CN110257507B - 一种孕期叶酸代谢、钙代谢及h型高血压联合检测方法 - Google Patents
一种孕期叶酸代谢、钙代谢及h型高血压联合检测方法 Download PDFInfo
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Abstract
本发明涉及一种孕期叶酸代谢、钙代谢及H型高血压联合检测方法，利用生物信息学知识和相关生物信息学软件，对公开数据库中所能检索到的多个SNP位点(rs1051266、rs1544410、rs1801131、rs1801133、rs2234693、rs9340799、rs731236、rs7975232、rs1801394)序列信息进行同源性分析、周边SNP分析、频度分析、碱基含量分析及SNP周边序列分析后，设计特异的多重PCR引物系统及多重LDR探针系统并进行优化。本发明利用多重PCR‑LDR技术结合遗传分析仪的毛细管电泳技术进行片段长度分析实现SNP的检测分型，其优势在于通量大，特异性及灵敏度高，结果稳定，重复性好，检测速度快的优点。
Description
技术领域
本发明涉及分子生物学基因技术领域，涉及一种孕期叶酸代谢、钙代谢及H型高血压联合检测方法。
背景技术
(一)孕期叶酸代谢能力
孕妇叶酸代谢能力基因检测的意义：孕妇早期缺乏叶酸是引起胎儿神经管畸形的主要原因，叶酸缺乏可引起神经管未能闭合，从而导致以脊柱裂和无脑畸形等为主的神经管畸形。生育期女性补充叶酸可以有效的预防神经管畸形，由于个体遗传差异导致个体间对于叶酸的代谢能力存在明显的差异，导致对叶酸的利用在个体间存在明显差异，如果使用
0.4mg/d的统一剂量补充叶酸存在叶酸补充相对不足的状况发生，尤其是存在叶酸代谢能力较弱的风险型个体中，存在叶酸补充不足的状况。叶酸代谢能力检测用于筛选由于遗传差异导致叶酸代谢能力较差而畸形生育风险较高的已怀孕或即将怀孕妇女，给予适宜的个体化的叶酸剂量补充指导建议，更好的干预或预防神经管畸形。图1为孕期叶酸代谢的原理图。
(二)孕期钙代谢能力
孕期钙代谢能力基因检测的作用：孕期钙代谢能力基因检测是基于个体的遗传差异来筛选在孕期骨质疏松风险较高的女性。维生素D的补充还可以降低孕妇和胎儿后续各种疾病的风险，如降低妊娠高血压、剖腹产、产后出血的可能性，降低胎儿产后患佝偻病的风险等。但如果不注意，服用了过多的维生素D，会造成人体中毒。钙补多了，容易造成高钙血症，甚至导致肾结石。因此，根据个体化差异风险推荐进行差异化的钙质和维生素D的合理补充，更好的保障孕妇和胎儿的健康。图2为维生素D与骨代谢图，图3为雌激素与骨代谢简图，图4为雌激素缺乏与骨代谢。
(三)H型高血压叶酸需求
H型高血压叶酸需求基因检测的作用中国高血压人群H型高血压患者超过70％；高血压合并高同型半胱氨酸是我国脑卒中高发的重要致病原因。2015年的CSPPT研究显示，高血压人群服用降压药依那普利叶酸复合制剂可以显著的降低第一次脑卒中的发病率约20％。由于叶酸代谢能力个体间存在显著的差异，导致代谢能力弱的个体的同型半胱氨酸水平较高，补充相同剂量的叶酸无法满足所有H型高血压个体的需求，通过评估叶酸代谢能力，推荐个性化的叶酸补充剂量非常必要。H型高血压叶酸需求基因检测用于评估H型高血压人群的叶酸代谢能力，针对代谢能力偏弱的个体在降压治疗的同时进行有个体化性的叶酸补充建议，可以更有效的预防脑卒中及其他叶酸缺乏的心脑血管疾病等。图5为叶酸摄入、叶酸代谢酶与同型半胱氨酸Hcy代谢图，图6为高血压、叶酸摄入、叶酸代谢酶、同型半胱氨酸与脑卒中关系图。
目前检测多重SNP基因突变位点的方法有主要为经典测序技术、Taqman技术、SnpShot技术、Massarray技术及PCR-LDR技术等，各种技术各有其特点。
多重PCR-LDR检测技术的原理是利用只有在检测点处的碱基互补配对时，连接酶的连接反应才能进行这一特性，同时对样本中的多个SNP位点进行检测分型。对待检测的多个SNP位点在公开数据库中检索其序列信息后进行同源性分析、周边SNP分析、频度分析、碱基含量分析及SNP周边序列分析后，设计特异的多重PCR引物系统及多重LDR探针系统并进行优化，按样本抽提及质控——核心PCR扩增——核心LDR系统——高通量的信号检测的分析流程进行检测。
