CN113215241A - 一种用于心脑血管疾病风险预警的检测引物组及其应用 - Google Patents
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Abstract
本发明提出了一种用于心脑血管疾病风险预警的检测引物组，包括83个引物对，每个引物对包括一个正向引物和一个反向引物，所述引物组的核苷酸序列如SEQIDNO:1～166所示。本发明引物组可同时检测18种心脑血管相关的疾病，83个SNP位点，可以做到心脑血管相关疾病的早发现，早预警，避免漏诊，并能帮助医生筛选出有效的治疗方案，对患者做出最正确的治疗决策，实现发病前的精准预警和发病后的精准用药。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种用于心脑血管疾病风险预警的检测引物组及其应用。
背景技术
基因是遗传的基本单元，携带有遗传信息的DNA或RNA序列，通过复制，把遗传信息传递给下一代，指导蛋白质的合成来表达自己所携带的遗传信息，从而控制生物个体的性状表达。资料证实10％～15％的癌症与遗传有关，糖尿病、心脑血管疾病等多种疾病都与遗传因素有关。单核苷酸多态性(SNP)，主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。个体携带的基因单核苷酸多态性(SNP)不同，其发病风险也不同，对药物的反应也各不相同。因此，通过个体基因单核苷酸多态性位点(SNP)的检测，可以做到疾病的早发现，早预警，并且能帮助医生筛选出有效的治疗方案，对患者做出最正确的治疗决策，实现发病前的精准预警和发病后的精准用药。
心脑血管相关疾病较多，现有的检测引物仅针对4种或7种心脑血管相关的疾病，对应的SNP位点也较少，不能对心脑血管疾病风险预警的大范围覆盖，容易出现漏诊，错过疾病的最佳治疗期。因此现有技术缺少一种特异性好、可实现多种心脑血管相关疾病以及多个SNP位点同时检测的引物组，这已成为了急需解决的问题。
发明内容
有鉴于此，本发明提出了一种可实现心脑血管疾病83个位点同时检测的引物组。
本发明的技术方案是这样实现的：一种用于心脑血管疾病风险预警的检测引物组，包括83个引物对，每个引物对包括一个正向引物和一个反向引物，所述引物组的核苷酸序列如SEQ ID NO:1～166所示。
一种用于心脑血管疾病风险预警的检测引物组的使用方法，包括如下步骤：
S1，提取样本DNA，取受试者的新鲜唾液，用QIAGEN试剂盒提取样本中的基因组DNA，然后用超声打断仪将基因组DNA破碎至500bp以下，并将打断后的DNA进行Q-Sep检测；
S2，文库构建，进行两步PCR扩增，PCR1扩增：选取引物组的全部引物对进行扩增，扩增程序为：94℃激活3min，94℃变性15sec，65℃退火20sec，72℃延伸15sec，变性-退火-延伸程序重复循环12次；72℃猝灭5min，4℃永久；PCR1扩增结束后，利用磁珠进行PCR1产物纯化；PCR2扩增：PCR1产物添加index标签序列，同时扩增，扩增程序为94℃激活3min，94℃变性15sec，62℃退火10sec，72℃延伸10sec，变性-退火-延伸程序重复循环10次；72℃5min，4℃永久；PCR2扩增结束后，利用磁珠进行PCR2产物分选与纯化；
S4，高通量测序：将所得的DNA文库通过测序平台进行测序，并对测序数据结果分析。
所述引物组中的每条引物在所述PCR1中的终浓度为0.1μmol/L。
一种用于心脑血管疾病风险预警的检测试剂盒，包括上述引物组。
一种用于心脑血管疾病风险预警的检测试剂盒在检测心脑血管疾病中的应用。
一种用于心脑血管疾病风险预警的检测试剂盒在制备治疗心脑血管疾病药品中的应用。
本发明的一种用于心脑血管疾病风险预警的检测引物组及其应用相对于现有技术具有以下有益效果：
(1)本发明引物组可同时检测18种心脑血管相关的疾病可以做到心脑血管疾病的早发现，早预警；对患病个体，帮助医生筛选出有效的治疗方案，对患者做出最正确的治疗决策，而且提高了治疗效果。
(2)本发明所述的引物组的各个引物之间相互干扰小，特异性强，扩增效果好，只需要2步扩增即可完成83个位点检测。
(3)本发明所述的引物组合物简化了操作步骤，可一次性进行双轮多重PCR，降低了实验成本，且具有快速、简便、准确性高、重复性好的优点。
(4)本发明所述的引物可以连接不同测序平台的Index，满足在不同测序平台上测序的要求。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例二的DNA质检图；
图2为本发明实施例四的文库质检图。
具体实施方式
下面将结合本发明实施方式，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本发明一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，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方式，都属于本发明保护的范围。
实施例一 引物组
1.引物设计
本发明针对心脑血管疾病设计的引物组覆盖83个目标位点，序列见表1。
表1引物组对应的SNP位点及其核苷酸序列
2.稀释Xinnao引物组混合物：①将每条引物的干粉按要求稀释至100μmol/L；②将各引物混合成引物混合液，其中每条引物的终浓度为0.1μmol/L。
实施例二 样本DNA提取
1.取样：采集受试者的新鲜唾液，受试者在取样前30min用清水漱口，期间不可饮水、进食等，30min后开始采集唾液，具体做法：用牙齿轻轻刮蹭口腔黏膜，少量多次的采集2ml新鲜唾液，与2ml唾液保存液均匀混合后，于常温保存待用。
2.提取样本DNA：每个样本取200μl，用QIAGEN试剂盒提取样本中的基因组DNA；保存于-20℃待用。
3.破碎基因组DNA：用超声打断仪将受试者的基因组DNA破碎至500bp以下，破碎条件为：20s ON；30s OFF；14cycle。
4.质控检测：将打断后的DNA进行Q-Sep检测，质检显示DNA片段的98％均在20-500bp内，满足建库要求。DNA质检图见附图中的图1。
实施例三 基因文库构建
1.第一轮PCR反应
按照以下体系配制PCR反应液，进行PCR反应：
表2 PCR1反应试剂
2.磁珠纯化：S1，将第一轮PCR反应产物冷却至25℃，加入1.0X BufferYC磁珠，混匀后静置5min；S2，将上述反应液短暂离心后置于磁力架上，待5min后，弃上清；S3，加入200μl 80％乙醇，漂洗磁珠，30s后，弃乙醇；S4，重复步骤S3一次；S5，将PCR管置于室温下，待磁珠干燥后，加入10.5μl ddH2O。混匀后静置5min；S6，将PCR管短暂离心后，置于磁力架上，待5min后，吸取9.5μl上清液至新的PCR管中。
3.第二轮PCR：使用具有不同index标记的第二轮引物，以第一轮纯化产物为模板进行扩增，按照以下体系配制PCR反应液，进行PCR反应：
表3 PCR2反应试剂
4.Buffer YC磁珠双轮片段筛选：S1，将第二轮PCR反应产物加入ddH2O补充至50μl，加入0.65X Buffer YC磁珠，混匀后，于室温静置5min；S2，将反应液置于磁力架上，待5min后吸取上清新的PCR管中；S3，在新的PCR管中加入0.2X Buffer YC磁珠，与反应液混匀，于25℃静置5min；S4，将反应液短暂离心后置于磁力架上，待5min后弃上清液；S5，加入200μl 80％乙醇漂洗磁珠，30s后弃乙醇；S6，重复步骤S5一次；S7，将PCR管置于室温，待磁珠干燥后加入11μl ddH2O，混匀后静置5min；S8，将PCR管短暂离心后置于磁力架上，待5min后，将上清液吸至新的PCR管中。
实施例四 质控检测
1.Qubit荧光定量仪检测结果，用Qubit 3.0测量文库浓度，结果如下表所示：
表4荧光定量仪检测结果
| 样本编号 | 浓度(ng/μl) | 体积(μl) | 总量(ng) |
| 1 | 3.85 | 10 | 38.50 |
2.Q-sep质检
对以上构建好的样本文库的进行Q-sep质检，质检结果显示均扩增出目的片段，文库满足测序要求。文库质检图见图2。
