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 PDF

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CN105713975A
CN105713975A CN201610185637.1A CN201610185637A CN105713975A CN 105713975 A CN105713975 A CN 105713975A CN 201610185637 A CN201610185637 A CN 201610185637A CN 105713975 A CN105713975 A CN 105713975A
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龙驹
潘志坚
唐维骏
庞婉容
邱盛盈
孙雷
樊祖茜
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QINZHOU MATERNAL AND CHILD HEALTH HOSPITAL
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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

一种快速检测–α2.4缺失型α地贫等位基因的试剂盒
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。
CN201610185637.1A 2016-03-29 2016-03-29 一种快速检测–α2.4缺失型α地贫等位基因的试剂盒 Pending CN105713975A (zh)

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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
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