CN116445608A - 检测耳聋相关基因突变的组合物、试剂盒、及用途 - Google Patents
检测耳聋相关基因突变的组合物、试剂盒、及用途 Download PDFInfo
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Abstract
本发明属于分子生物学检测领域,具体地,涉及检测耳聋相关基因的组合物,更具体地,涉及GJB2、SLC26A4、GJB3等9个基因的检测的组合物、试剂盒及用途。本发明提供一种检测耳聋相关基因突变的组合物,包括:如SEQ ID NO:1~34所示的寡核苷酸。使用本发明的组合物,能够使用一管PCR同时扩增9基因共覆盖27个SNP位点。能够快速准确的检测常见的耳聋相关基因突变。再一方面,使用本发明的组合物进行突变检测时,样品起始量仅需10ng。
Description
技术领域
本发明属于分子生物学检测领域,具体地,涉及检测耳聋相关基因的组合物,更具体地,涉及GJB2、SLC26A4、GJB3等9个基因的检测的组合物、试剂盒及用途。
背景技术
耳聋是一种临床上常见的疾病,发生率为1‰~3‰,遗传性因素引起的耳聋约占占先天性耳聋总人数的60%左右。2006年第二次残疾人抽样调查显示,我国残疾人总数为8000万,其中听力残疾者为2670万,1-7岁听障儿童约有80万。遗传性耳聋可通过常染色体隐性遗传、常染色体显性遗传或线粒体遗传等方式传给下一代,无论父母的单方或者双方是耳聋患者还是健康携带者,耳聋基因都会通过父母向子女遗传,子代都有一定的可能会罹患耳聋疾病。目前已知的非综合征型耳聋相关基因有70多个,与耳聋相关的遗传性综合征疾病有400多种。非综合征型是指仅有听觉系统症状,近几年研究认为其是单基因疾病,发病率为1/1000~1/800,遗传方式主要有常染色体显性、常染色体隐性、线粒体突变母系遗传、伴性染色体遗传。GJB2基因、SLC26A4基因、MT-RNR1(12SrRNA)基因和GJB3基因是常见的耳聋致病基因,也是针对新生儿、耳聋患者等进行预防性或诊断性筛查主要的靶基因。我国自2007年开始开展了规模化防控预警聋病的三级预防模式“新生儿听力及基因联合筛查”,可将听力损失的时间提早到出生后30天内,为临床上及早干预以及避免药物治聋提供了依据。GJB2基因(NM_004004,OMIM 121011)编码连接蛋白Cx26(Connexin26),定位于13q11-q12,包含2个外显子,编码226个氨基酸。GJB2基因突变后,Cx26蛋白调控的内淋巴液钾离子循环紊乱,导致感音性耳聋。SLC26A4基因(NM_000441,OMIM 605646)编码溶脂蛋白(Pendrin),定位于7q22.3,包含21个外显子,编码780个氨基酸,其功能为在实现细胞内外Cl-/I-或Cl-/HCO3-离子转运。SLC26A4基因突变后内淋巴液离子环境失衡,进而导致耳聋。MT-RNR1(12SrRNA)基因与20%~30%的药物性耳聋有关,中国人中常见的位点为1555A>G、1494C>T,这种突变使得线粒体DNA更易与氨基糖苷类药物结合,抑制线粒体氧化磷酸化过程造成耳毒性。GJB3基因是连接蛋白家族的成员,其突变可引起显性或隐性的耳聋。
因此,通过早期筛查可以避免“一针致聋”的悲剧。常见耳聋基因检测方法主要包括测序技术[Sanger测序和NGS测序]、荧光定量PCR、基因芯片技术、多重连接探针扩增技术、荧光原位杂交技术等。Sanger测序通量低、操作繁琐。NGS测序对点突变、20bp以内的插入缺失及微小重复效果较好,但对倒位、长片段重复等结构变异效果不佳。传统的PCR相关的方法检测位点少。而将用于检测不同目的片段的多重PCR建库结合在一起,可提升通量、降低成本,并提高诊断能力。
本领域需求一种产品,其能够提升通量,并且快速准确的检测常见的耳聋基因突变。
发明内容
有鉴于此,第一方面,本发明提供一种检测耳聋相关基因突变的引物池,所述引物池包括17对引物,如SEQ ID NO:1~34所示。
进一步地,所述组合物包括根据测序接头所设计的两轮PCR的引物对。
上述根据测序接头设计(即,第一、二轮PCR扩增引物对)根据不同的测序平台可以由本领域技术人员按照本领域常规方法确定。
