CN105713975A - 一种快速检测–α2.4缺失型α地贫等位基因的试剂盒 - Google Patents
一种快速检测–α2.4缺失型α地贫等位基因的试剂盒 Download PDFInfo
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Abstract
本发明公开了一种快速检测α2.4缺失型α地贫等位基因的试剂盒。该试剂盒包括扩增引物和荧光探针,所述的扩增引物为一对可以扩增α珠蛋白基因簇中α2.4等位基因的特征序列的引物2.4F和2.4R,其中:2.4F:5’CAGTCACACAAACTGTGAGTCCA3’;2.4R:5’GACATCACAAACGCAGGCAG3’;所述的荧光探针为:2.4Prob:5’HEXGGCCTGCCATAGGTGTTTACCAAGGGBHQ13’。该试剂盒对α2.4等位基因检测具有高度的灵敏性、稳定性、准确性以及较高的特异性。
Description
技术领域
本发明涉及医学检测技术领域,具体涉及一种快速检测-α2.4缺失型α地贫等位基因的试剂盒。
背景技术
申请人在日常工作中,发现-α2.4缺失型α地贫等位基因在广西有一定的分布几率(PangWR,SunL,LongJ,WengXJ,YeXH,WangJJ,LiaoY,TangWJ,FanZQ,WuSP,SongCL,WeiXY,ZhangCH.Identificationofthe-α2.4DeletioninOneFamilyandinOneHbHDiseasePatientinGuangxi,People'sRepublicofChina.[J].Hemoglobin.2016Mar17:1-4.),该基因型与东南亚缺失型杂合会导致HbH病,为降低该基因型的漏诊,有必要需建立一种可以快速检测-α2.4等位基因的检测试剂盒。
Taqman-PCR(荧光水解探针PCR)技术是一种广泛用于医学检测的技术,其优势在于检测精度高,通量大,且操作快速、简单,且大部分医院均已部署相关仪器设备。但目前尚未见有以荧光水解探针PCR技术开发快速快速检测-α2.4缺失型α地贫等位基因的试剂盒的相关报道。
发明内容
本发明要解决的技术问题是提供一种基于水解探针技术的快速检测-α2.4缺失型α地贫等位基因的试剂盒。该试剂盒对-α2.4等位基因检测具有高度的灵敏性、稳定性、准确性以及较高的特异性。
本发明所述的快速检测-α2.4缺失型α地贫等位基因的试剂盒,包括扩增引物和荧光探针,其特征在于:
所述的扩增引物为一对可以扩增α-珠蛋白基因簇中-α2.4等位基因的特征序列的引物2.4-F和2.4-R,其中:
2.4-F:5’-CAGTCACACAAACTGTGAGTCCA-3’;
2.4-R:5’-GACATCACAAACGCAGGCAG-3’;
所述的荧光探针为一条特异性检测2.4-F和2.4-R扩增产物的荧光探针2.4-Prob,具体为:
2.4-Prob:5’-HEX-GGCCTGCCATAGGTGTTTACCAAGGG-BHQ-1-3’。
本发明所述的荧光探针中,HEX指六氯-6-甲基荧光素,BHQ-1指荧光淬灭基团。
本发明所述的快速检测-α2.4缺失型α地贫等位基因的试剂盒还包括一些现有试剂盒中常规且必须的组分,如缓冲液、酶液、MgCl2和dNTP等,具体地,酶液即为DNA聚合酶,具体可采用康为世纪所生产的GoldStarTaqDNAPolymerase,缓冲液为与GoldStarTaqDNAPolymerase配套的缓冲液。
采用上述试剂盒快速检测-α2.4缺失型α地贫等位基因的方法,包括以下步骤:
1)抽提样本基因组DNA,制备DNA模板;
2)配制反应体系,具体为:
将引物2.4-F和2.4-R、荧光探针2.4-Prob以及PCR缓冲液、酶液、MgCl2、dNTP、水和DNA模板配制成反应体系;
3)样本检测:分别将配制的反应体系进行PCR扩增,记录每个PCR反应管内的荧光信号到达设定的域值时所经历的循环次数Cq(Cq值的大小可以反映所检测模板数的多少);
4)数据分析及结果判定:根据实时荧光PCR仪自带的分析软件分析判读结果,当HEX通道有Cq值读数时,则表示该样本携带-α2.4等位基因;当HEX通道没有Cq值读数时,则表示该样本不携带-α2.4等位基因。
上述方法的步骤1)中,采用现有常规方法制备DNA模板。
上述方法的步骤2)中,反应体系中各组分的浓度优选为:待检测DNA:1~3ng/μL;各引物:0.3~0.5μmol/L;探针浓度为0.3~0.5μmol/L;DNA模板:20~200ng;镁离子:1.5~1.9mmol/L;反应体系的终体积优选为20μL。
上述方法的步骤3)中,PCR扩增条件为:95℃预变性8~12分钟,然后95℃15~30秒,60℃退火50~70秒,39~49个循环,于60℃退火步骤末采集荧光信号。
与现有技术相比,本发明的特点在于:
1、本发明所述试剂盒可以实现单管单次反应直接完成-α2.4等位基因的检测,对-α2.4等位基因检测具有高度的灵敏性、稳定性、准确性以及较高的特异性;