相对于传统的SnpShot技术、Taqman技术与测序技术，多重PCR-LDR技术完全可以取代这些技术，且在检测的效率、准确性及检测成本上都有优异表现；而相对Massyarray技术而言，多重PCR-LDR技术在SNP检测中对样本及PCR产物都无需纯化，既降低了操作的复杂性、又避免了更多的PCR污染的问题，因此其也具有独特的优势。
目前尚没有报道多重PCR-LDR技术专门用于孕期叶酸代谢、孕期钙代谢及H型高血压用药的突变位点联合检测。
发明内容
鉴于现有技术中所存在的问题，本发明公开了一种孕期叶酸代谢、钙代谢及H型高血压联合检测方法，采用的技术方案是，包括以下步骤：
Step.1样本基因组DNA的获取：用Qiagen DNA抽提试剂盒提取全血样本的基因组DNA；
Step.2试剂保存：将收到的试剂盒放置于-20℃的环境中保存，避免反复冻融；
Step.3PCR扩增：取PCR Mix 5.5ul，PCR引物2ul，Taq酶1.2ul，基因组DNA 2ul，补水至15ul；PC扩增条件：预变性，95℃，10min；94℃30s，56℃90s，72℃1min，循环40次；终延伸，72℃，10min；PCR引物包括VDR，ESR1，MTHFR，MTRR，SLC19A1基因序列，引物组具有如SEQID No：1-14所示的核苷酸序列；
Step.4LDR连接：取Ligase Mix 0.5ul，探针0.5ul，连接酶1ul，PCR产物2ul，补水至5ul；预变性94℃，1min；94℃15s，50℃25s，循环40次；探针组具有如SEQ ID No：15-41所示的核苷酸序列；
Step.5毛细管电泳检测：取HIDI8.9ul，分子量内标0.1ul和连接产物2ul混合，95℃变性10min，冰浴冷却后上遗传分析仪3130XL，送样电压3KV，上样时间15sec；
Step.6结果分析：使用GeneMapper ID V3.2软件对毛细管电泳结果进行分析，统计9个SNP位点的分型结果。
优选的，所述SLC19A1的SNP位点为rs1051266，突变位置NM_194255.2:c.80A>G(p.His27Arg；A80G)。
优选的，所述VDR的SNP位点为rs1544410，突变位置S4((NM_001017535.1:c.1024+283G>A))；rs731236，突变位置S2(NM_001017535.1:c.1056T>C)；rs7975232，突变位置S1(NM_001017535.1:c.1025-49G>T)。
优选的，所述ESR1的SNP位点为rs9340799，突变位置S1(NM_001122742.1:c.453-351A>G)；rs2234693，突变位置S2(NM_001122742.1:c.453-397T>C)。
优选的，所述MTHFR的SNP位点为rs1801133，突变位置NM_005957.4:c.665C>T(p.Ala222Val；C677T)；rs1801131，突变位置NM_005957.4:c.1286A>C(p.Glu429Ala；A1298C)。
优选的，所述MTRR的SNP位点为rs1801394，突变位置NM_002454.2:c.66A>G(p.Ile22Met；A66G)。
优选的，所述探针组中的SEQ ID No 15、No 18、No 21、No 24、No 27、No30、No 33、No 36、No 39为荧光标记探针。
孕妇叶酸代谢能力基因检测涉及MTHFR C677T位点、MTHFR A1298C位点(MTHFR S2位点)、MTRR A66G位点(MTRR S2位点)和SLC19A1(RFC1)A80G位点(SLC19A1S1位点)。
MTHFR C677T位点