在一个具体的实施方案中,每条引物的5’端进一步分别含有部分测序接头序列,具体的,即可以在NO.1~17的5’端加上部分测序接头序列Adapter 1:5’-TCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’,在NO.18~34的5’端加上部分测序接头序列Adapter 2:5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
本发明的组合物一方面可用于构建耳聋相关基因扩增子文库,另一方面可用于检测耳聋相关基因突变。
一方面,使用本发明的组合物,能够使用一管PCR同时扩增GJB2基因(rs750188782、rs80338943、rs111033204、rs80338939、rs72474224、rs80338942)、SLC26A4基因(rs1057516953、rs111033380、rs121908363、rs201562855、rs111033305、rs111033220、rs200455203、rs111033318、rs121908362、rs192366176、rs111033313)、MT-RNR1(12SrRNA)基因(rs267606619、rs267606617)和GJB3基因(rs74315319、rs74315318)、GJB6基因(rs104894414)、POU3F4基因(rs267606974、rs267606975)、COCH基因(rs28938175)、TIME基因(rs28941781)、KCNQ4基因(rs80358277),共覆盖27个SNP位点。能够快速准确的检测常见的耳聋相关基因突变。
再一方面,第一轮PCR的高特异性保证了DNA模板的高效利用,第二轮PCR提高文库序列浓度,因此,在使用本发明的组合物进行突变检测时,样品起始量仅需10ng。
第二方面,本发明提供了一种使用本发明的组合物制备耳聋相关基因突变检测的试剂盒的用途。
第三方面,本发明提供了一种耳聋相关基因突变检测的试剂盒,包括如上所述的组合物。
进一步地,上述试剂盒中还具有提取所需试剂、扩增所需试剂以及测序所需试剂。
提取所需试剂是指从样品中提取所需核酸所需的试剂,例如商业化核酸提取试剂盒(可使用天根干血斑基因组DNA提取试剂盒,天根全血基因组DNA提取试剂盒等)。
扩增所需试剂包括第一轮PCR扩增所需的试剂,包括聚合酶、缓冲液、dNTP、扩增所需引物对。
所述聚合酶优选为适合于多重PCR扩增的聚合酶。
第一轮引物为测序接头Adapter+特异性引物。测序接头可以根据不同的测序平台而通过常规方法确定。在一个具体的实施方案中,第一轮PCR所包含的部分测序接头序列为:正向接头为5’-TCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’,反向接头为5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
第二轮PCR扩增所需试剂包括:聚合酶、缓冲液、dNTP、扩增所需引物对。
所述聚合酶优选为适合于高保真PCR扩增的聚合酶。
第二轮PCR的引物为测序接头序列Adapter+Index+测序接头序列Adapter。具体地,可以在P5端Index的5’端加上标签序列Adapter 3:5’-AATGATACGGCGACCACCGAGATCTACAC-3’,在P5端Index的3’端加上标签序列Adapter 4:ACACTCTTTCCCTACACGAC,在P7端Index的5’端加上标签序列Adapter 5:CAAGCAGAAGACGGCATACGAGAT,在P7端Index的3’端加上标签序列Adapter 6:GTGACTGGAGTTCAGACGTG。
Index序列可以根据Illumina测序仪官方推荐的Index序列,如表1所示。
表1
第二轮PCR涉及的Index序列为区分样本使用,即每1例样本对应一组Index序列组合,一组混合不同样本的上机测序的样本中,P5端Index唯一或者P7端Index唯一即可。
举例地,有20例样本,可使用P5端Index 20条与P7端Index 1条的20对组合。在配置体系时,需正确记录每份样本对应的Index,以便测序后数据处理。
第四方面,本发明提供一种耳聋相关基因扩增子文库的构建方法,所述方法包括:
1)使用本发明的引物池或试剂盒扩增样品中的耳聋相关基因,得到添加部分测序接头的耳聋相关基因靶向区域扩增子;
2)用含有Index的剩余测序接头对第一轮产物进行第二轮PCR扩增,得到含有Index和完整测序接头的耳聋相关基因靶向区域扩增子文库。
检测所适用的样品不限制于外周血,可以是新鲜组织、唾液和口腔拭子等可获得人基因组DNA样品的材料均可。