实施例五 测序
将样本的文库定量混合进行illumina测序，通过5’端添加的Index标签序列对测序数据进行区分，由此能够在满足检测要求的同时节约成本。得到相关数据，进行基因型分型，针对基因检测结果可以给出个体基因检测解读报告。
以上所述仅为本发明的较佳实施方式而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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<213> 人工序列(Artificial Sequence)
<400> 11
gtctcgtggg ctcggagatg tgtataagag acagaaggtt cggagaagtt tgca 54
<210> 12
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tcgtcggcag cgtcagatgt gtataagaga cagcactatg tgtggcacgg atccg 55
<210> 13
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gtctcgtggg ctcggagatg tgtataagag acagtagaca acatacgacc actct 55
<210> 14
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tcgtcggcag cgtcagatgt gtataagaga cagatagcta gcacgaaggc cccg 54
<210> 15
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gtctcgtggg ctcggagatg tgtataagag acagctactt tcgggccggt tacc 54
<210> 16
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tcgtcggcag cgtcagatgt gtataagaga cagcttgggt ggtgttaact gct 53
<210> 17
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gtctcgtggg ctcggagatg tgtataagag acagcttggg taagccactg ctca 54
<210> 18
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tcgtcggcag cgtcagatgt gtataagaga caggtgataa tcaagagatg tcgaa 55
<210> 19
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gtctcgtggg ctcggagatg tgtataagag acagcatcgg gccgacctta gtgac 55
<210> 20
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tcgtcggcag cgtcagatgt gtataagaga cagtcctgtg taacctggct tcg 53
<210> 21
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gtctcgtggg ctcggagatg tgtataagag acagacaaac cttggtaatg tctaatgt 58
<210> 22
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tcgtcggcag cgtcagatgt gtataagaga cagagcaaac ctcaaacatt aatggg 56
<210> 23
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gtctcgtggg ctcggagatg tgtataagag acaggtttca cccaccgaca acgac 55
<210> 24
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tcgtcggcag cgtcagatgt gtataagaga cagactgtcg ctttctagaa ttctaa 56
<210> 25
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gtctcgtggg ctcggagatg tgtataagag acagttccta ccgcccctca gatgc 55
<210> 26
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tcgtcggcag cgtcagatgt gtataagaga cagagcagcc gtcatctgtt taata 55
<210> 27
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gtctcgtggg ctcggagatg tgtataagag acaggggtgt ggtcattccg gtca 54
<210> 28
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tcgtcggcag cgtcagatgt gtataagaga caggacatgg ctagactcca cgc 53
<210> 29
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gtctcgtggg ctcggagatg tgtataagag acagtatcaa agagttgcgg ccggc 55
<210> 30
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
tcgtcggcag cgtcagatgt gtataagaga caggtctgtc accaatgtcc tag 53
<210> 31
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gtctcgtggg ctcggagatg tgtataagag acagtggcat cgagtagtcc tatt 54
<210> 32
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tcgtcggcag cgtcagatgt gtataagaga cagagcgata tgggactgat cact 54
<210> 33
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gtctcgtggg ctcggagatg tgtataagag acaggaagga tccgttagct ccagga 56
<210> 34
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tcgtcggcag cgtcagatgt gtataagaga cagatcgttt caaagccggg taa 53
<210> 35
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gtctcgtggg ctcggagatg tgtataagag acagataccc gaagtagagc tacaa 55
<210> 36
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
tcgtcggcag cgtcagatgt gtataagaga cagtcttgct tacctctgag agt 53
<210> 37
