CN112410419A - 基于淬灭探针的血清淀粉样蛋白a1基因型的检测方法和试剂盒 - Google Patents
基于淬灭探针的血清淀粉样蛋白a1基因型的检测方法和试剂盒 Download PDFInfo
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Abstract
本发明公开了一种淬灭探针,及其在SAA1基因检测中的应用;本发明还公开了一种用于SAA1基因的SNP位点检测的方法从而确定基因型的试剂盒;具体而言是利用巢式PCR技术和多通道淬灭探针技术,对SAA1基因型相关的2个基因多态性位点在一次反应中完成检测的方法及相应的试剂盒。本发明通过联合特异的SAA1巢式PCR和针对SNP的QP来检测SAA1基因型,为研究和未来的临床应用提供有力的工具。
Description
技术领域
本发明属于基因技术领域,涉及一种基于淬灭探针(Quenching probe,QP)的血清淀粉样蛋白A1(Serum amyloidA 1,SAA1)基因型的检测方法,包括应用于临床的试剂盒。
背景技术
人类血清淀粉样蛋白A1(Serum Amyloid A1,SAA1)是一种由104个氨基酸组成的急性期反应蛋白。其在天然状态时的分子量大约为12-14kDa,其编码基因位于人类第11号染色体。早期的研究认为SAA1是一种急性炎症蛋白,因为在急性炎症中,SAA1在血中的浓度可升高100-1000倍。此外,SAA1在生理状态下可与高密度脂蛋白(HDL)结合,在炎症期间通过调节高密度脂蛋白代谢而使其水平升高。但是,近年来大量研究表明:SAA1已经不仅是传统意义上的急性期炎症蛋白,而是作为天然固有免疫的调理素,SAA1可与IL-6、TNF-a等炎症性细胞因子相互作用,参与调节机体的固有免疫和获得性免疫。如已经发现SAA1在糖尿病、冠心病、类风湿性关节炎(RA)等慢性炎症疾病和自身免疫病的发生、发展中扮演了重要的角色。
基因多态性(Polymorphism)是指在一个生物群体中,同时存在两种或多种不连续的变异型或基因型或等位基因,亦称遗传多态性(Genetic Polymorphism)或基因多态性。基因单核苷酸多态性(Single Nuclear Polymorphism,SNP)是指基因序列内的单个碱基的不同,包括单个碱基的缺失,插入和置换。人类基因多态性与疾病发生和诊断与治疗有密切的关系,多态性的变化不仅在阐明机体对疾病、药物的易感性与耐受性、疾病临床表现的多样性,以及对药物治疗的反应性和预测转归上都起着重要的作用。通过基因多态性的研究,可从基因水平揭示个体间生物活性物质的功能及效应存在着差异的本质。因此,基因多态性研究已经成为临床医学及预防医学研究的新领域。
目前由于SAA1第3外显子两处SNP位点(rs1136743和rs1136747),形成了三种SAA1等位基因α、β和γ,组成了α+/+,β+/+,γ+/+,αβ,αγ和βγ共6种基因型。已有研究表明不同的SAA1基因型在人群中所占的比例存在差异性。例如,Yamada等对321名日本人SAA1基因型的研究发现,SAA1α、SAA1β和SAA1γ这3个等位基因在日本人中的分布频率分别为0.310,0.347,0.330。Ishii等发现,在127例RA患者中,发生淀粉样变者最常见的基因型是SAA1γ+/+,淀粉样沉积物的出现也和SAA1γ的基因频率高度相关。而在高加索人中,淀粉样变与SAA1α等位基因的频率呈正相关。有报道称地中海出血热的患者中,SAA1α+/+型的发病率是其他类型的7倍。Lung等发现SAA1基因型与NPC(nasopharyngeal carcinoma,鼻咽癌)风险较高显著相关,NPC患者的β+/+基因型频率比健康个体高2倍。
目前SAA1基因型研究普遍使用的是限制性内切酶片段长度检测(RFLP),聚合酶链式反应直接测序法(PCR direct sequencing)和等位基因特异PCR(allele-specific PCR)等。但多数方法步骤较为繁琐、复杂,不易进行大规模SNP分析。并且由于SAA1和SAA2基因的高度同源性,在检测SAA1的基因型的同时如何有效的排除SAA2的干扰,也是SAA1基因型检测面临的考验。因此,开发一种简便、可靠的SAA1基因型检测方法,对于SAA1基因型相关疾病的诊断和风险分析具有重要的临床意义。