在一个具体的实施方案中,步骤2)中的第一轮PCR扩增将上述组合物在一管中进行。
第五方面,本发明提供了一种根据上述任一项所述的方法获得的耳聋相关基因扩增子文库。
进一步地,在建库完成之后,对文库进行测序,以进行突变检测。
在一个具体的实施方案中,测序为第二代测序。在一个具体的实施方案中,所述第二代测序的测序平台不限制于Illumina相关测序平台测序,测序试剂盒读长不限制于150cycles、250cycles,适用于目前上市的各类型读长测序试剂盒。
本发明的有益效果为:
本发明采用多重PCR扩增子建库技术,同时对耳聋9个致病基因(GJB2、SLC26A4、MT-RNR1、GJB3、GJB6、POU3F4、COCH、TIME、KCNQ4)的27个致病位点(rs750188782、rs80338943、rs111033204、rs80338939、rs72474224、rs80338942、rs1057516953、rs111033380、rs121908363、rs201562855、rs111033305、rs111033220、rs200455203、rs111033318、rs121908362、rs192366176、rs111033313、rs267606619、rs267606617、rs74315319、rs74315318、rs104894414、rs267606974、rs267606975、rs28938175、rs28941781、rs80358277)进行捕获建库。采用两步PCR扩增方法来完成目标区域扩增和建库。该技术能够在保证扩增均一性的前提下,对SNP、Indel等几千甚至上万个突变位点进行快速靶向线性扩增后,使用主流测序平台进行测序与深度分析。试剂盒采用两轮PCR反应进行文库构建,具有文库构建周期短,比对率高,均一性好,重复性好,操作简便等优点,此外,该发明还可以采用多种样本进行文库构建,如唾液gDNA,血液gDNA等。上述该检测文库的构建方法,将整个流程简化,降低人力成本,降低检测数据量,降低成本。
附图说明
图1为本发明组合物使用方法流程图;
图2为文库质量检测中的文库样本使用LabChip微流控毛细管电泳系统进行文库片段长度和纯度测量示意图;
图3为GJB3目标位点区域reads用IGV软件展示;
图4为本发明组合物检测样本的目标位点测序深度。
具体实施方式
下面结合具体实施例对本发明做进一步阐释。本领域技术人员将会理解,下列所描述的实施例是本发明一部分实施例,而不是全部的实施例,仅用于说明本发明,而不应视为限制本发明的范围。所用试剂均为可以通过市售购买获得的常规产品。
实施例:
本发明包含多个扩增子,由1个反应管组成。试剂盒采用两轮PCR反应进行文库构建,具有文库构建周期短,比对率高,均一性好,重复性好,操作简便等优点,此外,该发明还可以采用多种样本进行文库构建,如唾液gDNA,血液gDNA等。
1.使用耳聋相关基因特异性引物
本发明所使用的扩增耳聋相关基因的特异性引物如表2所示,用于第一轮PCR反应。
表2
NO. | PCR1 forward primers | NO. | PCR1 reverse primers |
1 | TACCTGTTCAGCCTCATC | 18 | TCACAGATGGTGAGTACG |
2 | TTATCCCTTTCCCGTGTG | 19 | TGAGTCAGGAGTCACGA |
3 | GGGTTTTGATCTCCTCGA | 20 | CTCACCGTCCTCTTCATT |
4 | GGTGGAGTGTTTGTTCAC | 21 | GAGTTGGTGTTTGCTCAG |
5 | GGTGGAGTGTTTGTTGAC | 22 | CAAGGCCTCTTCCACTAA |
6 | ACTATGCCCCAAGAAGTC | 23 | TCCAGATGGGTAAAGCAG |
7 | CTGTGCTGTGTCTTCAAC | 24 | TCTGTGAGCTCATTGAGG |
8 | TACCGAGTCAAGGAATGG | 25 | ACAGGAAAGATACAGGCC |
9 | TGATAGACACTGCAGCTAG | 26 | TGAGCCTTAATAAGTGGGG |
10 | GGATTGCTCACCATTGTC | 27 | AGGAACACCACACTCAC |
11 | TCGTTGTCATCCAGTCTC | 28 | TGTGTCTTTCCTCCAGTG |