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gtctcgtggg ctcggagatg tgtataagag acagttccaa gccacttgtc ctt 53
<210> 38
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
tcgtcggcag cgtcagatgt gtataagaga cagtgacagt ctagagaacg cgc 53
<210> 39
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gtctcgtggg ctcggagatg tgtataagag acagaggcac gatggtcatg tagaa 55
<210> 40
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
tcgtcggcag cgtcagatgt gtataagaga caggtctggt gtgataatgt agcctg 56
<210> 41
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
gtctcgtggg ctcggagatg tgtataagag acaggagctc caaacttaca ttcccca 57
<210> 42
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
tcgtcggcag cgtcagatgt gtataagaga caggcttgtc atgattcatt ccggtg 56
<210> 43
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gtctcgtggg ctcggagatg tgtataagag acagtggtga gtggttcggc tgtt 54
<210> 44
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
tcgtcggcag cgtcagatgt gtataagaga cagtgaaagc ggacacatag tgct 54
<210> 45
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gtctcgtggg ctcggagatg tgtataagag acagtgtagt acccagaaca gcggc 55
<210> 46
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
tcgtcggcag cgtcagatgt gtataagaga cagcgactcc tgcaaacttc ggca 54
<210> 47
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
gtctcgtggg ctcggagatg tgtataagag acagacaatg tggaagtttg gtgttccac 59
<210> 48
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
tcgtcggcag cgtcagatgt gtataagaga cagactttcc cgcctatact ttgacga 57
<210> 49
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
gtctcgtggg ctcggagatg tgtataagag acaggccagt gaataagagc ctgga 55
<210> 50
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
tcgtcggcag cgtcagatgt gtataagaga cagagtgaat ggtactcatc tggcgatcg 59
<210> 51
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
gtctcgtggg ctcggagatg tgtataagag acagacccca acttggtctc ttagg 55
<210> 52
<211> 53
<212> DNA
<213> 人工序列(artificial sequence)
<400> 52
tcgtcggcag cgtcagatgt gtataagaga cagctgtgca ctccgagagc acc 53
<210> 53
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
gtctcgtggg ctcggagatg tgtataagag acaggtctca gctcacgatc acacta 56
<210> 54
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
tcgtcggcag cgtcagatgt gtataagaga cagtttccgt agctgcctgt atca 54
<210> 55
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
gtctcgtggg ctcggagatg tgtataagag acagcagcaa agcgtgatca caccg 55
<210> 56
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
tcgtcggcag cgtcagatgt gtataagaga cagccccata gttcctccta ggt 53
<210> 57
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
gtctcgtggg ctcggagatg tgtataagag acagccattg tgctcggcca at 52
<210> 58
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
tcgtcggcag cgtcagatgt gtataagaga caggacccac gcggaaactt atcc 54
<210> 59
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
gtctcgtggg ctcggagatg tgtataagag acagcaggca ctccccatta gtct 54
<210> 60
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
tcgtcggcag cgtcagatgt gtataagaga caggtgtaca taggctgatg cgg 53
<210> 61
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
gtctcgtggg ctcggagatg tgtataagag acagctgaag ggacagctcc ac 52
<210> 62
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
tcgtcggcag cgtcagatgt gtataagaga caggactcca gcgcgtcaag g 51
<210> 63
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
gtctcgtggg ctcggagatg tgtataagag acagaggatg ggcctcaatc tcaatca 57
<210> 64
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
tcgtcggcag cgtcagatgt gtataagaga cagttctcca ccaacaaccc gagt 54
