本发明公开了遗传性心律失常致病基因体外检测RNA探针:为如表1所述的9437条RNA探针。本发明还同时提供含有上述RNA探针的遗传性心律失常致病基因体外检测试剂盒。本发明还同时提供利用上述试剂盒进行的遗传性心律失常致病基因体外定性检测方法。本发明通过对靶基因捕获后再用二代测序技术具有创新性,只需要100多M的数据,就可以对46个基因(遗传性心律失常46个致病基因)进行近1000X深度的测序,成本更低,覆盖率更高。
1.遗传性心律失常致病基因体外检测RNA探针,其特征是:为以下所述的9437条RNA探针;ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="585"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" 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2.含有如权利要求1所述的RNA探针的遗传性心律失常致病基因体外检测试剂盒。技术领域
本发明涉及一种遗传性心律失常致病基因体外检测RNA探针及试剂盒,还包括相应的遗传性心律失常致病基因体外检测方法。
背景技术
在人类基因组计划完成十周年的今天,数百种遗传病可通过基因测序技术进行诊断,从而达到预防性干预和个体化治疗,减少病人及家属精神创伤和经济压力,延长病患生存时间的目的。基因诊断带给临床医学的发展革命性变化:癌症风险预测和癌症病人个性化用药指导;体外受精胚胎的基因筛查和无创产前筛查技术;新生儿及成人遗传病及其携带者筛查等。以遗传性心律失常为例,该病已经成为威胁人类健康和生命安全的主要疾病之一,不完全的统计数据表明每年我国心源性猝死人数超过54万,遗传性离子通道和结构异常导致心律失常的发病率分别为1/2000和1/500,这意味着全国目前有超过320万名患者。在缺乏有效早期诊断和诊断后干预手段的情况下,该类患者猝死率超过70%,但是在使用有效早期诊断产品并进行包括药物治疗和行为改善等干预手段后,死亡率可以降低到20%左右。通过早期对相关致病基因测序筛查,在明显临床症状显现前确诊的患者,可以通过药物控制和生活习惯干预来预防严重症状的发生,正确的治疗手段,既保护珍贵生命又能节省医疗费用。与美国如火如荼的基因检测行业相比,基因检测产业在我国发展较慢,通过基因测序技术实施个性化医疗在中国也刚刚起步,本行业迫切希望开发新技术从而能够给中国的基因检测行业注入活力,开启我国的5P个性化医疗时代。
发明内容
本发明要解决的技术问题是提供一种遗传性心律失常致病基因体外定性检测的方法及检测试剂盒和所用的RNA探针;本发明的检测方法旨在降低检测成本。为了解决上述技术问题,本发明提供遗传性心律失常致病基因体外检测RNA探针:为表1所述的9437条RNA探针;表1ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="291"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="481"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="481"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="481"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="483"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="483"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="483"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="482"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="483"/>ntent="drawing" 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wi="700"/>备注说明:上述RNA探针中,大写字母代表通用接头序列。本发明还同时提供含有上述RNA探针的遗传性心律失常致病基因体外检测试剂盒。