2、本发明所述试剂盒在检测时无需开管操作,可极大限度降低实验室PCR产物污染的可能;
3、采用水解探针的实验方案比现有其他方法所需时间少,对设备要求不高。
附图说明
图1为本发明实施例1中1#样本的扩增曲线;
图2为本发明实施例1中2#样本的扩增曲线。
具体实施方式
下面结合具体实施例对本发明作进一步的详述,以更好地理解本发明的内容,但本发明并不限于以下实施例。
实施例1:采用本发明所述试剂盒在已知基因型样本中的检测结果
1、试剂盒的组成:
扩增α-珠蛋白基因簇中-α2.4等位基因的特征序列的引物2.4-F和2.4-R:
2.4-F:5’-CAGTCACACAAACTGTGAGTCCA-3’(SEQIDNO:1);
2.4-R:5’-GACATCACAAACGCAGGCAG-3’(SEQIDNO:2);
异性检测2.4-F和2.4-R扩增产物的荧光探针2.4-Prob:
2.4-Prob:5’-HEX-GGCCTGCCATAGGTGTTTACCAAGGG-BHQ-1-3’(SEQIDNO:3)。
其它组成成分:
GoldStarTaqDNAPolymerase、与GoldStarTaqDNAPolymerase配套的缓冲液和dNTPs均购自康为世纪,MgCl2购自LifeTechnology。
按下述表1配制PCR反应体系:
表1:(mM表示mmol/L,μM表示μmol/L)
2、实施方法:
PCR反应所用仪器为Bio-Rad实时热循环仪CFX96。PCR反应程序为:95℃预变性8~12分钟,然后95℃15~30秒,60℃退火50~70秒,39~49个循环,于60℃退火步骤末采集荧光信号。
样本处理:采用Lab-AidDNAminiextractionkid(Bio-V,Xiamen)试剂盒抽提DNA,以双蒸水稀释至20~200ng/μL备用。DNA样本也可采用其他常规DNA提取方法提取。
样本检测:将1份正常野生型(αα/αα,N/N)样本(简称1#样本),以及1份用常规方法检测确定的已知携带-α2.4等位基因的待检样本(简称2#样本),根据上述反应体系和反应程序,于荧光定量PCR仪上进行扩增检测,记录Cq值。
3、样本来源:所有样本均来源自经常规Gap-PCR技术确定基因型的DNA样本。
4、数据分析及结果判定:根据实时荧光PCR仪自带的分析软件分析判读结果,当HEX通道有Cq值读数时,则表示该样本携带-α2.4等位基因;当HEX通道没有Cq值读数时,则表示该样本不携带-α2.4等位基因。
经检测分析,1#样本的HEX通道没有Cq值读数(扩增曲线如图1所示,Cq值读数由表2所示),表示该样本不携带-α2.4等位基因;2#样本的HEX通道有Cq值读数(扩增曲线如图2所示,Cq值读数由表3所示),表示该样本携带-α2.4等位基因。
表2:
表3:
实施例2:本发明所述试剂盒在随机基因型样本中的检测效果。
1、试剂盒的组成:
同实施例1。
2、实施方法:
同实施例1。
3、样本来源:
样本为19例随机样本(由钦州市妇幼保健院提供),以双蒸水稀释至20~200ng,作为待检样本),以及经Gap-PCR验证的1例阳性样本。
4、数据分析及结果判定:
根据实时荧光PCR仪自带的分析软件分析判读结果,当HEX通道有Cq值读数时,则表示该样本携带-α2.4等位基因;当HEX通道没有Cq值读数时,则表示该样本不携带-α2.4等位基因。结果如下述表4所示,共检出2例阳性样本(A01为阳性对照)。
表4:
Claims (3)
1.一种快速检测-α2.4缺失型α地贫等位基因的试剂盒,包括扩增引物和荧光探针,其特征在于:
所述的扩增引物为一对可以扩增α-珠蛋白基因簇中-α2.4等位基因的特征序列的引物2.4-F和2.4-R,其中:
2.4-F:5’-CAGTCACACAAACTGTGAGTCCA-3’;
2.4-R:5’-GACATCACAAACGCAGGCAG-3’;
所述的荧光探针为一条特异性检测2.4-F和2.4-R扩增产物的荧光探针2.4-Prob,具体为:
2.4-Prob:5’-HEX-GGCCTGCCATAGGTGTTTACCAAGGG-BHQ-1-3’。
2.根据权利要求1所述的试剂盒,其特征在于:所述的荧光探针中,HEX指六氯-6-甲基荧光素,BHQ-1指荧光淬灭基团。
3.根据权利要求1或2所述的试剂盒,其特征在于:所述的试剂盒还包括缓冲液、酶液、MgCl2和dNTP。
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CN107267648B (zh) * | 2017-08-04 | 2020-04-14 | 亚能生物技术(深圳)有限公司 | 一种快速检测地中海贫血常见突变基因的试剂盒 |
CN107245528B (zh) * | 2017-08-04 | 2020-04-21 | 亚能生物技术(深圳)有限公司 | 一种针对中国人群常见地贫突变基因的快速检测试剂盒 |
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