<210> 65
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
gtctcgtggg ctcggagatg tgtataagag acagtgggaa acaagagacg gacc 54
<210> 66
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
tcgtcggcag cgtcagatgt gtataagaga cagagtaccc cgggacactg c 51
<210> 67
<211> 54
<212> DNA
<213> 人工序列(artificial sequence)
<400> 67
gtctcgtggg ctcggagatg tgtataagag acagcccccg cttcttcatt gggg 54
<210> 68
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
tcgtcggcag cgtcagatgt gtataagaga cagtccagct cctaggtccg acc 53
<210> 69
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
gtctcgtggg ctcggagatg tgtataagag acagaggtca ttggaagctg gcgcg 55
<210> 70
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
tcgtcggcag cgtcagatgt gtataagaga caggcctgtc ttcccattgt ggt 53
<210> 71
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
gtctcgtggg ctcggagatg tgtataagag acagcagtct caggtgtgcc gcaca 55
<210> 72
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
tcgtcggcag cgtcagatgt gtataagaga caggccaggg ttcagacacg cac 53
<210> 73
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
gtctcgtggg ctcggagatg tgtataagag acagagaaca cacgcagctt cggt 54
<210> 74
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
tcgtcggcag cgtcagatgt gtataagaga cagaattaac atcgccgtca actt 54
<210> 75
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
gtctcgtggg ctcggagatg tgtataagag acagagtgtc accaaccacc tgattc 56
<210> 76
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
tcgtcggcag cgtcagatgt gtataagaga cagtggctgt tgcagtaact tt 52
<210> 77
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
gtctcgtggg ctcggagatg tgtataagag acagacaaag gttccaaaag cgcca 55
<210> 78
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
tcgtcggcag cgtcagatgt gtataagaga cagtccagca gaggtcttac ataat 55
<210> 79
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
gtctcgtggg ctcggagatg tgtataagag acagtgccac agcatagtca ctaag 55
<210> 80
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
tcgtcggcag cgtcagatgt gtataagaga caggcaaatg cctttgtacc atttcg 56
<210> 81
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
gtctcgtggg ctcggagatg tgtataagag acagtgtgaa cgaggtaggt accc 54
<210> 82
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
tcgtcggcag cgtcagatgt gtataagaga cagcaggcct gcccattacc tcat 54
<210> 83
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
gtctcgtggg ctcggagatg tgtataagag acaggcccct cgtatagctc tgtat 55
<210> 84
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
tcgtcggcag cgtcagatgt gtataagaga cagtgtacag tgcctggtaa ctcc 54
<210> 85
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
gtctcgtggg ctcggagatg tgtataagag acaggaaccg ctgcaacaat gagg 54
<210> 86
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
tcgtcggcag cgtcagatgt gtataagaga cagtgcaacc ccctatcaac agaaa 55
<210> 87
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
gtctcgtggg ctcggagatg tgtataagag acagaatggc agcacaattg gtaat 55
<210> 88
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
tcgtcggcag cgtcagatgt gtataagaga cagcctcagc agccgcctta ag 52
<210> 89
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
gtctcgtggg ctcggagatg tgtataagag acagtactac cagcggccct ccc 53
<210> 90
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
tcgtcggcag cgtcagatgt gtataagaga cagttttact cttttgccac cgca 54
<210> 91
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
gtctcgtggg ctcggagatg tgtataagag acaggccttg gtggtacttg cgttg 55
<210> 92
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
tcgtcggcag cgtcagatgt gtataagaga cagccaggtc cccatgctac ctaga 55