备注说明:该检测试剂盒中还含有如下物(均属于常规物):RNA酶抑制剂杂交缓冲液:10×SSPE,10×Denhardt’s溶液,10mMxa0EDTA,0.2%SDS洗脱液I:1×SSC,0.1%SDS洗脱液II:0.1×SSC,0.1%SDS重悬液:1Mxa0NaCl,10mMxa0Tris·HCl(pHxa07.5),1mMxa0EDTA中和液:0.1Mxa0NaOH溶解液:1Mxa0Trisxa0HCl(pHxa07.5)洗脱液W:2Mxa0NaCl,10mMxa0Tris·HCl(pHxa07.5),1mMxa0EDTA鲑鱼精DNAhumanxa0Cot-1DNADNA聚合酶,血液DNA提取试剂盒,Illumina的官方文库构建试剂盒。本发明还同时提供利用上述试剂盒进行的遗传性心律失常致病基因体外定性检测方法。本发明具体包括以下步骤:1、RNA探针制备1.1xa0RNA探针设计:从NCBI数据库获得46个遗传性心律失常致病基因的外显子区域,以此为基础按图1所示,分别整理出寡核苷酸微阵列芯片的所有目标序列,其中:设计原则:70%的重叠,外显子区域至少被覆盖3遍以上。N130:表示130bp长度的目标区域序列;5’接头:ATCGCACCAGCGTGT3’接头:CACTGCGGCTCCTCA;1)寡核苷酸微阵列芯片N130寡核苷酸片段截取方式如图1所示:2)ntent="drawing" img-format="tif" inline="yes" orientation="portrait" wi="700"/>3)、一个寡核苷酸微阵列芯片包含以上设计的所有序列,共9437条(如表1所示)。4)、委托公司合成,返回9437条寡核苷酸序列的混合液。1.2、PCR扩增寡核苷酸1.2.1以5ng起始量的寡核苷酸序列的混合液(上述步骤所得)为模板,进行首次PCR扩增。1)准备PCR反应预混液:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>2)将PCR反应管放于PCR仪上,运行下表所示程序:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>3)利用QIAquickxa0PCR纯化试剂盒(德国QIAGEN公司,货号为28104)纯化PCR产物,取5ulPCR产物2%凝胶电泳分析PCR产物片段大小,测定PCR产物浓度、纯度,计算扩增效率。1.2.2、以1.2.1所得的纯化后的PCR产物为模板,加T7再次PCR扩增:1)准备PCR反应预混液:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>2)将PCR反应管放于PCR仪上,运行下表所示程序:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>3)利用QIAquickxa0PCR纯化试剂盒纯化PCR产物,取5ulxa0PCR产物2%凝胶电泳分析PCR产物片段大小,测定PCR产物浓度、纯度,计算扩增效率。1.3以200ng起始量的1.2.2所得的纯化后的PCR产物为模板进行体外转录反应1.3.1、准备反应预混液:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>1.3.2、37°C恒温反应90分钟。1.3.3、RNeasy纯化试剂盒(德国QIAGEN公司,货号为74104)纯化体外转录产物,取5ulxa0PCR产物2%凝胶电泳分析转录产物片段大小,测定转录产物浓度、纯度,计算体外转录效率。1.3.4、体外转录产物即RNA探针,加入RNA酶抑制剂(Ambion公司),1U/ul,-70°保存。以下内容涉及检测方法。2杂交工作液配制2.1杂交缓冲液:10×SSPE,10×Denhardt’s溶液,10mMxa0EDTA,0.2%SDS;2.2洗脱液I:1×SSC,0.1%SDS;2.3洗脱液II:0.1×SSC,0.1%SDS;2.4重悬液:1Mxa0NaCl,10mMxa0Tris·HCl(pHxa07.5),1mMxa0EDTA;2.5中和液:0.1Mxa0NaOH;2.6溶解液:1Mxa0Trisxa0HCl(pHxa07.5);2.7洗脱液W:2Mxa0NaCl,10mMxa0Tris·HCl(pHxa07.5),1mMxa0EDTA;3、样品前处理(DNA文库构建)3.1血液DNA提取(市购的核酸提取试剂盒抽提DNA样品)3.2构建3.1所得的血液DNA样品基因组文库(Illumina"sxa0genomicxa0DNAxa0samplexa0preparationxa0kit)4捕获4.1捕获前准备:按下表准备文库混合液:500ng基因组文库,总量体积超过4.25ul的,真空浓缩至4.25ul。ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>RNA探针混合液准备:取RNA探针混合液500ng,加入1ul(20U)RNA酶抑制剂(Ambion),加水至探针混合液总体积为6ul,65°C预热2分钟。4.2杂交反应:1)7ul文库混合液放于PCR仪上,运行下表所示程序:步骤
