAAAG
HMGCR-R 5’-3’: GATCACACCATTGCACATTGC
HMGCR-T 5’-3’: ATTGTAATATAAAGGATTTtAAA
HMGCR-A 5’-3’: TGATTGTAATATAAAGGATTTaAA
ApoA5基因rs662799位点:
ApoA5-F 5’-3’: GGAGTTCAGCTTTTCCTCATGG
ApoA5-R 5’-3’: AGATGAGATGGCAAGAGGCATC
ApoA5-A 5’-3’: CGAAAGTaAGATTTGC
ApoA5-C 5’-3’: CAAATCTCACTTTCG。
本发明还提供了一种由上述引物、探针组成的同时检测rs7412、rs4149015、rs17244841、rs662799 基因多态性的试剂盒,该试剂盒组成如下表:
本发明所述试剂盒中PCR混合液引物对的浓度为 0.20μM;探针对的浓度为 0.1μM。
本发明所述试剂盒采用多重PCR-荧光探针法,实现在全封闭反应体系中进行核酸扩增与检测,用于PCR扩增反应体系为:36.5μl PCR混合液、0.5μl 的DNA聚合酶、3μl 模板DNA,总体积40μl。扩增程序为:95℃,5分钟,1个循环;95℃,45秒钟,58℃,1分钟,40个循环;38℃,5 秒钟,1个循环。
本发明的有益效果:本试剂盒采用多重PCR-荧光探针法,实现在全封闭反应体系中进行核酸扩增与检测,相对于金标法测序法和分子杂交法大幅度减少了检测时间,操作简便,灵敏度高,特异性高。本试剂盒同时检测rs7412(C>T)、rs4149015(G>A)、rs17244841(T>A)、rs662799(G>A)四个基因多态性,并设计了野生型等位基因作为对照点,可一次检测明确杂合或纯合基因型,指导洛伐他汀、普伐他汀、阿托伐他汀、辛伐他汀的个体化用药,保障用药安全。
附图说明
图1为实施例1 ApoE基因rs7412位点检测阳性对照品结果图。
图2为实施例1 ApoE基因rs7412位点检测阴性对照品结果图。
图3为实施例1 ApoE基因rs7412位点检测待测样本结果图。
图4为实施例1 SLCO1B1基因rs4149015位点检测阳性对照品结果图。
图5为实施例1 SLCO1B1基因rs4149015位点检测阴性对照品结果图。
图6为实施例1 SLCO1B1基因rs4149015位点检测待测样本结果图。
图7为实施例1 HMGCR基因rs17244841位点检测阳性对照品结果图。
图8为实施例1 HMGCR基因rs17244841位点检测阴性对照品结果图。
图9为实施例1 HMGCR基因rs17244841位点检测待测样本结果图。
图10为实施例1 ApoA5基因rs662799位点检测阳性对照品结果图。
图11为实施例1 ApoA5基因rs662799位点检测阴性对照品结果图。
图12为实施例1 ApoA5基因rs662799位点检测待测样本结果图。
具体实施方式
以下实施例仅用于进一步阐述本发明。应理解,这些实施例仅用于说明本发明所述检测方法及流程,而不用于限制本发明的范围。本领域技术人员在本发明基础上作的任何改动或修改都属于本发明所限定的范围内。
实施例1
1.待测样本DNA提取
采集1例外周静脉血样(EDTA抗凝),样本取血液200 μl,采用天根血液基因组DNA提取试剂盒(离心柱型)提取,具体操作步骤详见试剂盒说明书。
2.基因扩增试剂准备
从试剂盒中取出4管PCR混合液室温下避光解冻,旋涡振荡混匀后,8000rpm离心10s。从试剂盒中取出DNA聚合酶,8000rpm离心10s。按每反应36.5 μl PCR混合液加入0.5 μl的DNA聚合酶的比例混合,每种PCR混合液各配置3个反应液,共配置12个反应液, 将配制好的扩增试剂充分混匀,8000rpm离心10s,向设定的12个 PCR反应管中分别加入37μl配制好的扩增试剂,做好标记转移到样本处理区。
3.加样
配置的每种PCR反应液各有3管,分别加入3μl的样本DNA、对应的阳性质控品和对应的阴性质控品(去离子水)。
4.PCR扩增
宏石SLAN96-P实时荧光定量PCR仪,设置四种荧光检测通道的报告荧光、淬灭荧光;Passive Reference选择none。
荧光检测通道的设定
荧光PCR仪上设定如下程序:
扩增程序的设定
5.结果分析
a)所有阳性质控品CT值均<35,在控。
b)所有阴性质控品CT值均>39或undetermined,在控。
c)待测样本的结果如下表:
结果分析:待测样本的基因型为rs7412CC纯合,rs4149015GA杂合, rs17244841AA纯合,rs662799GA杂合。
用药建议:
1. rs7412CC纯合基因型患者与CT或TT基因型患者相比,接受阿托伐他汀治疗的CC基因型患者可能有更低的反应。
2. rs4149015GA杂合基因型的患者与GG基因型患者相比,AG基因型患者可能会增加普伐他汀血浆AUC,降低对他汀的反应。
3. rs17244841AA纯合基因型的患者接受他汀类药物治疗可能比AT或TT基因型患者更有可能产生反应。
4. rs662799GA杂合基因型的患者,与AA基因型患者相比,AG基因型和高脂血症患者服用阿托伐他汀、洛伐他汀或辛伐他汀治疗ldl -胆固醇的效果降低。
序列表
<110> 广西医大睿谷医学检验有限公司
<120> 一种个体化用药相关基因多态性检测试剂盒
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cctccgcgat gccgat 16
<210> 2
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cggccctgtt ccacca 16
<210> 3
<211> 14
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctgcagaagc gcct 14
<210> 4