<210> 93
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
gtctcgtggg ctcggagatg tgtataagag acagatgatg ctgtctgccg acc 53
<210> 94
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
tcgtcggcag cgtcagatgt gtataagaga cagggcgtca tcctcctttc gca 53
<210> 95
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
gtctcgtggg ctcggagatg tgtataagag acagggaagt ggacgtaggt ctcg 54
<210> 96
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
tcgtcggcag cgtcagatgt gtataagaga cagctgtaca gggcctgcta gtgg 54
<210> 97
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 97
gtctcgtggg ctcggagatg tgtataagag acagtcagca aaccacaaca gctaca 56
<210> 98
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 98
tcgtcggcag cgtcagatgt gtataagaga cagacatcct gagtgctagc catc 54
<210> 99
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 99
gtctcgtggg ctcggagatg tgtataagag acagcccttt ccatcttgga taga 54
<210> 100
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 100
tcgtcggcag cgtcagatgt gtataagaga cagtgagctt gcccatcatc agga 54
<210> 101
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 101
gtctcgtggg ctcggagatg tgtataagag acagtgcgtc ttctccttat gacaaat 57
<210> 102
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 102
tcgtcggcag cgtcagatgt gtataagaga cagtggcaaa atttttgcac acccc 55
<210> 103
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 103
gtctcgtggg ctcggagatg tgtataagag acaggcatgt ttgctttcag ggtgcat 57
<210> 104
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 104
tcgtcggcag cgtcagatgt gtataagaga cagagcacag tgtaggggaa agaac 55
<210> 105
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 105
gtctcgtggg ctcggagatg tgtataagag acagcgagga tgtactgtcc atactcgt 58
<210> 106
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 106
tcgtcggcag cgtcagatgt gtataagaga cagcatgccc agccagtata catg 54
<210> 107
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 107
gtctcgtggg ctcggagatg tgtataagag acaggagagg cgaggaagtc ctc 53
<210> 108
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 108
tcgtcggcag cgtcagatgt gtataagaga cagcagagga tgggcacatt cat 53
<210> 109
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 109
gtctcgtggg ctcggagatg tgtataagag acagacagaa ccacagagtg ctgt 54
<210> 110
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 110
tcgtcggcag cgtcagatgt gtataagaga cagaccaagc ctattgatgg tgttctt 57
<210> 111
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 111
gtctcgtggg ctcggagatg tgtataagag acagatgtgt tgcttgtcgc ttaac 55
<210> 112
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 112
tcgtcggcag cgtcagatgt gtataagaga caggagatgg gcatcctgtg accg 54
<210> 113
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 113
gtctcgtggg ctcggagatg tgtataagag acaggtgcct acacagggta gggg 54
<210> 114
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 114
tcgtcggcag cgtcagatgt gtataagaga cagtgtgctt atctgcaggg acg 53
<210> 115
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 115
gtctcgtggg ctcggagatg tgtataagag acagtctccc accctactcc atctt 55
<210> 116
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 116
tcgtcggcag cgtcagatgt gtataagaga cagtctgtgc ttgttcaaca gcga 54
<210> 117
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 117
gtctcgtggg ctcggagatg tgtataagag acagagcctt tcgactctgt tgcc 54
<210> 118
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 118
tcgtcggcag cgtcagatgt gtataagaga cagtggccat gtgatgctga taa 53
<210> 119
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 119
gtctcgtggg ctcggagatg tgtataagag acagcagcag tgacaggcca cagt 54