温度
时间
步骤1
95°C
5分钟
步骤2
65°C
∞
2)保持文库混合液65°C,向上步文库混合液中加入13ul的65°C预热的杂交缓冲液和6ul的65°C预热2分钟的RNA探针混合液(直接在PCR仪上操作),如下表所示:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>3)用枪头轻轻吸打混匀,盖紧PCR管盖,65°C,恒温反应24小时。得杂交反应液。4.3洗脱洗脱前准备:洗脱液II65°C金属浴或者水浴预热;洗磁珠:取50ul的M-280streptavidinxa0Dynabeads(Invitrogen公司,用之前反复颠倒混匀直至磁珠液体颜色均一),加入50ul(每次的量)的洗脱液W,洗三次,最后用200ul重悬液重悬。将反应24小时的杂交反应液加入到已经洗过三次并重悬的M-280streptavidinxa0Dynabead中,(始终保持杂交反应液在65°C),颠倒混匀3-5次,20°C恒温孵化30分钟(确保样本已经混匀)。简短的离心,磁力架吸附1分钟(或直至混合液变澄清),弃上清,拿出EP管。加入500ul的洗脱液I,重悬磁珠,漩涡混匀5秒,20°C恒温孵化15分钟,期间偶尔漩涡混匀一下。简短的离心,磁力架吸附1分钟(或直至混合液变澄清),弃上清,拿出EP管。加入500ul的65°C预热的洗脱液II,重悬磁珠,漩涡混匀5秒,65°C孵化10分钟,期间偶尔漩涡混匀一下。颠倒混匀,短暂离心,磁力架吸附1分钟(直至混合液变澄清),弃上清,拿出EP管。重复上两步骤(即,加入500ul的65°C预热的洗脱液II,离心)两遍(最后一遍洗脱结束后,要确保所有的洗脱液被彻底去除干净)。加入50ul的中和液,漩涡混匀5秒,20°C溶解10分钟。磁力架吸附2-3分钟(直至混合液变澄清),将上清液转移至含有70ul的溶解液中。脱盐浓缩:杂交产物用Qiagen公司的DNA纯化试剂盒纯化,20ul洗脱,最终得20ul的杂交产物。4.4富集扩增1)准备PCR反应预混液如下表所示:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>2)将PCR反应管放于PCR仪上,运行下表所示程序:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>3)用QIAquickxa0PCRxa0Purificationxa0Kit(QIAGEN),按照操作说明纯化富集扩增产物。5按标准程序进行Illuminaxa0hiseq2000上机。备注说明:至该步骤5,每个待测的血液DNA已经能得到针对此46个致病基因的序列。6数据分析。本发明步骤1中,遗传性心律失常46个致病基因列表如表2所示:表2ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="568"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="547"/>本发明步骤1.1RNA探针的设计原则70%的重叠,外显子区域至少被覆盖3遍以上,委托公司合成(Genetimesxa0Technologyxa0Inc)。本发明步骤1.2PCR扩增寡核苷酸微阵列芯片用的酶可通过市购方式获得,如Q5DNA聚合酶,NEB,货号:M0493L,试剂盒包含Q5Hotstartxa0Highxa0Fidelityxa0buffer(缓冲液)、Q5Hotstartxa0Highxa0Fidelityxa0DNAxa0polymerase(聚合酶)。本发明步骤1.2PCR扩增的引物由英潍捷基(上海)贸易有限公司合成,HPLC纯化,序列如下:引物名称
引物序列(5"--3")
引物A
CTGGGAATCGCACCAGCGTGT
引物B
CGTGGATGAGGAGCCGCAGTG
引物C(加T7)
GAATTCTAATACGACTCACTATAGGCATCGCACCAGCGTGT
