CN113088571A - 一种scn5a基因检测试剂盒及检测方法 - Google Patents
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Abstract
本发明公开了一种SCN5A基因检测试剂盒及检测方法,检测方法包括如下步骤:A、DNA提取;B、基因组DNA进行质检;C、构建扩增子靶向捕获文库;D、对制备好的文库进行上机测序;E、对数据结果进行标准分析;F、数据质控;G、变异检测结果,本发明操作简单,成本低,覆盖了SCN5A基因全编码区、编码区前后50bp及chr3:38691021等位点,同时可进行多对特异性引物的扩增,相比传统的一代测序技术降低了操作的复杂度,在保持与二代测序技术相同测序深度的同时又降低了成本。
Description
技术领域
本发明涉及基因检测技术领域,具体为一种SCN5A基因检测试剂盒及检测方法。
背景技术
心源性猝死是指急性症状发作后1小时内发生的以意识突然丧失为特征的由心脏原因引起的自然死亡,占猝死的大部分,死者生前可有也可无心血管疾病。心源性猝死具有起病急、进展快、病死率高等特点。全球每年有1700万人因心血管疾病而死亡,心源性猝死占了25%左右。女性的心源性猝死发生率为1.40/1000人/年,男性为6.68/1000人/年。约50%的心脏骤停发生在无已知的心脏病个体中,但大部分患者有未诊断的缺血性心脏病。据估计,我国每年心源性猝死的人超过50万,且愈趋年轻化。心源性猝死者绝大多数患有器质性心脏病,主要包括冠心病、肥厚型和扩张型心肌病、心脏瓣膜病、心肌炎、非粥样硬化性冠状动脉异常、浸润性病变、传导异常(QT间期延长综合征、心脏阻滞)和严重室性心律失常等。另外,洋地黄和奎尼丁等药物中毒亦可引起。大多数心脏性猝死则是室性快速心律失常所致。一些暂时的功能性因素,如心电不稳定、血小板聚集、冠状动脉痉挛、心肌缺血及缺血后再灌注等使原有稳定的心脏结构异常发生不稳定情况。某些因素如自主神经***不稳定、电解质失调、过度劳累、情绪压抑及用致室性心律失常的药物等,都可触发心源性猝死。心源性猝死遇难者一级亲属的家庭筛查是一项非常重要的干预措施,它不仅可以识别有风险的家属,对可用的治疗作出建议,还可以充分预防猝死。在高达50%心律失常性猝死综合征遇难者的亲属中,可作出遗传性致心律失常性疾病的诊断。
长QT综合征(long QT syndrome,LQTS)是一种家族遗传性电活动紊乱心脏病。QT间期是指心电图上QRS波群起点至T波终点的总时限,代表了心室除极及复极的总时间,是电兴奋在心室传导的反映。LQTS的临床表现主要为尖端扭转性室性心动过速引起的反复晕厥和猝死。大多数病人的症状发生在运动、情绪紧张、激动时,晕厥一般持续1-2min,少部分病人的猝死发生在睡眠时。心电图是诊断LQTS的重要依据,大部分患者心电图上QT间期延长(男性≥470ms,女性≥480ms),但也有少部分患者QT间期可正常,称之为“QT间期正常”或“隐匿型”LQTS。LQTS是最早发现的离子通道病,致病基因及突变位点也相应较多,涉及Na+通道、K+通道、Ca2+通道及某些亚单位,甚至是对离子通道起调控作用的蛋白。针对以下情况,专家推荐进行LQTS的相关基因检测:基于病史、家族史及心电图表型被心脏专家高度怀疑LQTS的患者;无症状的特发QT间期延长者;排除继发性QT间期延长因素的患者。
Brugada综合征(Brugada syndrome,BrS)是一类易引起心源性猝死的离子通道病。心电图特征为右束支传导阻滞,V1~V3胸前导联ST段抬高,QT间期正常。BrS的致病基因涉及Na+通道、K+通道、Ca2+通道及它们的调节亚单位,这其中以Na+通道最为多见。鉴于BrS可导致猝死等严重心脏事件,专家共识推荐BrS家族成员及其相关亲属应行特定突变检测。临床怀疑BrS的患者应行SCN5A基因检测。
