CN111808943B - 一种精神类个体化用药基因检测方法 - Google Patents
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Abstract
本发明提供一种精神类个体化用药基因检测方法，通过分析NCBI数据库和临床上精神病人常用药物的使用和代谢及药效的个体差异，筛选出与41种抗精神病药物密切相关基因的32个多态性位点，并制备出检测该32个多态性位点的引物组合物和使用该组合物的检测试剂。通过二代测序解决了现有技术中存在的检测精神病用药基因位点时灵敏度低、操作复杂、成本高等问题。
Description
技术领域
本发明应用于基因检测技术领域，具体涉及一种精神类个体化用药基因检测方法。
背景技术
最近几年，精神类疾病已逐渐成为影响人类健康和正常学习生活的重大疾病之一，至今 为止，精神病患者几乎占了全球总人数的1％，其在我国的发病率约为17％，并呈逐年上升 的趋势。精神病不仅在思维和认知层面表现异常，还伴有冲动、攻击行为、活动过度、管理 困难等一系列行为异常，由于其自身的复杂性，精神病已经成为社会巨大的医疗负担。目前 控制精神障碍的主要手段是药物治疗，但是药物治疗存在较为明显的个体差异，主要表现为 无明显疗效或者疗效不稳定，甚至引发一系列的不良反应，如肝功能受损、粒细胞减少、锥 体外系反应(EPS)、代谢内分泌失调等等。临床数据表明，在精神分裂症患者中，约有1/3～ 1/2的患者对典型和非典型抗精神病药的反应不佳；此外，在抑郁症的治疗中，仅有不到45％ 的患者在足量足疗程的抗抑郁药物治疗下临床症状能完全缓解。而导致这些疗效差异和不良 反应的主要原因可能与体内代谢酶、受体或者转运体的突变有关。例如药物氯氮平，它是非 典型抗精神病药物的先导，在众多抗精神病药物使用中占有较高比例，特别对难治性精神病 患者使用率更高。氯氮平的疗效范围较宽，不同个体对氯氮平的临床反应表现出较大的差异， 主要是因为细胞色素P450 1A2(CYP1A2)酶的活性存在较大的个体差异，此酶主要催化氯氮 平的去甲基代谢生成去甲氯氮平，对其体内代谢起主要作用，研究表明它的基因位点突变会 导致精神分裂症患者对氯氮平的临床疗效降低；还有研究显示中国汉族精神分裂症患者的 CYP2D6的基因多态性与新型非典型抗精神病药物利培酮及其代谢物9-羟基利培酮血药浓度 有一定相关性，进而对利培酮的药效产生影响。
因此，利用基因检测手段，明确个体的相关药物代谢酶的基因多态性，依据个体基因特 性指导用药和给出相应的治疗方案，最终实现由“对症下药”到“对人下药”即个体化用药 的转变，这对提高药效和减少药物不良反应事件发生具有极其重要的作用，且具有一定的社 会意义和经济意义。
目前针对基因多态性检测的技术很多，普遍使用的技术包括基于Tm值和溶解曲线的荧 光定量PCR法和一代测序等等，这些技术有的操作繁琐，有的无法实现多位点的同时检测， 使得目前在临床上对于多态性检测技术应用仍不理想，一直无法满足临床检测需求。随着高 通量测序技术的发展，二代测序(NGS)越来越多的被应用基因检测项目，第二代测序技术 以其可高效率地完成大量序列的检测，同时精度可以达到99.99％。二代测序技术在保证检测 精度的同时还可以一次对成千上万个样本进行处理。
发明内容
本发明针对现有技术中存在的检测精神病用药基因位点时灵敏度低、操作复杂、成本高 等问题，提供一种精神类个体化用药基因检测方法。本发明基于二代测序技术，具有高灵敏 度、低成本、操作简单、通量高等特点，可以同时一次性检测32个相关基因位点。
本发明解决其技术问题是采用以下技术方案实现的：
一种精神类个体化用药基因检测方法，包括以下步骤：
1)取含有EDTA抗凝剂血液样本进行核酸提取；
2)预混CYP2D6(rs1080985、rs35742686、rs3892097、rs5030655)、ABCB1(rs1128503)、 UGT2B7(rs12233719)、CYP2C19(rs12248560、rs4244285、rs4986893)、EPM2A(rs1415744)、 OPRM1(rs1799971)、DRD2(rs1799978)、ANKK1(rs1800497)、NAT2(rs1801280)、 UGT2B15(rs1902023)、UGT1A4(rs2011425)、EPHX1(rs2234922)、CYP2B6(rs2279343)、 ADORA2A(rs2298383)、SCN2A(rs2304016)、SACM1L(rs2742435)、POLG(rs3087374)、 SCN1A(rs3812718)、BMP5(rs41271330)、CYP1A2(rs4646425、rs762551)、FKBP5(rs4713916)、 MC4R(rs489693)、GNB3(rs5443)、HTR1A(rs6295)、OPRD1(rs678849)、RGS4(rs951439) 32个基因位点对应的32对扩增引物；
3)将步骤1)得到的DNA和步骤2)预混好的引物混合液一起加入到多重PCR反应液里，进行多重PCR扩增反应；
4)扩增产物利用产物纯化试剂盒进行纯化，完成测序文库制备；