本发明步骤1.3体外转录用的酶可通过市购方式获得,如体外转录试剂(Ambion,货号:AM1314),试剂盒包含体外转录酶、10×缓冲液、RNA酶抑制剂。本发明步骤1.3体外转录用的NTP混合液通过市购方式获得,如Roche,货号:11685597910。本发明步骤2所述的杂交缓冲液是由10×SSPE,10×Denhardt’s溶液,10mMxa0EDTA,0.2%SDS组成,洗脱液I是由1×SSC,0.1%SDS组成,洗脱液II由0.1×SSC,0.1%SDS组成,重悬液由1Mxa0NaCl,10mM·Trisxa0HCl(pHxa07.5),1mMxa0EDTA组成,中和液由0.1Mxa0NaOH组成,溶解液由1Mxa0Tris·HCl(pHxa07.5)组成,洗脱液W由2Mxa0NaCl,10mMxa0Tris·HCl(pHxa07.5),1mMxa0EDTA组成,所有的试剂组份(10×SSPE,10×Denhardt’s溶液,10mMxa0EDTA,SDS,1Mxa0Tris·HCl)均可通过市购方式获得,如Sigma。本发明步骤3样品前处理所用到的核酸提取试剂盒均可通过市购方式获得,如全血DNA抽提用QIAGENxa0DNeasyxa0Blood&Tissuexa0Kit;货号:69504。本发明步骤3样品前处理所用到的文库准备试剂盒均可通过市购方式获得,如Illumina基因组DNA文库制备试剂盒;货号:1005361。本发明步骤4捕获前准备所述的文库混合液准备,鲑鱼精DNA通过市购方式获得,如购自Stratagene的Sonicatedxa0salmonxa0spermxa0DNAxa0Kit,货号:201190,用于封闭。本发明步骤4捕获前准备所述的文库混合液准备,Humanxa0Cot-1DNA购自Invitrogen,货号:15279-011,用于封闭。本发明步骤4捕获用的磁珠可通过市购方式获得,如M-280streptavidinxa0Dynabeads,购自Invitrogen,货号:11205D。本发明步骤4富集扩增所用的酶通过市购方式获得,如NEB的Q5扩增试剂盒,货号:E0555L。本发明步骤4富集扩增的引物由英潍捷基(上海)贸易有限公司合成,HPLC纯化,序列如下:引物名称
引物序列(5"-3")
引物1.1
AATGATACGGCGACCACCGAGATCTACAC
引物2.1
CAAGCAGAAGACGGCATACGAGAT
本发明所有的纯化试剂盒均通过市购方式获得,如PCR产物纯化试剂盒QIAquickxa0PCR纯化试剂盒(QIAGEN,货号:28106),RNA纯化试剂盒RNeasyxa0minixa0Kit(QIAGEN,货号:74104),PCR产物纯化浓缩试剂盒MinElutexa0PCRxa0Purificationxa0Kit(QIAGEN,货号:28004)。本发明涉及到的检测方法一次便可完成几十个致病基因的靶向捕获,相对于全外显子组测序来说,本发明只需对目标致病基因进行捕获,从而节约了成本,也简化了后续数据分析,非常具有开发成临床检测项目的潜力。本发明通过设计与目的片段互补的带有生物素标记的RNA探针,与靶向基因序列杂交,然后与抗生物素链酶亲和素抗体结合后用磁珠吸附,完成对遗传性心律失常致病基因靶向序列的捕获。本发明中使用的二代测序方法一次便可完成几十个致病基因的检测,相对于全外显子组测序来说,本发明只需对目标致病基因进行捕获,从而节约成本,也简化了后续数据分析。综上所述,本发明能够特异性捕获46个已知的遗传性心律失常致病基因,覆盖约621个外显子及外显子内含子结合区域,捕获区约为124.2kb,结合二代基因测序筛查部分临床诊断明确的遗传性心律失常。通过基因筛查,可以了解受检者的家族史,有效预测患病风险,建立健康合理的生活习惯,避免因心律失常所导致的不明原因的猝死,达到及早预防的目的,同时也可以给临床医生提供确诊的科学依据。本发明能够有效地去除非目标区域,只对遗传性心律失常致病基因序列进行测序,从而大大简化实验步骤,节约测序成本。
附图说明
图1是寡核苷酸微阵列芯片N130寡核苷酸片段截取方式图。图2是KCNH2基因Sanger测序结果。上图为标准序列(野生型),下图为患者样本测序结果,箭头示c.2499A>G杂合突变。图3是KCNQ1基因Sanger测序结果。上图为正常人序列(野生型),下图为患者样本测序结果,箭头示c.798T>C杂合突变。图4是ANK2基因Sanger测序结果。上图为正常人序列(野生型),下图为患者样本测序结果,箭头示c.11232C>T杂合突变。图5是SNTA1基因Sanger测序结果。上图为正常人序列(野生型),下图为患者样本测序结果,箭头示c.771C>G杂合突变。
具体实施方式
应用实例1:一种遗传性心律失常致病基因体外定性检测的方法临床样本:1个患者样本来自浙江大学医学院附属第一医院,临床诊断为QT间期延长综合征(临床诊断评分≥4分和/或QTc≥480ms)。QT是心电图上的Q波和T波,可以称为复极延迟综合征(是指心电图上QT间期延长,伴有T波和(或)u波形态异常)。应用实例2:一种遗传性心律失常致病基因体外定性检测的方法临床样本:1例正常人样本。一、实验方法与步骤1、RNA探针制备1.1xa0RNA探针设计:获得遗传性心律失常46个致病基因的外显子区域,以这46个致病基因的外显子序列为基础,按图1所示分别整理出寡核苷酸微阵列芯片的所有目标序列,其中:设计原则:70%的重叠,外显子区域至少被覆盖3遍以上。