5，10-亚甲基四氢叶酸还原酶(MTHFR)是甲硫氨酸-叶酸代谢中的一个关键酶，它将体内的5,10-亚甲基四氢叶酸还原为5-甲基四氢叶酸，同型半胱氨酸在5-甲基四氢叶酸以及维生素B12的参与下，生成活性甲基，进而直接提供给体内所需甲基的大分子。正常的MTHFR活性对于防止同型半胱氨酸积聚有重要作用，MTHFR基因突变造成MTHFR活性降低可使同型半胱氨酸专病为甲硫氨酸的过程出现障碍，造成叶酸水平降低以及高同型半胱氨酸血症。
677C>T多态性将MTHFR丙氨酸转变为缬氨酸，位于酶活性区域。杂合型酶活性降至65％，纯合型降至30％。
T等位基因的母亲比677C/C基因母亲生育Down综合征的患儿的风险高，与CC型野生型相比，TT基因型的中国女性生育神经管畸形的患儿的OR为3.35，表明携带TT基因型是孕妇生育神经管畸形患儿的危险因素；中国女性杂合基因型CT也是神经管畸形的遗传危险因素。携带T等位基因的父母，其子代患非综合性唇腭裂的危险性是不携带T等位基因父母的子代的2.420倍；母子都是TT突变纯合子，子代患非综合性唇腭裂的危险性是母子为非TT纯合子的4.162倍；母亲MTHFR基因C677T多态性与其子代先天性心脏病的发生有关，母亲携带纯合突变基因型TT是其子代先天性心脏病发生的重要危险因素。
MTHFR A1298C位点(MTHFR S2位点)
A1298C是MTHFR基因的又一多态性位点，这一位点突变将谷氨酸转变为丙氨酸，杂合型酶活性降至83％，纯合型降至61％。相对AA基因型，CC和CA基因型的中国女性生育NTDs患儿的风险较高。677C>T和1298A>C杂合型会出现更高的同型半胱氨酸和更低的血浆叶酸浓度。母亲677CT/1298AC基因型与677CC/1298AA型相比，子代出现非综合性唇腭裂的风险OR为4.43。
MTRR A66G位点(MTRR S2位点)
甲硫氨酸合酶还原酶(MTRR)是一种黄素蛋白相关的酶，能维持甲硫氨酸合成酶的活性状态，对维持体内甲硫氨酸循环起重要的调节作用。
该酶的基因存在66A→G的多态性，导致蛋氨酸被异亮氨酸替代，酶活性显著降低。这会导致甲硫氨酸合成酶活性降低，体内同型半胱氨酸堆积。母亲MTRRA66G突变时，生育Down综合征的风险增加，而同时还MTHFR基因677C-T突变者时，生育Down综合征患者的风险大大更高。
SLC19A1(RFC1)A80G位点(SLC19A1S1位点)
还原叶酸载体1(RFC1)与叶酸受体(FR)相互配合、协同作用，共同完成叶酸从组织到细胞内的转运。RFC1的基因点突变可引起叶酸转运缺陷，进而影响叶酸代谢RFC1对正在发育的胚胎，特别是叶酸穿过胎盘的运输有重要影响，因此RFC1的基因改变可能影响胎儿正常的组织发育。
RFC1GG基因型的母亲子代发生NTDs危险高于AA基因型；母亲孕早期不增补叶酸，生育神经管畸形的危险高于增补叶酸的母亲；母亲孕期未增补叶酸，母亲GG型和GA型的子代发生神经管畸形的风险比AA型高。
孕期钙代谢能力基因检测涉及VDR基因和ESR1基因。
VDR基因
维生素D是人体内钙稳态和骨代谢的主要调节因子之一。VDR基因的编码蛋白是介导1,25-(OH)2D3发挥生物学效应的维生素D受体。维生素D不直接作用于靶器官，而是通过与维生素D受体(VDR)结合发挥作用。VDR是由VDR基因编码，因此VDR基因是研究骨代谢性疾病遗传基础的重要基因之一。
VDR基因存在多个单核苷酸多态性位点，研究显示不同的VDR基因型与骨密度(bone mineral density,BMD)、骨量丢失和肠道钙吸收、和维生素D缺乏性佝偻病发生等密切相关。研究发现VDR基因BsmI、TaqI和ApaI限制性酶切多态性与孕妇的骨密度有明显相关性，发现携带有BBAAtt的孕妇个体骨密度最高，而bbaaTT个体中骨密度最低。
ESR1基因
ESR1基因编码雌激素受体1(ESR1)。这种核受体具有转录因子的活性，当被雌激素活化后，能与DNA结合，调控基因的转录。它的主要功能是促进性别和生殖功能发育，同时它也参与人体多种生理过程。
研究发现ESR1基因PvuII和XbaI限制性酶切多态性与孕妇的骨密度具有相关性，PP基因型携带者骨密度较高，pp型携带者骨密度较低；X基因型携带者的骨密度降低更加明显。携带者需要加强钙质和维生素D的补充，对可能患病风险进行更好的预防。