5)将步骤4)制备完成的测序文库定量后进行二代测序(NGS)；
6)测序结束后,利用生物信息统计法，根据每个位点的引物进行拆分，得到每个位点对 应的片段的扩增序列总条数，再统计出野生型纯合/杂合/突变纯合序列条数占对应位点扩增序 列总条数的百分比，通过计算出的百分比进行基因分型判读。
进一步的，所述扩增引物中包含用于二代测序的芯片接头序列，所述32个基因位点的引 物对序列如下：
用于检测基因CYP2D6位点rs1080985的引物对PG1如SEQ ID NO.1-2所示，
用于检测基因CYP2D6位点rs35742686的引物对PG2如SEQ ID NO.3-4所示，
用于检测基因CYP2D6位点rs3892097的引物对PG3如SEQ ID NO.5-6所示，
用于检测基因CYP2D6位点rs5030655的引物对PG4如SEQ ID NO.7-8所示，
用于检测基因ABCB1位点rs1128503的引物对PG5如SEQ ID NO.9-10所示，
用于检测基因UGT2B7位点rs12233719的引物对PG6如SEQ ID NO.11-12所示，
用于检测基因CYP2C19位点rs12248560的引物对PG7如SEQ ID NO.13-14所示，
用于检测基因CYP2C19位点rs4244285的引物对PG8如SEQ ID NO.15-16所示，
用于检测基因CYP2C19位点rs4986893的引物对PG9如SEQ ID NO.17-18所示，
用于检测基因EPM2A位点rs1415744的引物对PG10如SEQ ID NO.19-20所示，
用于检测基因OPRM1位点rs1799971的引物对PG11如SEQ ID NO.21-22所示，
用于检测基因DRD2位点rs1799978的引物对PG12如SEQ ID NO.23-24所示，
用于检测基因ANKK1位点rs1800497的引物对PG13如SEQ ID NO.25-26所示，
用于检测基因NAT2位点rs1801280的引物对PG14如SEQ ID NO.27-28所示，
用于检测基因UGT2B15位点rs1902023的引物对PG15如SEQ ID NO.29-30所示，
用于检测基因UGT1A4位点rs2011425的引物对PG16如SEQ ID NO.31-32所示，
用于检测基因EPHX1位点rs2234922的引物对PG17如SEQ ID NO.33-34所示，
用于检测基因CYP2B6位点rs2279343的引物对PG18如SEQ ID NO.35-36所示，
用于检测基因ADORA2A位点rs2298383的引物对PG19如SEQ ID NO.37-38所示，
用于检测基因SCN2A位点rs2304016的引物对PG20如SEQ ID NO.39-40所示，
用于检测基因SACM1L位点rs2742435的引物对PG21如SEQ ID NO.41-42所示，
用于检测基因POLG位点rs3087374的引物对PG22如SEQ ID NO.43-44所示，
用于检测基因SCN1A位点rs3812718的引物对PG23如SEQ ID NO.45-46所示，
用于检测基因BMP5位点rs41271330的引物对PG24如SEQ ID NO.47-48所示，
用于检测基因CYP1A2位点rs4646425的引物对PG25如SEQ ID NO.49-50所示，
用于检测基因CYP1A2位点rs762551的引物对PG26如SEQ ID NO.51-52所示，
用于检测基因FKBP5位点rs4713916的引物对PG27如SEQ ID NO.53-54所示，
用于检测基因MC4R位点rs489693的引物对PG28如SEQ ID NO.55-56所示，
用于检测基因GNB3位点rs5443的引物对PG29如SEQ ID NO.57-58所示，
用于检测基因HTR1A位点rs6295的引物对PG30如SEQ ID NO.59-60所示，
用于检测基因OPRD1位点rs678849的引物对PG31如SEQ ID NO.61-62所示，
用于检测基因RGS4位点rs951439的引物对PG32如SEQ ID NO.63-64所示。
进一步的，所述用于二代测序的芯片接头序列，分别为：AATGATACGGCGACCACCG AGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT和CAAGCAGAAGACGGCATAC GAGATTACCGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC。
进一步的，所述扩增引物中包含用于序列分析时的index序列，一次拆分序列和二次拆 分序列分别为TACCGAAT和TCGACGTC。