N130:表示130bp长度的目标区域序列;5’接头:ATCGCACCAGCGTGT;3’接头:CACTGCGGCTCCTCA;1)寡核苷酸微阵列芯片N130寡核苷酸片段截取方式如图1所述:2)ntent="drawing" img-format="tif" inline="yes" orientation="portrait" wi="700"/>3)一个寡核苷酸微阵列芯片包含以上设计的所有序列,共9437条(如表1所示)。4)委托公司合成(Genetimesxa0Technologyxa0Inc);得9437条寡核苷酸序列的混合液。5)合成来的芯片质控:需要对方出示芯片纯度,浓度,合成报告,要求总量不少于600ng(不计损耗可进行120次PCR),不少于12K条序列。所有探针序列见表1;1.2xa0PCR扩增寡核苷酸微阵列芯片1.2.1以5ng起始量的寡核苷酸微阵列芯片(即,以5ng起始量的寡核苷酸序列的混合液(上述步骤所得)为模板,用进行首次PCR扩增。1)准备PCR反应预混液:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>2)将PCR反应管放于PCR仪上,运行下表所示程序:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>3)QIAquickxa0PCRxa0Purificationxa0Kit(QIAGEN)纯化PCR产物,取5ulxa0PCR产物2%凝胶电泳分析PCR产物片段大小,测定PCR产物浓度、纯度,计算扩增效率。4)PCR产物质控要求:片段大小160-200bp,OD260/280在1.8-2.0之间。1.2.2以1.2.1所得的纯化后PCR产物为模板,加T7再次PCR扩增1)准备PCR反应预混液:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>2)将PCR反应管放于PCR仪上,运行下表所示程序:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>3)QIAquickxa0PCRxa0Purificationxa0Kit(QIAGEN)纯化PCR产物,取5ulxa0PCR产物2%凝胶电泳分析PCR产物片段大小,测定PCR产物浓度、纯度,计算扩增效率。4)PCR产物质控要求:片段大小160-200bp,OD260/280在1.8-2.0之间,扩增效率≥300倍。1.3以200ng起始量的1.2.2所得的纯化后的PCR产物为模板进行体外转录反应1.3.1准备反应预混液:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>1.3.2xa037°C恒温反应90分钟。1.3.3xa0RNeasyxa0minixa0Kit(QIAGEN)纯化体外转录产物,取5ulPCR产物2%凝胶电泳分析转录产物片段大小,测定转录产物浓度、纯度,计算体外转录效率。1.3.4体外转录产物即RNA探针,加入SUPERase-Inxa0Rnasexa0inhibitor(Ambion),1U/ul,-70°保存。2杂交工作液配制2.1杂交缓冲液:10×SSPE,10×Denhardt’s溶液,10mMxa0EDTA,0.2%SDS配制1000ul:500ul(20×SSPE)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0200ul(50×Denhardt)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0200ul(1%SDS)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa020ul(0.5Mxa0EDTA)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0xa080ul(H₂O)备注说明:1%SDS为:1g的SDS溶解在100ml水中而得。以下类同。