CN112195235A - 一种检测铁调素基因tmprss6 snp位点的试剂盒及应用 - Google Patents
一种检测铁调素基因tmprss6 snp位点的试剂盒及应用 Download PDFInfo
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Abstract
本发明公开了一种检测铁调素基因TMPRSS6 SNP位点的试剂盒及应用，包括用于检测铁调素TMPRSS6基因的rs855791位点和rs4820268位点及用于扩增以上两个位点在内的核苷酸序列的引物及扩增片段。一种检测铁调素基因TMPRSS6 SNP位点的试剂盒在孕期保健工作中预测孕期铁代谢能力和妊娠糖尿病检测中应用。本试剂盒可检测TMPRSS6的rs855791和rs4820268位点，提供的检测方法简单、快速、准确，所需设备普及率高。该检测结果可以实现妊娠糖尿病的精准施治与个性化医疗，应用于孕期贫血和妊娠糖尿病的诊疗路径的优化，可指导临床精准施治、早期预防。
Description
技术领域
本发明涉及生物医学检测技术领域，具体涉及与一种检测铁调素基因TMPRSS6SNP位点的试剂盒及应用。
背景技术
单核苷酸多态性(SNP)是指在基因组上由单个核苷酸的变异所引起的DNA序列的多态性。铁调素基因TMPRSS6的两个关键SNP位点的变异可影响体内铁蛋白(SF)水平，铁蛋白(SF)在非感染、炎症状态下可反映机体储存铁元素的状态，是临床常用的铁营养状况评价指标，铁调素基因SNP表型异常时铁代谢路径发生变化，铁蛋白水平则不再与铁营养状况呈单纯的正相关关系，会呈现出铁缺乏状态下的异常升高。而SF升高与糖尿病、高血压、代谢综合征及心血管疾病等有关。
妊娠糖尿病(GDM)是指妊娠期间发生或首次发现的不同程度的糖耐量异常。到妊娠中、晚期，孕妇体内抗胰岛素样物质增加，如胎盘生乳素、雌激素、孕酮、皮质醇和胎盘胰岛素酶等使孕妇对胰岛素的敏感性随孕周增加而下降。为维持正常糖代谢水平，胰岛素需求量必须相应增加。对于胰岛素分泌受限的孕妇，妊娠期不能代偿这一生理变化而使血糖升高，使原有糖尿病加重或出现GDM。在我国，GDM的发生率高达16％－19％，是巨大儿、剖宫产、小于胎龄儿、妊娠高血压、难产等多种不良妊娠结局的主要诱因，是孕期健康管理的难点和热点，得到了妇产科临床广泛的关注。目前，GDM的临床管理路径是孕中期通过糖耐量检查确诊后开展血糖控制，以期降低血糖控制不良的后果。临床缺乏更为积极有效的GDM早期筛查预测的生物标志物和预防性干预的办法。
从分子水平对铁调素基因TMPRSS6的活性进行检测，可以区分个体铁代谢能力，预测妊娠糖尿病的发生，可为GDM提供预防性干预，将GDM孕期管理的干预关口提前，让高危患者的治疗、预后得到改善。
针对铁调素基因进行SNP检测的临床适用的方法尚未得到开发和应用。目前通过许多生物化学方法已经能确定个体特定基因位点的SNP，如限制性片段长度多态性(restriction fragment length polymorphism，RFLP)、基因微阵列检测技术(microarray)、聚合酶链式反应技术(polymerase chain reaction，PCR)、高通量测序(next-generation sequencing)等，PCR技术是临床医学领域最常用的SNP检测手段，包含PCR-桑格(Sanger)测序、反转录PCR、飞行时间质谱检测等，其中对目标区域进行PCR扩增后再进行Sanger测序是行业的金标准，适用范围广，检测结果快且设备在医院检验科内普及率极高。
现有技术的缺点：(1)缺少利用铁调素基因检测结果对妊娠糖尿病风险评估的应用以及产品；
(2)普适性的风险建议以及干预路径缺乏个性化以及精准性。
发明内容