SCN5A(sodium channel,voltage-gated,type V)为钠通道α基因,编码钠通道α,是人类电压门控制钠通道基因家族一员。心脏钠通道介导心脏中钠离子内流形成心肌细胞动作电位的快递上升相,为兴奋传导起重要作用。该基因多态性对钠离子道道蛋白的数目及动力学特征产生负面影响,使钠离子内流受阻,影响了心肌细胞动作电位的形成,易导致心肌细胞去极化异常,心律失常,易导致房颤、LQT、BrS等心脏疾病的发生,增加了猝死的风险性。
SCN5A基因的突变是导致心源性猝死的原因之一,基因诊断也成为目前确诊心源性猝死的重要手段。然而目前公认的心源性猝死致病基因变异仅针对的是已发现的热点突变,仍有部分变异未作为心源性猝死防控或治疗的靶点。(然而,并不是前述基因的任何位点的突变都与心源性猝死相关;通过检测现有的少数基因突变位点来诊断心源性猝死亦难免造成假阴性的检测结果。)本发明首先阐明了以人类SCN5A基因全编码区及部分非编码区为新的筛查靶点来对心源性猝死进行早期诊断或预后预测的可行性。
发明内容
本发明的目的在于提供一种SCN5A基因检测试剂盒及检测方法,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:一种SCN5A基因检测方法,检测方法包括如下步骤:
A、DNA提取;
B、基因组DNA进行质检;
C、构建扩增子靶向捕获文库;
D、对制备好的文库进行上机测序;
E、对数据结果进行标准分析;
F、数据质控;
G、变异检测结果。
优选的,所述步骤A中具体方法如下:取全血样本200微升,采用萃取方法提取全基因组DNA,使用Nanodrop2000对DNA型质量检测和浓度测定;其中,A260/280比值在1.8~2.0,A260/230比值在1.7~1.9判定为合格,最后将合格的DNA样品统一稀释至100纳克/微升。
优选的,所述步骤B中具体为:Qubit对DNA浓度进行精准定量,一般OD值在1.8~2.0之间,DNA总量大于40ng,满足建库标准的DNA样本将用于后续建库测序。
优选的,所述步骤C中具体方法如下:利用多重PCR技术和SCN5A基因特异性多重PCR引物,同时对基因组DNA上的多个目标区域进行扩增,得到扩增子,然后通过PCR的方式将二代测序接头添加到扩增子的两侧,得到扩增子文库,进行二代测序,获取目标区域的序列信息。
优选的,所述步骤D中采用illumina nova进行上机器测序,采用PE150,每样本数据量不低于1G。
优选的,所述步骤G中,基于与基因组参考序列比对的bam结果,采用samtools、GATK软件寻找SNP和InDel,然后运用ANNOVAR软件对SNP,InDel位点进行注释,确定突变位点对应的基因信息、功能信息、有害性。
优选的,按照文库构建流程进行操作,具体流程如下:
1)取1μl文库使用Qubit dsDNA HS Assay Kit进行定量,记录文库浓度,文库浓度约在10-50ng/μl;
2)取1μl样品使用Agilent 2100Bioanalyzer system(Agilent DNA1000Kit)进行文库片段长度测定,文库长度约在300-450bp之间。
优选的,所述步骤F测序得到的原始数据中,会存在少量reads包含接头信息、低质量碱基或未检出的碱基,为了保证信息分析质量,需要对Raw reads进行初步过滤,得到Clean reads,后续分析都基于Clean reads进行;其中,数据过滤的内容主要如下:
(1)以8bp滑窗的方式,剪切掉平均碱基质量值小于20的序列;
(2)去掉序列尾部的接头序列;
(3)如果序列首碱基或尾碱基小于20,则会直接剪切掉该碱基;
(4)通常情况下载,去除完后若剩余序列长度小于40,则丢弃该对序列。
优选的,一种SCN5A基因检测试剂盒,包括上述任意一项所述的检测方法。
与现有技术相比,本发明的有益效果是:本发明操作简单,成本低,覆盖了SCN5A基因全编码区、编码区前后50bp及chr3:38691021等位点,同时可进行多对特异性引物的扩增,相比传统的一代测序技术降低了操作的复杂度,在保持与二代测序技术相同测序深度的同时又降低了成本。