进一步的，各个所述引物的摩尔比为PG1：PG2：PG3：PG4：PG5：PG6：PG7：PG8： PG9：PG10：PG11：PG12：PG13：PG14：PG15：PG16：PG17：PG18：PG19：PG20：PG 21：PG22：PG23：PG24：PG25：PG26：PG27：PG28：PG29：PG30：PG31：PG32＝0.1～0. 2:0.1～0.2:0.15～0.2:0.3～0.5:0.15～0.2:0.1～0.2:0.1～0.2:0.15～0.2:0.2～0.3:0.4～0.5:0.25～0.3:0.1～0.3: 0.1～0.2:0.1～0.2:0.1～0.2:0.3～0.5:0.1～0.2:0.1～0.2:0.25～0.3:0.3～0.4:0.1～0.3:0.3～0.4:0.1～0.2:0. 3～0.4:0.2～0.25:0.1～0.2:0.1～0.2:0.1～0.2:0.1～0.2:0.15～0.2:0.2～0.3:0.1～0.3。
进一步的，各个所述引物的摩尔比为PG1：PG2：PG3：PG4：PG5：PG6：PG7：PG8： PG9：PG10：PG11：PG12：PG13：PG14：PG15：PG16：PG17：PG18：PG19：PG20：PG21： PG22：PG23：PG24：PG25：PG26：PG27：PG28：PG29：PG30：PG31：PG32＝ 0.15:0.15:0.175:0.4:0.175:0.15:0.15:0.175:0.25:0.45:0.275:0.2:0.15:0.15:0.15:0.4:0.15:0.15:0.275:0 .35:0.2:0.35:0.15:0.35:0.215:0.15:0.15:0.15:0.15:0.175:0.25:0.2。
进一步的，所述步骤3)中PCR反应液的配置为：Multiplex PCR Buffer，25μL；Multiplex PCR Enzyme Mix，0.25μL；Primer Mix，13.63μL；基因组DNA(模板)，150ng；灭菌去离子 水。
进一步的，所述步骤3)中多重PCR扩增反应条件为：
预变性：94℃，1分钟；
35个扩增循环：变性：94℃，30秒；退火：60℃，30秒；延伸：72℃，30秒；
延伸：72℃，5分钟。
进一步的，步骤4)中测序前产物纯化的步骤为：
1)将低温储存的磁珠置于室温回温5～10min；
2)将所述PCR产物瞬离；
3)将瞬离后的PCR产物加入56uL磁珠，吹吸混匀15次，室温放置5min；
4)将PCR管放置于磁力架，2min后弃上清；
5)每管加250uL 80％乙醇溶液洗涤，30s后弃上清，反复两次；
6)吸除管内剩余乙醇，室温静置10min，待磁珠晾干；
7)每管加50uL洗脱液，吹吸混匀15次，室温放置5min；
8)放置磁力架5min，收集30uL洗脱下来的液体于新的离心管中，检测浓度，并用洗脱液稀释至1-5ng/uL再上机测序。
与现有技术相比，本发明的有益效果在于：
(1)本发明实现了对41种抗精神病药物密切相关基因的32个多态性位点同时进行检测， 将32对精神用药基因位点的引物对预混包装，方便检测，实现了一个反应体系的多基因变异 分析，降低成本的同时大大提高了检测通量和效率；
(2)本发明具有高灵敏度、低成本、操作简便、检测通量高等特点，可以快速精准的对 精神病患者个体化用药基因多态性进行检测。
具体实施方式
以下结合具体实施例对本发明技术方案作进一步详细说明。应当理解，下列实施例仅为 示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述 内容所实现的技术均涵盖在本发明旨在保护的范围内。
另外，除非另有特别说明，本发明中用到的各种原材料、试剂、仪器和设备均可通过市 场购买获得或现有方法制备得到。
实施例1：检测试剂的制备
1、药物和检测基因靶点的选择
发明人通过分析NCBI数据库和临床上精神病人常用药物的使用和代谢及药效的个体差 异，筛选出与41种抗精神病药物密切相关基因的32个多态性位点，并制备出检测该32个 多态性位点的引物组合物和使用该组合物的检测试剂，建立准确的检测方法用以实现有效的 用药指导。41种抗精神病药物分别是卡马西平、苯妥英、丙戊酸、拉莫三嗪、美芬妥因、万 拉法新、帕罗西汀、西酞普兰、阿米替林、氯丙咪嗪、安非他酮、莫达非尼、丙咪嗪、艾司 西酞普兰、去甲替林、地昔帕明、氟西汀、舍曲林、多塞平、氟伏沙明、文拉法辛、米氮平、 利培酮、奥氮平、氯氮平、奋乃静、氟哌啶醇、喹硫平、阿立哌唑、齐拉西酮、氨磺必利、氯丙嗪、硫利达嗪、氟奋乃静、奥沙西泮、地西泮、劳拉西泮、舒马普坦、曲马多、丁丙诺 啡和咖啡因。药物和基因关联分析如表1：
表1药物和基因关联分析
用于特异性的检测样本中的CYP2D6、ABCB1、UGT2B7、CYP2C19、EPM2A、 OPRM1、DRD2、ANKK1、NAT2、UGT2B15、UGT1A4、EPHX1、CYP2B6、ADORA2A、 SCN2A、SACM1L、POLG、SCN1A、BMP5、CYP1A2、FKBP5、MC4R、GNB3、 HTR1A、OPRD1、RGS4等基因的相关的32个位点的基因多态性，如表2所示：
表2 PCR反应体系中检测位点
2、检测靶点扩增引物设计