发明内容
针对现有技术存在的的缺陷,本发明的目的是提供一种用于SAA1基因的SNP位点检测从而确定基因型的试剂盒;具体而言是利用巢式PCR技术和多通道淬灭探针技术,对SAA1基因型相关的2个基因多态性位点在一次反应中完成检测的方法及相应的试剂盒。
使用淬灭探针(Quenching probe,QP)在检测目标基因的SNP方面非常有效。通过添加特定的引物组,产生基因扩增反应,然后使用QP通过荧光法快速简便地检测特定基因的排列。本发明通过联合特异的SAA1巢式PCR和针对SNP的QP来检测SAA1基因型,为研究和未来的临床应用提供了有力的工具。
本发明提供了一种淬灭探针,其核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示:
CCAGACACCCCCAGGTC(SEQ ID NO.5)
CTGATCACTTCTGCAGC(SEQ ID NO.6)。
本发明还提供了一种引物序列,所述引物序列包括:
特异性扩增引物序列:
TGGGAGGTGGAGGTTGCGATG(SEQ ID NO.1)
AGGAAGGAGGGATGAAAACACTGGG(SEQ ID NO.2)
特异性巢式引物序列:
GGAACTATGATGCTGCCAAAA(SEQ ID NO.3)
GCTCGTCTCCCTCCTGACTG(SEQ ID NO.4)
本发明还提供了一种扩增体系,其包括:
PCR引物混合液;PCR巢式引物混合液;淬灭探针混合液;PCR反应液;模板;ddH2O。
其中,所述PCR引物混合液:SAA1正向引物、SAA1反向引物,其引物序列如SEQ IDNO.1和SEQ ID NO.2所示;所述PCR引物混合液的终浓度为0.01-0.2μM;优选地,为0.02μM。
其中,所述PCR巢式引物混合液:巢式正向引物、巢式反向引物,其引物序列如SEQID NO.3和SEQ ID NO.4所示;所述PCR巢式引物混合液的终浓度为0.01-1.0μM;优选地,为0.2μM或0.6μM。
其中,所述淬灭探针混合液:QP1-FAM:rs1136743、QP1-TMARA:rs1136747,其引物序列如SEQ ID NO.5和SEQ ID NO.6所示;所述淬灭探针混合液的终浓度为0.01-1.0μM;优选地,为0.6μM。
其中,所述PCR反应液:PCR缓冲液、Taq酶、dNTPs和Mg2+;所述PCR反应液的体积为7.0-10.0μL;优选地,为10.0μL。
所述PCR反应液具体指PCR缓冲液、Taq酶、dNTPs和Mg2+及ddH2O配制而成的溶液。
所述Taq酶的使用量为0.1-2.0U;优选地,为2.0U。
所述dNTPs在PCR反应液(PCR体系)的终浓度为0.02-1.0μM;优选地,为0.2μM。
所述Mg2+的体积在PCR反应液(PCR体系)的终浓度为1.0-4.0μM;优选地,为2.5μM。
其中,所述模板:阳性对照或阴性对照或基因组DNA;所述模板的体积为0.2-5.0μL;优选地,为1μL。
所述阳性对照具体指克隆于pMDTM19-T载体(或pMDTM18-T载体)的用SEQ ID NO.1和SEQ ID NO.2扩增的人基因组产物(分别由SAA1α+/+、SAA1β+/+和SAA1γ+/+样本作为扩增模板)。
所述阴性对照具体指pMDTM19-T载体(或pMDTM18-T载体)自连产物。
所述基因组DNA具体指从正常人或病患全血样本或组织中利用分子生物学抽提方法获得的人基因组脱氧核糖核酸。
本发明还提供了一种试剂盒,其包括所述淬灭探针和/或引物序列、样本处理液、所述扩增体系、阳性对照、阴性对照。
其中,所述样本处理液为全血PCR裂解液,为ddH2O或TE或NaOH溶液。
其中,所述阳性对照指克隆于pMDTM19-T载体(或pMDTM18-T载体)的用SEQ ID NO.1和SEQ ID NO.2扩增的人基因组产物(分别由SAA1α+/+、SAA1β+/+和SAA1γ+/+样本作为扩增模板)。累计有α+/+、β+/+、γ+/+三种类型阳性对照。