<210> 120
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 120
tcgtcggcag cgtcagatgt gtataagaga cagtggatct gagtcatgac tgc 53
<210> 121
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 121
gtctcgtggg ctcggagatg tgtataagag acagctctgg ttccagcata gtga 54
<210> 122
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 122
tcgtcggcag cgtcagatgt gtataagaga cagggcagtt gtagaatttc tagaa 55
<210> 123
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 123
gtctcgtggg ctcggagatg tgtataagag acagtggtgc atgctgtagg ccc 53
<210> 124
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 124
tcgtcggcag cgtcagatgt gtataagaga caggggaggc ctctatgggt tta 53
<210> 125
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 125
gtctcgtggg ctcggagatg tgtataagag acagttccac aagaactcag tcacaac 57
<210> 126
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 126
tcgtcggcag cgtcagatgt gtataagaga cagacccaat gtatcagcag caagt 55
<210> 127
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 127
gtctcgtggg ctcggagatg tgtataagag acaggtgtgt gtgtgtgtgt gtgt 54
<210> 128
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 128
tcgtcggcag cgtcagatgt gtataagaga cagtggtgtt ggcccagact g 51
<210> 129
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 129
gtctcgtggg ctcggagatg tgtataagag acagagagag ttggagtcaa gacc 54
<210> 130
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 130
tcgtcggcag cgtcagatgt gtataagaga cagtttgcaa gtgttcttta aagccgtgg 59
<210> 131
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 131
gtctcgtggg ctcggagatg tgtataagag acagaggaag tagctctggc tacc 54
<210> 132
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 132
tcgtcggcag cgtcagatgt gtataagaga cagactgtga tgctggtctg cat 53
<210> 133
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 133
gtctcgtggg ctcggagatg tgtataagag acagggagga ctcccatcac tctc 54
<210> 134
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 134
tcgtcggcag cgtcagatgt gtataagaga cagtaaacag tcagtccgcg gtc 53
<210> 135
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 135
gtctcgtggg ctcggagatg tgtataagag acaggtgcac acaccctaag acctc 55
<210> 136
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 136
tcgtcggcag cgtcagatgt gtataagaga caggctgttg gtgcagtgaa ggt 53
<210> 137
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 137
gtctcgtggg ctcggagatg tgtataagag acagtgcaga gtgcccactg atgg 54
<210> 138
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 138
tcgtcggcag cgtcagatgt gtataagaga cagtcttgaa cctggcagac ttt 53
<210> 139
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 139
gtctcgtggg ctcggagatg tgtataagag acagtgagag ctgcttctat gaacca 56
<210> 140
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 140
tcgtcggcag cgtcagatgt gtataagaga cagatcccca ataacacagc catc 54
<210> 141
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 141
gtctcgtggg ctcggagatg tgtataagag acagaatcag tgcaacctag aatgca 56
<210> 142
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 142
tcgtcggcag cgtcagatgt gtataagaga cagtcatatg gtggaatcac aaaactc 57
<210> 143
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 143
gtctcgtggg ctcggagatg tgtataagag acagccaccg gggatgatga tcattc 56
<210> 144
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 144
tcgtcggcag cgtcagatgt gtataagaga cagaacacac accagctctc aaa 53
<210> 145
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 145
gtctcgtggg ctcggagatg tgtataagag acagactttg tgtagtatgg acgtta 56
<210> 146
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 146