针对上述问题，本发明的目的在于提供一种检测铁调素基因TMPRSS6 SNP位点的试剂盒及应用，以应用于孕期贫血和妊娠糖尿病的诊疗路径的优化，可指导临床精准施治、早期预防，改进临床孕期贫血诊疗路径和妊娠糖尿病早期预测及干预路径。
本发明提供如下技术方案：一种检测铁调素基因TMPRSS6 SNP位点的试剂盒：包括用于检测铁调素TMPRSS6基因的rs855791位点和rs4820268位点及用于扩增以上两个位点在内的核苷酸序列的引物及扩增片段。
所述的rs855791位点核苷酸序列：>chr22:37066728-37067261534bp。
rs855791位点正向引物序列：TGACCTCAGGTGTTCCGTC，即SEQ ID No.1。
rs855791位点反向引物序列：AGGCTTCAGCAGGCTGATG，即SEQ ID No.2。
rs855791位点扩增片段：即SEQ ID No.3。
rs4820268位点序列：>chr22:37073257-37073650 394bp。
rs4820268位点正向引物序列：AGGGAGATTGATGGTTGCAC，即SEQ ID No.4。
rs4820268位点反向引物序列：CACAGTTTGCAGAGCCACAT，即SEQ ID No5。
rs4820268位点扩增片段：即SEQ ID No 6。
所述试剂盒还包括PCR反应常用的酶和试剂，如dNTPs、DNA聚合酶、Mg2+、PCR反应缓冲液，还可以含有用于检测分型使用的限制性内切酶及缓冲液。
该试剂盒在孕期保健工作中预测孕期铁代谢能力和妊娠糖尿病检测中应用。
与现有技术相比，本发明的有益效果如下：本试剂盒可检测TMPRSS6的rs855791和rs4820268位点，提供的检测方法简单、快速、准确，所需设备普及率高。
该检测结果可以实现妊娠糖尿病的精准施治与个性化医疗，应用于孕期贫血和妊娠糖尿病的诊疗路径的优化，可指导临床精准施治、早期预防。
具体实施方式
下面将结合本发明实施例对本发明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。
实施例：
一种检测铁调素基因TMPRSS6 SNP位点的试剂盒：包括用于检测铁调素TMPRSS6基因的rs855791位点和rs4820268位点及用于扩增以上两个位点在内的核苷酸序列的引物及扩增片段。
rs855791位点引物序列及扩增片段见下表1
表1
rs4820268位点引物序列及扩增片段见下表2
表2
所述试剂盒还包括PCR反应常用的酶和试剂，如dNTPs、DNA聚合酶、Mg2+、PCR反应缓冲液，还可以含有用于检测分型使用的限制性内切酶及缓冲液。
2.研究对象：本研究收集了398例孕中期的血液样本，使用基因微阵列技术对样本进行了TMPRSS6上的rs855791以及rs4820268位点基因分型，还进行了各类血生化指标的检测，并且调查了各个受试人的民族、性别、年龄、文化水平、既往病史、饮食状况等信息。
3.实验方法：样本获取，收集2mL唾液样本，注意避免产生过多气泡，收集后与保存液混合均匀，常温保存和运输，保存在4℃中不超过一周。
4.样本进入实验室后，使用全自动核酸提取仪，进行DNA的分离和纯化。提纯后DNA进入DNA质检环节，采用定量仪器对其进行定量和质控，使用TECAN多功能酶标仪进行定量，使用Nanodrop对浓度以及A260/A230等指标进行检测质控，质控合格样本进入芯片检测环节。
5.核酸扩增富集：低起始量的DNA通过Illumina Hybridization Oven恒温扩增仪，使DNA总量富集至1000～2000倍。再经过DNA片段化、沉淀、重悬等操作后，使用全自动液体处理工作站将片段化后DNA重悬液点样至Illumina定制化芯片上，放置于IlluminaHybridization Oven中过夜杂交。