H型高血压叶酸需求基因检测涉及MTHFR C677T位点、MTHFR A1298C位点和MTRRA66G位点。
MTHFR C677T位点
5,10-亚甲基四氢叶酸还原酶(MTHFR)是甲硫氨酸-叶酸代谢中的一个关键酶，它将体内的5,10-亚甲基四氢叶酸还原为5-甲基四氢叶酸，同型半胱氨酸在5-甲基四氢叶酸以及维生素B12的参与下，生成活性甲基，进而直接提供给体内所需甲基的大分子。正常的MTHFR活性对于防止同型半胱氨酸积聚有重要作用，MTHFR基因突变造成MTHFR活性降低可使同型半胱氨酸专病为甲硫氨酸的过程出现障碍，造成叶酸水平降低以及高同型半胱氨酸血症。677C>T多态性将MTHFR的特定位置的丙氨酸转变为缬氨酸，位于酶活性区域。杂合型携带者酶活性降至65％，纯合型降至30％。
研究显示，TT等位基因型与CC或CT基因型相比较，与高水平的同型半管氨酸水平显著相关，TT型携带者的同型半胱氨酸水平是CC或CT型的约两倍；同样与CC型比较，Meta分析显示TT基因型也促进静脉血栓的形成和增加冠心病的发病风险。
MTHFR A1298C位点
A1298C是MTHFR基因的又一多态性位点，这一位点突变将谷氨酸转变为丙氨酸，杂合型酶活性降至83％，纯合型降至61％。研究显示在C677T等位基因型为CC的个体中，A1298C多态性中CC携带者的同型半胱氨酸水平显著的高于AA基因携带者。
MTRR A66G位点
甲硫氨酸合酶还原酶(MTRR)是一种黄素蛋白相关的酶，能维持甲硫氨酸合成酶的活性状态，对维持体内甲硫氨酸循环起重要的调节作用。该酶的基因存在66A→G的多态性，导致蛋氨酸被异亮氨酸替代，酶活性显著降低。这会导致甲硫氨酸合成酶活性降低，体内同型半胱氨酸堆积。
本发明的有益效果：本发明利用多重PCR-LDR技术结合遗传分析仪的毛细管电泳技术进行片段长度分析实现SNP的检测分型，其优势在于通量大，特异性及灵敏度高，结果稳定，重复性好，检测速度快的优点。
附图说明
图1为本发明的孕期叶酸代谢的原理图；
图2为本发明的维生素D与骨代谢图；
图3为本发明的雌激素与骨代谢简图；
图4为本发明的雌激素缺乏与骨代谢图；
图5为本发明的叶酸摄入、叶酸代谢酶与同型半胱氨酸Hcy代谢图；
图6为本发明的高血压、叶酸摄入、叶酸代谢酶、同型半胱氨酸与脑卒中关系图；
图7为本发明的电泳峰图；
图8为本发明的孕期叶酸代谢能力检测图；
图9为本发明的叶酸代谢能力评价与孕期叶酸剂量补充建议图；
图10为本发明的孕期钙代谢能力基因检测图；
图11为本发明的孕期钙代谢能力基因检测报告分析与用药建议；
图12为本发明的H型高血压叶酸需求基因检测；
图13为本发明的叶酸代谢能力评价与叶酸剂量补充建议。
具体实施方式
实施例1
本发明的一种孕期叶酸代谢、钙代谢及H型高血压联合检测方法，利用生物信息学知识和相关生物信息学软件，对公开数据库中所能检索到的多个SNP位点(rs1051266、rs1544410、rs1801131、rs1801133、rs2234693、rs9340799、rs731236、rs7975232、rs1801394)序列信息进行同源性分析、周边SNP分析、频度分析、碱基含量分析及SNP周边序列分析后，设计特异的多重PCR引物系统及多重LDR探针系统并进行优化。采用的方法包括以下步骤：
Step.1样本基因组DNA的获取：用Qiagen DNA抽提试剂盒提取全血样本的基因组DNA；
Step.2试剂保存：将收到的试剂盒放置于-20℃的环境中保存，避免反复冻融；Step.3PCR扩增：取PCR Mix 5.5ul，PCR引物2ul，Taq酶1.2ul，基因组DNA 2ul，补水至15ul，PCRMix含有dNTP,Mg2+；PC扩增条件：预变性，95℃，10min；94℃30s，56℃90s，72℃1min，循环40次；终延伸，72℃，10min；PCR引物如表1所示。
| SNP位点 | 序列 | SEQ ID No |
| rs1051266-F | AGCGGTGGAGAAGCAGGTG | 1 |