其中,所述阴性对照指pMDTM19-T载体(或pMDTM18-T载体)自连产物。
本发明还提供了所述淬灭探针、或引物序列、或试剂盒、或扩增体系在制备SNP位点检测、SAA1基因型检测、基因多态性的试剂中的应用。
所述位点为rs1136743和rs1136747位点。
本发明还提供了所述淬灭探针、或引物序列、或试剂盒、或扩增体系在基于SAA1基因型或基因多态性与临床疾病风险预测中的应用。
本发明还提供了所述淬灭探针、或引物序列、或试剂盒、或扩增体系在预防/诊断/治疗与SAA1基因型或基因多态性相关疾病中的应用。
所述疾病包括糖尿病、冠心病、类风湿性关节炎(RA)等慢性炎症疾病和自身免疫病,以及淀粉样病变、地中海出血热、鼻咽癌等疾病。
其中,所述自身免疫病包括类风湿关节炎、强直性脊柱炎、结节病、克罗恩病、溃疡性结肠炎等。
所述淬灭探针与特异的巢式PCR联合使用在所述应用中。
其中,巢式PCR反应条件:95℃,5min→8个循环(95℃,15s;64℃,15s;72℃,1min)→50个循环(95℃,10s;60℃,10s;72℃,15s)。
本发明还提供了一种基于淬灭探针的SAA1基因型的检测方法,所述方法包括以下步骤:
(1)合成如上所述的引物及探针;
(2)采用QIAamp DNA BloodMini kit进行全血或组织样本DNA抽提,并测定其浓度、通过O.D.260/280及O.D.260/230鉴定其质量,对合格样品用ddH2O稀释至10~20ng/μL;
(3)按照表3配制相应的扩增体系,并按照如下设定程序运行:95℃,5min→8个循环(95℃,15s;64℃,15s;72℃,1min)→50个循环(95℃,10s;60℃,10s;72℃,15s)→熔解曲线荧光分析{40℃,1min→85℃,10s(每摄氏度收集3个荧光信号点)}。
结果判定:根据熔解曲线中荧光变化峰值出现时对应的熔解温度(meltingtemperature,Tm),来判定SNP位点的对应的碱基类型。由于2条淬灭探针完全匹配的是T碱基,因此对应SNP位点上若是T碱基,则应检测到相对高Tm的峰型,而C碱基应检测得到相对低Tm的峰型。具体6种SAA1基因型检测得到的对应Tm和峰型图详见表4和图1。
本发明还提供了所述检测方法在SAA1基因型检测中的应用。
本发明的有益效果在于:本发明提供了一种简便、可靠的SAA1基因型检测方法,对于SAA1基因型相关疾病的诊断和风险分析具有重要的临床意义。本发明通过联合特异的SAA1巢式PCR和针对SNP的QP来检测SAA1基因型,为研究和未来的临床应用提供了有力的工具。本发明提供的方法在密闭体系中实现了检测、避免了产物污染的风险;在同一体系中同时对两个SNP位点同步检测,可避免了试剂及模板的浪费、对来源有限的特殊病例医疗样本尤为重要;利用双色荧光有效避免了熔解曲线由于扩增体系盐浓度微弱变化过程中引发的漂移而导致的结果误判。
附图说明
图1为六种SAA1基因型使用淬灭探针方法检测所得到的结果。
具体实施方式
结合以下具体实施例和附图,对本发明作进一步的详细说明,本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求书为保护范围。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人的分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)所述的条件,或者按照制造厂商所建议的条件。
实施例1:引物和探针的设计及合成
针对SAA1基因rs1136743和rs1136747位点与SAA1基因型相关的基因多态性位点,设计对应SAA1基因特异性扩增引物序列(SEQ ID No:1和SEQ ID No:2)和特异的巢式引物(SEQ ID No:3至SEQ ID No:4),具体如表1:
表1
编号 | 序列(5’-3’) | 用途 |