附图说明
图1为本发明标准分析流程图;
图2为本发明文库构建流程图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
在本发明的描述中,需要说明的是,术语“上”、“下”、“内”、“外”“前端”、“后端”、“两端”、“一端”、“另一端”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本发明和简化描述,而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本发明的限制。此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性。
在本发明的描述中,需要说明的是,除非另有明确的规定和限定,术语“安装”、“设置有”、“连接”等,应做广义理解,例如“连接”,可以是固定连接,也可以是可拆卸连接,或一体地连接;可以是机械连接,也可以是电连接;可以是直接相连,也可以通过中间媒介间接相连,可以是两个元件内部的连通。对于本领域的普通技术人员而言,可以具体情况理解上述术语在本发明中的具体含义。
请参阅图1-2,本发明提供一种技术方案:一种SCN5A基因检测方法,检测方法包括如下步骤:
A、DNA提取;
B、基因组DNA进行质检;
C、构建扩增子靶向捕获文库;
D、对制备好的文库进行上机测序;
E、对数据结果进行标准分析;
F、数据质控;
G、变异检测结果。
本发明中,所述步骤A中具体方法如下:取全血样本200微升,采用萃取方法提取全基因组DNA,使用Nanodrop2000对DNA型质量检测和浓度测定;其中,A260/280比值在1.8~2.0,A260/230比值在1.7~1.9判定为合格,最后将合格的DNA样品统一稀释至100纳克/微升。
本发明中,所述步骤B中具体为:Qubit对DNA浓度进行精准定量,一般OD值在1.8~2.0之间,DNA总量大于40ng,满足建库标准的DNA样本将用于后续建库测序。
本发明中,所述步骤C中具体方法如下:利用多重PCR技术和SCN5A基因特异性多重PCR引物,同时对基因组DNA上的多个目标区域进行扩增,得到扩增子,然后通过PCR的方式将二代测序接头添加到扩增子的两侧,得到扩增子文库,进行二代测序,获取目标区域的序列信息。
本发明中,所述步骤D中采用illumina nova进行上机器测序,采用PE150,每样本数据量不低于1G。
本发明中,所述步骤G中,基于与基因组参考序列比对的bam结果,采用samtools、GATK软件寻找SNP和InDel,然后运用ANNOVAR软件对SNP,InDel位点进行注释,确定突变位点对应的基因信息、功能信息、有害性。
本发明中,按照文库构建流程进行操作,具体流程如下:
1)取1μl文库使用Qubit dsDNA HS Assay Kit进行定量,记录文库浓度,文库浓度约在10-50ng/μl;
2)取1μl样品使用Agilent 2100Bioanalyzer system(Agilent DNA 1000Kit)进行文库片段长度测定,文库长度约在300-450bp之间。
本发明中,所述步骤F测序得到的原始数据中,会存在少量reads包含接头信息、低质量碱基或未检出的碱基,为了保证信息分析质量,需要对Raw reads进行初步过滤,得到Clean reads,后续分析都基于Clean reads进行;其中,数据过滤的内容主要如下:
(1)以8bp滑窗的方式,剪切掉平均碱基质量值小于20的序列;
(2)去掉序列尾部的接头序列;
(3)如果序列首碱基或尾碱基小于20,则会直接剪切掉该碱基;
(4)通常情况下载,去除完后若剩余序列长度小于40,则丢弃该对序列。
其中,SCN5A基因特异性多重PCR引物来自于下述表1和表2