| rs1051266-R | GGTAGGGGGTGATGAAGCTC | 2 |
| rs1544410-F | AGTGTGCAGGCGATTCGTAG | 3 |
| rs1544410-R | AATGTTGAGCCCAGTTCACG | 4 |
| rs1801131-F | TGAAGAGCAAGTCCCCCAAG | 5 |
| rs1801131-R | CATTCCGGTTTGGTTCTCCC | 6 |
| rs1801133-F | CCCTATTGGCAGGTTACCCC | 7 |
| rs1801133-R | CGGAAGAATGTGTCAGCCTC | 8 |
| rs2234693&rs9340799-F | TGTTCTGTGTTGTCCATCAG | 9 |
| rs2234693&rs9340799-R | AGGAATATACAATTATTTCAGAACC | 10 |
| rs731236&rs7975232-F | GGATGTACGTCTGCAGTGTG | 11 |
| rs731236&rs7975232-R | TTGAGTGTCTGTGTGGGTGG | 12 |
| rs1801394-F | TCACTGTTACATGCCTTGAAGTG | 13 |
| rs1801394-R | TACAGTGAAGATCTGCAGAAAATC | 14 |
表1PCR引物序列
Step.4LDR连接：取Ligase Mix 0.5ul，探针0.5ul，连接酶1ul，PCR产物2ul，补水至5ul；预变性94℃，1min；94℃15s，50℃25s，循环40次；探针组具有如SEQ ID No：15-41所示的核苷酸序列；探针序列如表2所示，其中带有“*”号的为荧光标记探针。
表2探针组序列
Step.5毛细管电泳检测：取HIDI8.9ul，分子量内标0.1ul和连接产物2ul混合，95℃变性10min，冰浴冷却后上遗传分析仪3130XL，送样电压3KV，上样时间15sec；图7为电泳峰图。
Step.6结果分析：使用GeneMapper ID V3.2软件对毛细管电泳结果进行分析，统计9个SNP位点的分型结果。表3为基因分型结果。图8-图13为孕期叶酸代谢、钙代谢及H型高血压检测报告及建议。
| SNP位点 | 基因 | 所检突变位点位置 | 基因型 |
| rs1544410 | VDR | S4(NM_001017535.1:c.1024+283G>A) | GG |
| rs731236 | VDR | S2(NM_001017535.1:c.1056T>C) | AA |
| rs9340799 | ESR1 | S1(NM_001122742.1:c.453-351A>G) | AA |
| rs2234693 | ESR1 | S2(NM_001122742.1:c.453-397T>C) | AG |
| rs7975232 | VDR | S1(NM_001017535.1:c.1025-49G>T) | AC |
| rs1801133 | MTHFR | NM_005957.4:c.665C>T(p.Ala222Val；C677T) | CT |
| rs1801131 | MTHFR | NM_005957.4:c.1286A>C(p.Glu429Ala；A1298C) | AC |
| rs1801394 | MTRR | NM_002454.2:c.66A>G(p.Ile22Met；A66G) | AA |
| rs1051266 | SLC19A1 | NM_194255.2:c.80A>G(p.His27Arg；A80G) | AG |
表3基因分型结果
上述虽然对本发明的具体实施例作了详细说明，但是本发明并不限于上述实施例，在本领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下做出各种变化，而不具备创造性劳动的修改或变形仍在本发明的保护范围以内。
Claims (6)