本发明利用NCBI公布的CYP2D6(rs1080985、rs35742686、rs3892097、rs5030655)、ABCB1(rs1128503)、UGT2B7(rs12233719)、CYP2C19(rs12248560、rs4244285、rs4986893)、EPM2A(rs1415744)、OPRM1(rs1799971)、DRD2(rs1799978)、ANKK1(rs1800497)、 NAT2(rs1801280)、UGT2B15(rs1902023)、UGT1A4(rs2011425)、EPHX1(rs2234922)、 CYP2B6(rs2279343)、ADORA2A(rs2298383)、SCN2A(rs2304016)、SACM1L(rs2742435)、 POLG(rs3087374)、SCN1A(rs3812718)、BMP5(rs41271330)、CYP1A2(rs4646425、 rs762551)、FKBP5(rs4713916)、MC4R(rs489693)、GNB3(rs5443)、HTR1A(rs6295)、 OPRD1(rs678849)、RGS4(rs951439)等32基因位点序列信息，设计多重PCR扩增引物， 本发明经过大量实验筛选，优选的扩增引物如表3：
表3 PCR扩增引物序列
上述扩增引物中包含用于二代测序的芯片接头序列，分别为：AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT和CAAGCAGAAGACGGCA TACGAGATTACCGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC。
上述扩增引物中同时包含用于序列分析时的index序列，优选的一次拆分序列和二次拆 分序列分别为TACCGAAT和TCGACGTC。
3、测序文库构建
(1)本发明采用多重PCR一步法进行测序文库的制备。引物混合体系中的各引物对的 用量如表4：
表4引物混合体系
| 引物名称 | 引物浓度(μM) | 引物用量(μl) | 引物名称 | 引物浓度(μM) | 引物用量(μl) |
| rs1080985-F | 10 | 0.15 | rs1080985-R | 10 | 0.15 |
| rs1128503-F | 10 | 0.15 | rs1128503-R | 10 | 0.15 |
| rs12233719-F | 10 | 0.175 | rs12233719-R | 10 | 0.175 |
| rs12248560-F | 10 | 0.4 | rs12248560-R | 10 | 0.4 |
| rs1415744-F | 10 | 0.175 | rs1415744-R | 10 | 0.175 |
| rs1799971-F | 10 | 0.15 | rs1799971-R | 10 | 0.15 |
| rs1799978-F | 10 | 0.15 | rs1799978-R | 10 | 0.15 |
| rs1800497-F | 10 | 0.175 | rs1800497-R | 10 | 0.175 |
| rs1801280-F | 10 | 0.25 | rs1801280-R | 10 | 0.25 |
| rs1902023-F | 10 | 0.45 | rs1902023-R | 10 | 0.45 |
| rs2011425-F | 10 | 0.275 | rs2011425-R | 10 | 0.275 |
| rs2234922-F | 10 | 0.2 | rs2234922-R | 10 | 0.2 |
| rs2279343-F | 10 | 0.15 | rs2279343-R | 10 | 0.15 |
| rs2298383-F | 10 | 0.15 | rs2298383-R | 10 | 0.15 |
| rs2304016-F | 10 | 0.15 | rs2304016-R | 10 | 0.15 |
| rs2742435-F | 10 | 0.4 | rs2742435-R | 10 | 0.4 |
| rs3087374-F | 10 | 0.15 | rs3087374-R | 10 | 0.15 |
| rs35742686-F | 10 | 0.15 | rs35742686-R | 10 | 0.15 |
| rs3812718-F | 10 | 0.275 | rs3812718-R | 10 | 0.275 |
| rs3892097-F | 10 | 0.35 | rs3892097-R | 10 | 0.35 |
| rs41271330-F | 10 | 0.2 | rs41271330-R | 10 | 0.2 |
| rs4244285-F | 10 | 0.35 | rs4244285-R | 10 | 0.35 |
| rs4646425-F | 10 | 0.15 | rs4646425-R | 10 | 0.15 |
| rs4713916-F | 10 | 0.35 | rs4713916-R | 10 | 0.35 |
| rs489693-F | 10 | 0.215 | rs489693-R | 10 | 0.215 |
| rs4986893-F | 10 | 0.15 | rs4986893-R | 10 | 0.15 |