tcgtcggcag cgtcagatgt gtataagaga cagtggtgga cgccgtatca ttgga 55
<210> 147
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 147
gtctcgtggg ctcggagatg tgtataagag acagttatgt tggaactgct aagcag 56
<210> 148
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 148
tcgtcggcag cgtcagatgt gtataagaga caggctgtgc acagagacat aacat 55
<210> 149
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 149
gtctcgtggg ctcggagatg tgtataagag acagtctctt cccccagata ccga 54
<210> 150
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 150
tcgtcggcag cgtcagatgt gtataagaga cagactttat gccaagcaca caact 55
<210> 151
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 151
gtctcgtggg ctcggagatg tgtataagag acagacatct agttcatact ccagtcaaa 59
<210> 152
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 152
tcgtcggcag cgtcagatgt gtataagaga cagacttgaa tagtgtgtct gctgctg 57
<210> 153
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 153
gtctcgtggg ctcggagatg tgtataagag acagaccagg actacccaaa agg 53
<210> 154
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 154
tcgtcggcag cgtcagatgt gtataagaga cagggaaagg ggccacatgt ggg 53
<210> 155
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 155
gtctcgtggg ctcggagatg tgtataagag acaggtgcag aggactggat gccc 54
<210> 156
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 156
tcgtcggcag cgtcagatgt gtataagaga caggggcagg ggagtgactg at 52
<210> 157
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 157
gtctcgtggg ctcggagatg tgtataagag acagtgagcc ctccaagtct ctatga 56
<210> 158
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 158
tcgtcggcag cgtcagatgt gtataagaga cagcacattt gcagtggctt tggtaa 56
<210> 159
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 159
gtctcgtggg ctcggagatg tgtataagag acagacacag gacgtctcac catga 55
<210> 160
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 160
tcgtcggcag cgtcagatgt gtataagaga cagaaaagtg tggcccaacc ctg 53
<210> 161
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 161
gtctcgtggg ctcggagatg tgtataagag acagctagct gggatcaccg cacc 54
<210> 162
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 162
tcgtcggcag cgtcagatgt gtataagaga cagtcctggg ttacaagcaa aaca 54
<210> 163
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 163
gtctcgtggg ctcggagatg tgtataagag acagtccttc atggcagaaa ggctt 55
<210> 164
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 164
tcgtcggcag cgtcagatgt gtataagaga cagttgccat gtttgtctcg ggt 53
<210> 165
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 165
gtctcgtggg ctcggagatg tgtataagag acagtgtgtg tgtgtgtgtg tgtg 54
<210> 166
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 166
tcgtcggcag cgtcagatgt gtataagaga cagtggaggg aatgggagtt gtgac 55
Claims (6)
1.一种用于心脑血管疾病风险预警的检测引物组，其特征在于，包括83个引物对，每个引物对包括一个正向引物和一个反向引物，所述引物组的核苷酸序列如SEQ ID NO:1～166所示。
2.如权利要求1所述的一种用于心脑血管疾病风险预警的检测引物组的使用方法，包括如下步骤：
S1，提取样本DNA，取受试者的新鲜唾液，用QIAGEN试剂盒提取样本中的基因组DNA，然后用超声打断仪将基因组DNA破碎至500bp以下，并将打断后的DNA进行Q-Sep检测；
S2，文库构建，进行两步PCR扩增，PCR1扩增：选取引物组的全部引物对进行扩增，扩增程序为：94℃激活3min，94℃变性15sec，65℃退火20sec，72℃延伸15sec，变性-退火-延伸程序重复循环12次；72℃5min，4℃永久；PCR1扩增结束后，利用磁珠进行PCR1产物纯化；PCR2扩增：PCR1产物添加index标签序列，同时扩增，扩增程序为94℃激活3min，94℃变性15sec，62℃退火10sec，72℃延伸10sec，变性-退火-延伸程序重复循环10次；72℃猝灭5min，4℃永久；PCR2扩增结束后，利用磁珠进行PCR2产物分选与纯化；
S4，高通量测序：将所得的DNA文库通过测序平台进行测序，并对测序数据结果分析。
3.如权利要求2所述的一种用于心脑血管疾病风险预警的检测引物组的使用方法，其特征在于，所述引物组中的每条引物在所述PCR1中的终浓度为0.1μmol/L。
4.一种用于心脑血管疾病风险预警的检测试剂盒，其特征在于，包括权利要求1所述的引物组。
5.如权利要求4所述的一种用于心脑血管疾病风险预警的检测试剂盒在检测心脑血管疾病中的应用。
6.如权利要求4所述的一种用于心脑血管疾病风险预警的检测试剂盒在制备治疗心脑血管疾病药品中的应用。
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