2.2洗脱液I:1×SSC,0.1%SDS配制10ml:500ul(20×SSC)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa01ml(1%SDS)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa08.5ml(H₂O)2.3洗脱液II:0.1×SSC,0.1%SDS配制10ml:50ul(20×SSC)xa0xa0xa0xa0xa0xa0xa0xa0xa01ml(1%SDS)xa0xa0xa0xa0xa0xa0xa0xa0xa08.95ml(H₂O)2.4重悬液:1Mxa0NaCl,10mMxa0Tris·HCl(pHxa07.5),1mMxa0EDTA配制10ml:5ml(2Mxa0NaCl)xa0xa0xa0xa0xa0xa0xa0xa0xa0100ul(1Mxa0Tris·HCl(pHxa07.5))xa0xa0xa0xa0xa0xa0xa0xa0xa020ul(0.5Mxa0EDTA)xa0xa0xa0xa0xa0xa0xa0xa0xa04880ul(H₂O)2.5中和液:0.1Mxa0NaOH配置1ml:100ul(1Mxa0NaOH)加900ul水。2.6溶解液:1Mxa0Tris·HCl(pHxa07.5)2.7洗脱液W:2Mxa0NaCl,10mMxa0Tris·HCl(pHxa07.5),1mMxa0EDTA配制10ml:5ml(4Mxa0NaCl)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa0100ul(1Mxa0Tris·HCl(pHxa07.5))xa0xa0xa0xa0xa0xa0xa0xa0xa0xa020ul(0.5Mxa0EDTA)xa0xa0xa0xa0xa0xa0xa0xa0xa0xa04880ul(H₂O)3样品前处理(DNA文库构建)。3.1获取临床样本的全血样品不少于200ml,用QIAGENxa0DNeasyxa0Blood&Tissuexa0Kit(货号:69504)提取血液DNA,每个样品DNA总量推荐3-5ug。3.2用Illumina基因组DNA文库构建试剂盒对2个DNA样品分别进行基因组文库构建,按照Illumina标准操作规程操作,文库片段范围要求250bp~350bp,总量不少于500ng。4捕获4.1捕获前准备:按下表所示准备文库混合液:500ng基因组文库,总量体积超过4.25ul的,真空浓缩至≤4.25ul。ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>RNA探针混合液准备:取RNA探针混合液500ng,加入1ul(20U)RNA酶抑制剂(Ambion),加水至混合至探针混合液总体积为6ul,65°C预热2分钟。4.2杂交反应:1)7ul文库混合液放于PCR仪上,运行下表所示程序:步骤
温度
时间
步骤1
95°C
5分钟
步骤2
65°C
∞
2)保持文库混合液65°C,向上步文库混合液中加入13ul的65°C预热的杂交缓冲液和6ul的65°C预热2分钟的RNA探针混合液(直接在PCR仪上操作),如下表所示:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>3)用枪头轻轻吸打混匀,盖紧PCR管盖,65°C,恒温反应24小时。4.3洗脱洗脱前准备:洗脱液II65°C金属浴或者水浴预热:1洗磁珠:取50ul的M-280streptavidinxa0Dynabeads(用之前反复颠倒混匀直至磁珠液体颜色均一),加入50ul的洗脱液W,洗三次,最后用200ul重悬液重悬。2将反应24小时候的杂交反应液加入到已经洗过三次的M-280streptavidinxa0Dynabead中,(始终保持杂交反应液在65°C),颠倒混匀3-5次,20°C恒温孵化30分钟(确保样本已经混匀)。3简短的离心,磁力架吸附1分钟(或直至混合液变澄清),弃上清,拿出EP管。4加入500ul的洗脱液I,重悬磁珠,漩涡混匀5秒,20°C恒温孵化15分钟,期间偶尔漩涡混匀一下。5简短的离心,磁力架吸附1分钟(或直至混合液变澄清),弃上清,拿出EP管。6加入500ul的65°C预热的洗脱液2,重悬磁珠,漩涡混匀5秒,65°C孵化10分钟,期间偶尔漩涡混匀一下。7颠倒混匀,简短离心,磁力架吸附1分钟(直至混合液变澄清),弃上清,拿出EP管。8重复上两步骤两遍(最后一遍洗脱结束后,要确保所有的洗脱液被彻底去除干净)。9加入50ul的中和液,漩涡混匀5秒,20°C溶解10分钟。10磁力架吸附2-3分钟(直至混合液变澄清),将上清液转移至含有70ul的溶解液中。11脱盐浓缩:杂交产物用MinElutexa0PCRxa0Purificationxa0Kit(QIAGEN)纯化浓缩,20ul洗脱,最终得道20ul的杂交产物。