| rs5030655-F | 10 | 0.15 | rs5030655-R | 10 | 0.15 |
| rs5443-F | 10 | 0.15 | rs5443-R | 10 | 0.15 |
| rs6295-F | 10 | 0.15 | rs6295-R | 10 | 0.15 |
| rs678849-F | 10 | 0.175 | rs678849-R | 10 | 0.175 |
| rs762551-F | 10 | 0.25 | rs762551-R | 10 | 0.25 |
| rs951439-F | 10 | 0.2 | rs951439-R | 10 | 0.2 |
(2)将待测样本的DNA和按表4预混好的引物混合液一起加入到多重PCR反应液里面， 进行多重PCR扩增反应。本发明中多重PCR扩增反应采用TaKaRa生产的Multiplex PCRAssay Kit Ver.2试剂盒进行扩增。多重PCR反应液的配制如表5：
表5 PCR反应液
(3)本发明中PCR扩增条件为：
(4)扩增产物利用产物纯化试剂盒(本发明中使用MECKMAN COULTER的AgencourtAMPure XP 60mL kit试剂盒)进行纯化，完成测序文库制备。
4、二代测序及结果分析
本发明专利中使用的检测平台为Iillumina公司生产CN500高通量测序仪，制备完成的测 序文库定量后进行二代测序(NGS)；测序结束后,利用生物信息统计法，根据每个位点的引物 进行拆分，得到每个位点对应的片段的扩增序列总条数，再统计出野生型纯合/杂合/突变纯合 序列条数占对应位点扩增序列总条数的百分比，通过计算出的百分比进行基因分型判读。
实施例2：精神类个体化用药基因检测
本实施例收集了2例患者外周血，分别编号为样本1和样本2，并采用一代测序进行对 照验证结果的准确性。2例外周样本来自重庆市第九人民医院，均使用含有EDTA抗凝剂的 釆血管采集血液标本5ml。样本室温静置30分钟，1500～2000rpm离心10分钟，分别收集血浆和血液中细胞于无菌螺口塑料管中。
1、样本处理
将样本利用商业化试剂盒进行核酸提取，本发明采用美基生物生产的HiPureBlood DNA Mini Kit。在1.5ml离心管中，加入25μl Proteinase K，转移10-250μl抗凝血液至装有蛋 白酶的离心管中。轻轻振荡混匀，加入250μl Buffer AL至样品中，颠倒3-5次混匀，最高 速度涡旋30秒混匀，70℃水浴10分钟，其间涡旋混匀一次。加入250μl无水乙醇至样 品中，涡旋30秒混匀，短暂离心收集管壁的液滴。把DNA柱装在新的收集管中，转移混合液至柱子中。10000×g离心1分钟，丢弃收集管和流出液。把DNA柱装在新的收集管中， 加入500μl Buffer DW1至柱子上，颠倒混匀几次，10000×g离心30-60秒，倒弃流出液， 把柱子重新装回收集管中。加入650μl Buffer DW2(已用乙醇稀释)至柱子中，10000×g离 心30-60秒，倒弃流出液，把柱子重新装回收集管中。10000×g离心2分钟，可彻底去除柱 子中残留的乙醇。将柱子转移至新的1.5ml离心管中。加入30-100μl预热至70℃ Elution Buffer或Buffer TE至柱子的膜中央，放置3分钟后，10000×g离心1分钟。再加入30-100 μl预热至70℃ Elution Buffer或Buffer TE至柱子的膜中央，放置3分钟，10000×g离 心1分钟，丢弃DNA结合柱，检测DNA液体的浓度，把DNA保存2-8℃，长期保存需保 存于-20℃。
2、PCR反应
反应体系配制按照实施例1中的PCR反应液配制反应体系，将2个样本提取的DNA加入配制好的反应体系中，加入的模板量约150ng，并进行多重PCR扩增反应。
3、产物测序前纯化
本实验采用贝克曼的商业化试剂盒进行PCR产物测序前纯化，步骤如下：
1)将存放于4℃冰箱的磁珠置于室温回温5-10min；
2)将第二轮PCR产物瞬离(若壁上无残留则不需要此步骤)；
3)将瞬离后的PCR产物加入56uL磁珠，吹吸混匀15次，室温放置5min；
4)将PCR管放置于磁力架，2min后弃上清；
5)每管加250uL 80％乙醇，30s后弃上清；反复两次；
6)吸除管内剩余乙醇；室温静置10min，待磁珠晾干；
7)每管加50uL洗脱液，吹吸混匀15次，室温放置5min；
8)放置磁力架5min；收集30uL洗脱下来的液体于新的离心管中，检测浓度，并用洗脱液稀释至1-5ng/uL再上机测序。
4、测序结果
反应结束后下机数据用生物信息法统计结果并进行基因分型的结果判读。样本1和样本 2采用本发明的检测方法与一代测序结构完全一致，检测结果见表6。
表6检测结果
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例。对 于本技术领域的技术人员来说，在不脱离本发明技术构思前提下所作任何修改、等同替换、 改进等，均应包含在本发明的保护范围之内。
序列表