表1
表2
其他目的位点 | ForwardPrimer(Fp) | ReversePrimer(Rp) |
chr3:38691021 | CCTCGGGGAGGAAAGTTgg | GTAGGATGCAGGGATCGCTC |
综上所述,本发明操作简单,成本低,覆盖了SCN5A基因全编码区、编码区前后50bp及chr3:38691021等位点,同时可进行多对特异性引物的扩增,相比传统的一代测序技术降低了操作的复杂度,在保持与二代测序技术相同测序深度的同时又降低了成本。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
Claims (9)
1.一种SCN5A基因检测方法,其特征在于:检测方法包括如下步骤:
A、DNA提取;
B、基因组DNA进行质检;
C、构建扩增子靶向捕获文库;
D、对制备好的文库进行上机测序;
E、对数据结果进行标准分析;
F、数据质控;
G、变异检测结果。
2.根据权利要求1所述的一种SCN5A基因检测方法,其特征在于:所述步骤A中具体方法如下:取全血样本200微升,采用萃取方法提取全基因组DNA,使用Nanodrop2000对DNA型质量检测和浓度测定;其中,A260/280比值在1.8~2.0,A260/230比值在1.7~1.9判定为合格,最后将合格的DNA样品统一稀释至100纳克/微升。
3.根据权利要求1所述的一种SCN5A基因检测方法,其特征在于:所述步骤B中具体为:Qubit对DNA浓度进行精准定量,一般OD值在1.8~2.0之间,DNA总量大于40ng,满足建库标准的DNA样本将用于后续建库测序。
4.根据权利要求1所述的一种SCN5A基因检测方法,其特征在于:所述步骤C中具体方法如下:利用多重PCR技术和SCN5A基因特异性多重PCR引物,同时对基因组DNA上的多个目标区域进行扩增,得到扩增子,然后通过PCR的方式将二代测序接头添加到扩增子的两侧,得到扩增子文库,进行二代测序,获取目标区域的序列信息。
5.根据权利要求1所述的一种SCN5A基因检测方法,其特征在于:所述步骤D中采用illumina nova进行上机器测序,采用PE150,每样本数据量不低于1G。
6.根据权利要求1所述的一种SCN5A基因检测方法,其特征在于:所述步骤G中,基于与基因组参考序列比对的bam结果,采用samtools、GATK软件寻找SNP和InDel,然后运用ANNOVAR软件对SNP,InDel位点进行注释,确定突变位点对应的基因信息、功能信息、有害性。
7.根据权利要求4所述的一种SCN5A基因检测方法,其特征在于:按照文库构建流程进行操作,具体流程如下:
1)取1μl文库使用Qubit dsDNA HS Assay Kit进行定量,记录文库浓度,文库浓度约在10-50ng/μl;
2)取1μl样品使用Agilent 2100Bioanalyzer system(Agilent DNA 1000Kit)进行文库片段长度测定,文库长度约在300-450bp之间。
8.根据权利要求1所述的一种SCN5A基因检测方法,其特征在于:所述步骤F测序得到的原始数据中,会存在少量reads包含接头信息、低质量碱基或未检出的碱基,为了保证信息分析质量,需要对Raw reads进行初步过滤,得到Clean reads,后续分析都基于Cleanreads进行;其中,数据过滤的内容主要如下:
(1)以8bp滑窗的方式,剪切掉平均碱基质量值小于20的序列;
(2)去掉序列尾部的接头序列;
(3)如果序列首碱基或尾碱基小于20,则会直接剪切掉该碱基;
(4)通常情况下载,去除完后若剩余序列长度小于40,则丢弃该对序列。
9.一种SCN5A基因检测试剂盒,其特征在于:所述基因检测试剂盒包括权利要求1-8任意一项所述的检测方法。
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