12 | CTGAGCAACTGTGACTTG | 29 | CTGATTGGACCCCAGTAA |
13 | GTGAACGTTCCCAAAGTG | 30 | AGTGGTGAAGCCAGTATC |
14 | GGGTTCTTTGACGACAAC | 31 | CTGAGGCTCCATGAAGTT |
15 | CTACCCCAGAAAACTACGA | 32 | CTCAGAGCGGTCAAGTTA |
16 | CCATTGCCAGGATCACT | 33 | CGATCTTGTCAATGCTGG |
17 | ATCGAGGTGAGTGTCAAG | 34 | CAGTCAGAGATCATGGCA |
2.本试剂盒文库构建流程如图1所示。
(1)样品处理
使用商业化核酸提取试剂盒(可使用天根干血斑基因组DNA提取试剂盒,天根全血基因组DNA提取试剂盒等)提取DNA。
(2)第一轮多重PCR反应
按表3配置第一轮PCR反应体系,引物为包含SEQ ID NO:1~34的混合物:
表3
PCR反应组分 | 体积(10μL体系) |
4×VAHTS Multi-PCR Mix | 2.5 |
PrimerMix | 6 |
DNA模版 | 1 |
ddH2O | 0.5 |
按表4所示程序进行第一轮多重PCR扩增反应。
表4
(3)第二轮接头序列PCR反应
按表5配置第二轮PCR反应体系:
表5
按表6所示程序进行第二轮PCR扩增反应。
表6
(4)混库后上机测序
将每个样本经过两轮PCR后的产物各取1ul进行混合完成文库构建。之后对文库进行质量检测,如图1所示,取1μl文库样本使用LabChip微流控毛细管电泳系统进行文库片段长度和纯度测量。其中,横坐标为时间,纵坐标为相对荧光单位,即为采集到的信号强度。文库主要片段平均大小为445bp。
3、本发明组合物测试样本的检测结果
采用本发明提供的建库试剂盒对16例样本进行了耳聋相关基因突变筛查。目标区域覆盖率为100%,最低目标位点测序深度大于500×,目标位点测序深度见图4。突变检测结果见表7。对于分析所得到的突变结果,经Sanger测序全部得到验证,敏感性100%,阴性结果全部一致,特异性100%。表明本发明方法及试剂盒可以用于耳聋相关基因的突变检测。
表7、突变检测结果
对比例、本发明对比例组合物检测
为了进一步说明本发明组合物的优越性,本发明还提供了别的一些跟耳聋相关的基因(GJB2、SLC26A4、MT-RNR1、GJB3、GJB6、POU3F4、COCH、MATN3、FGFR3)为靶标而设计的组合物用于检测。按照实施例2所述方法对16例样本进行了耳聋相关基因突变筛查。对于分析所得到的突变结果,经Sanger测序全部得到验证,敏感性85.3%,特异性91.0%。表明其检测效果逊于本发明的组合物。
Claims (8)
1.一种检测耳聋相关基因突变的组合物,包括:
如SEQ ID NO:1~34所示的寡核苷酸。
2.根据权利要求1所述的组合物,其特征在于,所述组合物进一步包括根据测序接头设计的引物对。
3.根据权利要求1所述的组合物,其特征在于,每条引物的5’端进一步分别含有部分测序接头序列,在NO.1~17的5’端加上部分测序接头序列Adapter 1:5’-TCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’,在NO.18~34的5’端加上部分测序接头序列Adapter 2:
5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’。
4.一种如权利要求1~3中任一项所述的组合物制备耳聋基因突变检测的试剂盒的用途。
5.一种耳聋基因突变检测的试剂盒,包括如权利要求1~3中任一项所述的组合物。
6.根据权利要求5所述的试剂盒,其特征在于,所述试剂盒还包括提取所需试剂、扩增所需试剂以及测序所需试剂。
7.一种耳聋相关基因文库的构建方法,所述方法包括:
1)使用如权利要求1~3中任一项所述的组合物扩增样品中的耳聋相关基因,得到添加部分测序接头的耳聋相关基因靶向区域扩增子;
2)用含有Index的剩余测序接头对第一轮产物进行第二轮PCR扩增,得到含有Index和完整测序接头的耳聋相关基因靶向区域扩增子文库。
8.根据权利要求7所述的方法获得的耳聋相关基因扩增子文库。
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