6.基因微阵列检测：杂交结束后，使用TECAN全自动液体处理工作站，经过一系列自动化操作和酶催化反应后，ATCG碱基会被标记成不同颜色组合。通过autoloader全自动机械臂，将待检测的芯片转移至Illumina iScan芯片扫描仪上，经过内置的亚微米分辨率的超高清照相机，系统将对芯片上不同颜色、不同强度的光学信号进行解码，最终生成芯片检测的原始数据文件。
7：数据提取：从原始数据文件中提取出本实施例所需要的rs855791以及rs4820268位点的基因型信息。
8.统计结果如下表所示
首先评估哈迪-温伯格平衡的基因型分布，然后将人群根据基因型进行分组，使用卡方检验进行分析，计算不同组间理论人数与实际人数之间的方差，计算卡方值，评估组间是否存在显著性差异。使用分类变量和均值的频率和计数汇总数据，并使用连续变量的标准差进行统计。使用广义线性回归分析TMPRSS6变异体与反映体铁状态(包括血清铁，转铁蛋白饱和度)的参数的相关性，使用最小二乘法对线性关系进行求解，得到最佳拟合线性方程，并调整年龄，BMI，通过获取每周摄入的红肉(主要包括牛肉，羊肉，猪肉和加工肉)的频率。
9.研究结果表明了rs855791和rs4820268两个位点的分型对血清铁以及转铁蛋白饱和度都有着显著的影响，并进一步影响GDM的风险。因此利用这两个位点评估GDM的风险具有重要的临床意义。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。。
Claims (9)
1.一种检测铁调素基因TMPRSS6 SNP位点的试剂盒，其特征在于：包括用于检测铁调素TMPRSS6基因的rs855791位点和rs4820268位点及用于扩增以上两个位点在内的核苷酸序列的引物及扩增片段。
2.根据权利要求1所述的一种检测铁调素基因TMPRSS6 SNP位点的试剂盒，其特征在于：所述的rs855791位点核苷酸列：>chr22:37066728-37067261534bp。
3.根据权利要求1所述的一种检测铁调素基因TMPRSS6 SNP位点的试剂盒，其特征在于：所述的rs855791位点正向引物序列：TGACCTCAGGTGTTCCGTC，即SEQIDNo.1；
所述的rs855791位点反向引物序列：AGGCTTCAGCAGGCTGATG，即SEQIDNo.2。
4.根据权利要求1所述的一种检测铁调素基因TMPRSS6 SNP位点的试剂盒，其特征在于：所述的rs855791位点扩增片段：即SEQ ID No.3。
5.根据权利要求1所述的一种检测铁调素基因TMPRSS6 SNP位点的试剂盒，其特征在于：所述的rs4820268位点序列：>chr22:37073257-37073650394bp。
6.根据权利要求1所述的一种检测铁调素基因TMPRSS6 SNP位点的试剂盒，其特征在于：所述的rs4820268位点正向引物序列AGGGAGATTGATGGTTGCAC，即SEQ ID No.4；rs4820268位点反向引物序列：CACAGTTTGCAGAGCCACAT，即SEQ ID No5。
7.根据权利要求1所述的一种检测铁调素基因TMPRSS6 SNP位点的试剂盒，其特征在于：所述的rs4820268位点扩增片段序列：即SEQ ID No 6。
8.根据权利要求1所述的一种检测铁调素基因TMPRSS6 SNP位点的试剂盒，其特征在于：所述试剂盒还包括PCR反应常用的酶和试剂，包含dNTPs、DNA聚合酶、Mg2+、PCR反应缓冲液，还含有用于检测分型使用的限制性内切酶及缓冲液。
9.一种检测铁调素基因TMPRSS6 SNP位点的试剂盒在孕期保健工作中预测孕期铁代谢能力和妊娠糖尿病检测中应用。
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