1.一种检测叶酸代谢、钙代谢及H型高血压SNP位点的引物组，其特征在于，所述引物组包括VDR，ESR1，MTHFR，MTRR，SLC19A1基因SNP点位的引物组和探针组，引物组如SEQ ID No：1-14所示的核苷酸序列；探针组如SEQ ID No：15-41所示的核苷酸序列；
所述探针组中的SEQ ID No 15、SEQ ID No 18、SEQ ID No 21、SEQ ID No 24、SEQ IDNo 27、SEQ ID No 30、SEQ ID No 33、SEQ ID No 36、SEQ ID No 39为荧光标记探针；SEQID No 1：AGCGGTGGAGAAGCAGGTG；
SEQ ID No 2：GGTAGGGGGTGATGAAGCTC；
SEQ ID No 3：AGTGTGCAGGCGATTCGTAG；
SEQ ID No 4：AATGTTGAGCCCAGTTCACG；
SEQ ID No 5：TGAAGAGCAAGTCCCCCAAG；
SEQ ID No 6：CATTCCGGTTTGGTTCTCCC；
SEQ ID No 7：CCCTATTGGCAGGTTACCCC；
SEQ ID No 8：CGGAAGAATGTGTCAGCCTC；
SEQ ID No 9：TGTTCTGTGTTGTCCATCAG；
SEQ ID No 10：AGGAATATACAATTATTTCAGAACC；
SEQ ID No 11：GGATGTACGTCTGCAGTGTG；
SEQ ID No 12：TTGAGTGTCTGTGTGGGTGG；
SEQ ID No 13：TCACTGTTACATGCCTTGAAGTG；
SEQ ID No 14：TACAGTGAAGATCTGCAGAAAATC；
SEQ ID No 15：ATTTCTTCTGCGATGGCCTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 16：TTTTTTTTTTTTTTTTCATGTACCACAGCTTGCTCACAT；
SEQ ID No 17：TTTTTTTTTTTTTTTTTTCATGTACCACAGCTTGCTCACAC；
SEQ ID No 18：AGACCACACTCAGGGTCTCTTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 19：TTTTTTTTTTTTTTTTTTAGACCAATGCTCATCCCAACTCT；
SEQ ID No 20：TTTTTTTTTTTTTTTTTTTTAGACCAATGCTCATCCCAACTCC；
SEQ ID No 21：CTTCACTGGTCAGCTCCTCCTTTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 22：TTTTTTTTTTTTTTTTTTTTAACGAAGACTTCAAAGACACTTT；
SEQ ID No 23：TTTTTTTTTTTTTTTTTTTTTTAACGAAGACTTCAAAGACACTTG；
SEQ ID No 24：GTTTTATGCTTTGTCTCTGTTTTTTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 25：TTTTTTTTTTTTTTTTTTTTTTTCTGAGTTCCAAATGTCCCAGCT；
SEQ ID No 26：TTTTTTTTTTTTTTTTTTTTTTTTTCTGAGTTCCAAATGTCCCAGCC；
SEQ ID No 27：CTCCCGCAGACACCTTCTCCTTTTTTTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 28：TTTTTTTTTTTTTTTTTTTTTTTTAAGCTGCGTGATGATGAAATCGG；
SEQ ID No 29：TTTTTTTTTTTTTTTTTTTTTTTTTTAAGCTGCGTGATGATGAAATCGA；