SEQ ID No:1 | TGGGAGGTGGAGGTTGCGATG | SAA1正向引物 |
SEQ ID No:2 | AGGAAGGAGGGATGAAAACACTGGG | SAA1反向引物,测序引物 |
SEQ ID No:3 | GGAACTATGATGCTGCCAAAA | 巢式正向引物 |
SEQ ID No:4 | GCTCGTCTCCCTCCTGACTG | 巢式反向引物 |
针对SAA1基因rs1136743和rs1136747位点设计特异的不同荧光标记的淬灭探针(SEQ ID No:5和SEQ ID No:6),具体如表2:
表2
编号 | 针对位点 | 标记和序列(5’-3’) | 用途 |
SEQ ID No:5 | rs1136743 | FAM-CCAGACACCCCCAGGTC | SNP检测 |
SEQ ID No:6 | rs1136747 | TAMRA-CTGATCACTTCTGCAGC | SNP检测 |
实施例2、样本DNA提取
采用QIAamp DNA Blood Mini kit进行全血或组织样本DNA抽提;在赛默飞NanoDrop 2000超微量紫外分光光度计完成DNA浓度的测定;进行样本测定时,需要记录浓度、O.D.260/280(被检测物质核酸与蛋白的光密度比值,要求大于等于1.8)以及O.D.260/230(被检测物质核酸与残留有机物的光密度比值,要求大于等于1.7)三个数值,以便浓度未达到要求查找可能的污染原因,同时也要求实验人员必须对DNA样品进行分装以至于实验过程减少样本冻融的次数,保证DNA的高质量。然后用去离子水将抽提好的样本浓度稀释为10-20ng/μL即可,以满足后续基因分型样本质控的基本要求。
实施例3、生物学实验
表3
1、巢式PCR反应条件:95℃,5min→8个循环(95℃,15s;64℃,15s;72℃,1min)→50个循环(95℃,10s;60℃,10s;72℃,15s)。
2、熔解曲线荧光分析方法:40℃,1min→85℃,10s(每摄氏度收集3个荧光信号点)。
根据熔解曲线中荧光变化峰值出现时对应的熔解温度(melting temperature,Tm),来判定SNP位点的对应的碱基类型。由于2条淬灭探针完全匹配的是T碱基,因此对应SNP位点上若是T碱基,则应检测到相对高Tm的峰型,而C碱基应检测得到相对低Tm的峰型。具体6种SAA1基因型检测得到的对应Tm和峰型图详见表4和图1。
表4
实施例4、淬灭探针方法与PCR直接测序法SAA1基因型检测结果的比较
为了评价本发明的淬灭探针(QP)方法在SAA1基因分型中的临床应用,将巢式PCR引物对和本发明两个QPs配合到临床样本的检测中。提取外周血基因组DNA,同时进行QP方法的检测和直接测序检测。在对10名志愿者的QP方法学的测序检测和PCR直接测序检测中,每例样本的SAA1基因型列于表5。QP方法与PCR直接测序方法的基因分型结果均一致,说明本发明的QP方法对SAA1基因分型是可靠的。因此,QP方法为SAA1基因分型和临床应用提供了一种快速可靠的策略。
表5
综上所述,本发明为人群中SAA1基因型及其基因多态性与临床疾病风险的预测提供了有效的检测手段,也为个体化基因组学的研究提供了有效工具。以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变形或修改,这并不影响本发明的实质内容。
本发明保护内容不限于以上实施例。在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求书为保护范围。
SEQUENCE LISTING
<110> 德赛诊断系统(上海)有限公司
<120> 基于淬灭探针的血清淀粉样蛋白A1基因型的检测方法和试剂盒
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<170> PatentIn version 3.3
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Claims (12)