4.4富集扩增1)准备PCR反应预混液如下表所示:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>2)将PCR反应管放于PCR仪上,运行下表所示程序:ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>3)用QIAquickxa0PCR纯化试剂盒(QIAGEN),按照操作说明纯化富集扩增产物5按标准程序进行Illumina上机二、结果分析1)针对Illuminaxa0HiSeq2000测序仪产生的原始数据,并首先采用BWA比对算法软件将全基因组原始读长序列(read)与参考基因组进行初步比对;备注说明:9437条探针,混合后的作用是捕获46个目的基因,所得到的结果是46个目的基因的数据,这也是和全外显子测序相比的一个优势,只需要对这46个基因的序列数据进行分析即可。2)重新比对与重新校准处理,分别采用三种重要的工具:(1)碱基质量得分重新矫正;(2)indel临近区域局部重新匹配比对处理;(3)屏蔽重复读长序列。屏蔽重复测序读长的处理目的是考虑处理在pair-end测序双端存在的由PCR所导致的序列冗余,同时用于处理重复读长在indel区域内所获得边界存在不同匹配的情形(1)高效率的与已知位点(indel,SNP)进行重新匹配与比对处理,可对低覆盖度区域进行操作(仅对已知indel进行重新匹配)。(2)采用碱基错配方式用以判定位点是否需要重新匹配比对,并进行全面的局部重新匹配处理。3)第二阶段(phasexa02)异体识别与基因型计算,首先针对矫正重新匹配的序列进行比对结果的合并处理,将所有全基因组测序的比对结果进行合并用于后续变异体和基因型的识别。4)多重样本SNP与Indel识别采用基于贝叶斯基因型似然比模型算法的通用基因型识别算法对SNP和indel进行识别。贝叶斯基因型似然比模型算法的原理是同时估计在一个包含N个样本的群体或单独个体样本中最可能出现的基因型和等位基因频率,并准确计算出每个核苷酸位置上的变异体和基因型的后验概率。通用基因型识别算法用来识别统计学上所判断的非参考序列等位基因的变异体(SNP、Indel)位置和基因型。两个样本测序后的数据统计如表3所示:表3ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>通过贝叶斯基因型似然比模型算法得到的突变如表4所示:表4ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>备注说明:其余基因经检测没有发生突变。对其中KCNH2基因的c.2499A>Gxa0p.N633S突变用金标准Sanger法进行验证,发现与本发明检测到的突变类型完全一致,均为外显子7上的杂合突变,如图2所示;对其中KCNQ1基因的c.798T>Cxa0p.L266P突变用金标准Sanger法进行验证,发现与本发明检测到的突变类型完全一致,均为外显子6上的杂合突变,如图3所示。应用实例3、将应用实例1中的患者的全血样品改成另一个患者B的全血样品,按照应用实例所述方法进行检测。测序后的数据统计如表5所示:表5ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>通过贝叶斯基因型似然比模型算法得到的突变如表6所示:表6ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="700"/>备注说明:其余基因经检测没有发生突变。对其中ANK2基因的c.11232C>Txa0p.T3744N突变用金标准Sanger法进行验证,发现与本发明检测到的突变类型完全一致,均为外显子42上的杂合突变,如图4所示;对其中SNTA1基因的c.771C>Gxa0p.A257G突变用金标准Sanger法进行验证,发现与本发明检测到的突变类型完全一致,均为外显子4上的杂合突变,如图5所示。综上结果显示所述:本发明的探针可用于新一代测序,检测遗传性恶性心律失常患者的突变位点,为医生进一步对遗传性恶性心律失常患者进行针对性治疗提供理论指导。本发明经过大量的实验验证,遗传性恶性心律失常患者的全血样品按照本发明所述方法进行检测,均能检测到相应的突变类型。本发明的优势:疾病基因突变位置不是固定的,因此无法用taqman探针法进行检测,必须用测序法。目前,一代测序每次只能检测500-1000bp长度,基因如果达到几百kb,检测会变的非常昂贵,且检测花费时间很长。直接利用全基因组二代测序,需要几百X的覆盖率,总测序数据量需要100G以上,成本过高,同时大量无用的测序结果,也会使分析变的非常困难。因此,本发明通过对靶基因捕获后再用二代测序技术具有创新性,只需要100多M的数据,就可以对该46个基因进行近1000X深度的测序,成本更低,覆盖率更高。目前市面上没有恶性心律失常的同类检测产品。最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="479"/>ntent="drawing" img-format="tif" inline="no" orientation="portrait" wi="649"/>