SEQ ID No 30：ATCAGCGCGGCGTCCTGCACTTTTTTTTTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 31：TTTTTTTTTTTTTTTTTTTTTTTTTTACAGGCGGTCCTGGATGGCCTCG；
SEQ ID No 32：TTTTTTTTTTTTTTTTTTTTTTTTTTTTACAGGCGGTCCTGGATGGCCTCA；
SEQ ID No 33：GCAGGCCTGTCTGTGGCCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 34：TTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGAGCCTGAGTATTGGGAATGT；
SEQ ID No 35：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGAGCCTGAGTATTGGGAATGC；
SEQ ID No 36：GCCGCCAGGACCGGAGCTCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 37：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAGCAAAGGTAGCACACGAGGT；
SEQ ID No 38：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAGCAAAGGTAGCACACGAGGC；
SEQ ID No 39：GCCCAGCTGAGAGCTCCTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT；
SEQ ID No 40：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGGTGGGATTGAGCAGTGAGGT；
SEQ ID No 41：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGGTGGGATTGAGCAGTGAGGG。
2.根据权利要求1所述的一种用于检测叶酸代谢、钙代谢SNP点位的引物组，其特征在于：所述SLC19A1的SNP位点为rs1051266，突变位置NM_194255.2:c.80A>G(p.His27Arg；A80G)。
3.根据权利要求1所述的一种检测叶酸代谢、钙代谢及H型高血压SNP位点的引物组，其特征在于：所述VDR的SNP位点为rs1544410，突变位置S4:NM_001017535.1:c.1024+283G>A；rs731236，突变位置S2:NM_001017535.1:c.1056T>C；rs7975232，突变位置S1:NM_001017535.1:c.1025-49G>T。
4.根据权利要求1所述的一种检测叶酸代谢、钙代谢及H型高血压SNP位点的引物组，其特征在于：所述ESR1的SNP位点为rs9340799，突变位置S1:NM_001122742.1:c.453-351A>G；rs2234693，突变位置S2:NM_001122742.1:c.453-397T>C。
5.根据权利要求1所述的一种检测叶酸代谢、钙代谢及H型高血压SNP位点的引物组，其特征在于：所述MTHFR的SNP位点为rs1801133，突变位置NM_005957.4:c.665C>T(p.Ala222Val；C677T)；rs1801131，突变位置NM_005957.4:c.1286A>C(p.Glu429Ala；A1298C)。
6.根据权利要求1所述的一种检测叶酸代谢、钙代谢及H型高血压SNP位点的引物组，其特征在于：所述MTRR的SNP位点为rs1801394，突变位置NM_002454.2:c.66A>G(p.Ile22Met；A66G)。
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