1.一种淬灭探针,其特征在于,其核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示:
CCAGACACCCCCAGGTC(SEQ ID NO.5);
CTGATCACTTCTGCAGC(SEQ ID NO.6)。
2.一种引物序列,其特征在于,所述引物序列包括:
特异性扩增引物序列:
TGGGAGGTGGAGGTTGCGATG(SEQ ID NO.1);
AGGAAGGAGGGATGAAAACACTGGG(SEQ ID NO.2);
特异性巢式引物序列:
GGAACTATGATGCTGCCAAAA(SEQ ID NO.3);
GCTCGTCTCCCTCCTGACTG(SEQ ID NO.4)。
3.一种扩增体系,其特征在于,其包括:
PCR引物混合液;PCR巢式引物混合液;淬灭探针混合液;PCR反应液;模板;ddH2O;
其中,所述PCR引物混合液:SAA1正向引物、SAA1反向引物,其引物序列如权利要求2所述的SEQ ID NO.1和SEQ ID NO.2所示;所述PCR引物混合液的终浓度为0.01-0.2μM;
其中,所述PCR巢式引物混合液:巢式正向引物、巢式反向引物,其引物序列如权利要求2所述的SEQ ID NO.3和SEQ ID NO.4所示;所述PCR巢式引物混合液的终浓度为0.01-1.0μM;
其中,所述淬灭探针混合液:QP1-FAM:rs1136743、QP1-TMARA:rs1136747,其引物序列如权利要求1所述的SEQ ID NO.5和SEQ ID NO.6所示;所述淬灭探针混合液的终浓度为0.01-1.0μM;
其中,所述PCR反应液:PCR缓冲液、Taq酶、dNTPs和Mg2+;所述PCR反应液的体积为7.0-13.0μL;
其中,所述模板:阳性对照或阴性对照或基因组DNA;所述模板的体积为0.2-5.0μL。
4.一种试剂盒,其特征在于,其包括如权利要求1所述的淬灭探针、如权利要求2所述的引物序列、如权利要求3所述的扩增体系、样本处理液、阳性对照、阴性对照。
5.如权利要求1所述的淬灭探针、或如权利要求2所述的引物序列、或如权利要求3所述的扩增体系、或如权利要求4所述的试剂盒在制备SNP位点检测、或SAA1基因型检测、或基因多态性检测的试剂中的应用。
6.如权利要求1所述的淬灭探针、或如权利要求2所述的引物序列、或如权利要求3所述的扩增体系、或如权利要求4所述的试剂盒在基于SAA1基因型或基因多态性预测临床疾病风险中的应用。
7.如权利要求1所述的淬灭探针、或如权利要求2所述的引物序列、或如权利要求3所述的扩增体系、或如权利要求4所述的试剂盒在预防/诊断/治疗与SAA1基因型或基因多态性相关疾病中的应用。
8.如权利要求6或7所述的应用,其特征在于,所述疾病包括糖尿病、冠心病、类风湿性关节炎RA、自身免疫病、淀粉样病变、地中海出血热、鼻咽癌。
9.如权利要求6或7所述应用,其特征在于,所述淬灭探针与特异的巢式PCR联合使用在所述应用中。
11.如权利要求10所述的方法,其特征在于,其中,所述扩增体系中的巢式PCR反应条件为:95℃,5min→8个循环(95℃,15s;64℃,15s;72℃,1min)→50个循环(95℃,10s;60℃,10s;72℃,15s)。
12.一种基于淬灭探针的SAA1基因型的检测方法,其特征在于,所述方法包括以下步骤:
(1)合成如权利要求1所述的探针及如权利要求2所述的引物序列;
(2)采用QIAamp DNA Blood Mini kit进行全血或组织样本DNA抽提,并测定其浓度,通过O.D.260/280及O.D.260/230鉴定其质量,对合格样品用ddH2O稀释至10~20ng/μL;
(3)配制如权利要求3所述的扩增体系,并按照如下设定程序运行:95℃,5min→8个循环(95℃,15s;64℃,15s;72℃,1min)→50个循环(95℃,10s;60℃,10s;72℃,15s)→熔解曲线荧光分析{40℃,1min→85℃,10s(每摄氏度收集3个荧光信号点)}。
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