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctgcagaagt gcctg 15
<210> 5
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccatattctc atatatgcat cctcaca 27
<210> 6
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gctctcttct gaaataaagt acagaccc 28
<210> 7
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgtgtataca ggtaaaag 18
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tgtgtataca agtaaaagt 19
<210> 9
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tgataaaact aacaggtatt caagatacaa ag 32
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gatcacacca ttgcacattg c 21
<210> 11
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
attgtaatat aaaggatttt aaa 23
<210> 12
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgattgtaat ataaaggatt taaa 24
<210> 13
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggagttcagc ttttcctcat gg 22
<210> 14
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
agatgagatg gcaagaggca tc 22
<210> 15
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cgaaagtaag atttgc 16
<210> 16
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
caaatctcac tttcg 15
Claims (7)
1.一种个体化用药相关基因多态性检测的特异性引物探针组合,其特征在于,所述引物探针组合包括针对rs7412、rs4149015、rs17244841、rs662799基因多态性的特异性寡核苷酸引物和特异性荧光标记MGB探针,具体如下:
rs7412引物探针序列为:
上游引物5’-3’:CCTCCGCGATGCCGAT
下游引物5’-3’:CGGCCCTGTTCCACCA
探针一5’-3’:CTGCAGAAGcGCCT
探针二5’-3’:CTGCAGAAGtGCCTG
rs4149015引物探针序列为:
上游引物5’-3’:CCATATTCTCATATATGCATCCTCACA
下游引物5’-3’:GCTCTCTTCTGAAATAAAGTACAGACCC
探针一5’-3’:TGTGTATACAgGTAAAAG
探针二5’-3’:TGTGTATACAaGTAAAAGT
rs17244841引物探针序列为:
上游引物5’-3’:TGATAAAACTAACAGGTATTCAAGATACAAAG
下游引物5’-3’:GATCACACCATTGCACATTGC
探针一5’-3’:ATTGTAATATAAAGGATTTtAAA
探针二5’-3’:TGATTGTAATATAAAGGATTTaAA
rs662799引物探针序列为:
上游引物5’-3’:GGAGTTCAGCTTTTCCTCATGG
下游引物5’-3’:AGATGAGATGGCAAGAGGCATC
探针一5’-3’:CGAAAGTaAGATTTGC
探针二5’-3’:CAAATCTCACTTTCG。
2.一种个体化用药相关基因多态性检测试剂盒,其特征在于,包括如权利要求1所述的特异性引物探针组合的PCR混合液,所述PCR混合液分别为rs7412、rs4149015、rs17244841、rs662799 PCR混合液,所述PCR混合液还包括Tris-HCl、KCl、MgCl2、dNTPs。
3.如权利要求2所述的一种个体化用药相关基因多态性检测试剂盒,其特征在于,所述PCR混合液中引物对的浓度为 0.20μM;探针对的浓度为 0.1μM。
4.如权利要求2所述的一种个体化用药相关基因多态性检测试剂盒,其特征在于,还包括DNA聚合酶和阳性质控品A、阳性质控品B、阳性质控品C、阳性质控品D,所述的阳性质控品A为含2个rs7412等位基因位点的质粒混合液,阳性质控品B为含2个rs4149015等位基因位点的质粒混合液;阳性质控品C为含2个rs17244841等位基因位点的质粒混合液,阳性质控品D为含2个rs662799等位基因位点的质粒混合液。
5.如权利要求4所述的个体化用药相关基因多态性检测试剂盒,其特征在于,所述试剂盒用于PCR扩增反应体系为:36.5μl PCR混合液、0.5μl 的DNA聚合酶、3μl 模板DNA,总体积40μl。
6.如权利要求5所述的个体化用药相关基因多态性检测试剂盒,其特征在于,所述试剂盒用于PCR扩增反应体系的扩增程序为:95℃,5分钟,1个循环;95℃,45秒钟,58℃,1分钟,40个循环;38℃,5 秒钟,1个循环。
7.如权利要求2-6任一项所述的一种个体化用药相关基因多态性检测试剂盒的应用,其特征在于,所述的应用为指导洛伐他汀、普伐他汀、阿托伐他汀、辛伐他汀的个体化用药。
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