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<212> DNA
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<210> 34
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 35
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
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aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
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<210> 36
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<212> DNA
<213> 人工序列(Artificial Sequence)
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caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
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<210> 37
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 38
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
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caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
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<210> 39
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 40
<211> 82
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgcatc tgagttgcca ca 82
<210> 41
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgact tcatttgaac cacagc 86
<210> 42
<211> 88
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcatatg taaaacctgc acaatgtg 88
<210> 43
<211> 82
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcaccc aggcagtgct at 82
<210> 44
<211> 84
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatctcttg aggtatccta acca 84
<210> 45
<211> 88
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcgtag gtacaaagag cctatcct 88
<210> 46
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatccaggt ttcagaattt gtaaac 86
<210> 47
<211> 84
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctttc ccacaagacc agca 84
<210> 48
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcctttt caaccctcag aat 83
<210> 49
<211> 84
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctacg ttgcttccct gtgt 84
<210> 50
<211> 80
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgcagg agctagcagc 80
<210> 51
<211> 84
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtctgat gctcaaaggg tgag 84
<210> 52
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatctcatt catttcagtc atttga 86
<210> 53
<211> 88
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccacc tctctggact cctacatt 88
<210> 54
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatccacaa gggtttgtag agatta 86
<210> 55
<211> 89
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccatc atgtcttaat tctgttgtc 89
<210> 56
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcagctg agtcaggatg tatgag 86
<210> 57
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtccaag agcatcatct gcg 83
<210> 58
<211> 84
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcacacg ctcattcatg gagt 84
<210> 59
<211> 86
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcattt ggaagaagac cgagtg 86
<210> 60
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcttgtt gttgtcgtcg ttg 83
<210> 61
<211> 88
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcaagg tgtccttctt accatagt 88
<210> 62
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcatgag aactgcctcc aat 83
<210> 63
<211> 82
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttc 60
gacgtcttcc tgctgtgtgg ct 82
<210> 64
<211> 85
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
caagcagaag acggcatacg agattaccga atgtgactgg agttcagacg tgtgctcttc 60
cgatcgtttg aagttttaag ttcct 85
Claims (4)
1.一种精神类个体化用药基因检测的引物组合物，其特征在于，所述引物组合物
为用于检测rs1080985、rs35742686、rs3892097、rs5030655、rs1128503、rs12233719、rs12248560、rs4244285、rs4986893、rs1415744、rs1799971、rs1799978、rs1800497、rs1801280、rs1902023、rs2011425、rs2234922、rs2279343、rs2298383、rs2304016、rs2742435、rs3087374、rs3812718、rs41271330、rs4646425、rs762551、rs4713916、rs489693、rs5443、rs6295、rs678849、rs951439的32个SNP位点的32对扩增引物；
所述扩增引物中包含用于二代测序的芯片接头序列，序列如下：
用于检测基因CYP2D6位点rs1080985的引物对PG1如SEQ ID NO.1-2所示，
用于检测基因CYP2D6位点rs35742686的引物对PG2如SEQ ID NO.3-4所示，
用于检测基因CYP2D6位点rs3892097的引物对PG3如SEQ ID NO.5-6所示，
用于检测基因CYP2D6位点rs5030655的引物对PG4如SEQ ID NO.7-8所示，
用于检测基因ABCB1位点rs1128503的引物对PG5如SEQ ID NO.9-10所示，
用于检测基因UGT2B7位点rs12233719的引物对PG6如SEQ ID NO.11-12所示，
用于检测基因CYP2C19位点rs12248560的引物对PG7如SEQ ID NO.13-14所示，
用于检测基因CYP2C19位点rs4244285的引物对PG8如SEQ ID NO.15-16所示，
用于检测基因CYP2C19位点rs4986893的引物对PG9如SEQ ID NO.17-18所示，
用于检测基因EPM2A位点rs1415744的引物对PG10如SEQ ID NO.19-20所示，
用于检测基因OPRM1位点rs1799971的引物对PG11如SEQ ID NO.21-22所示，
用于检测基因DRD2位点rs1799978的引物对PG12如SEQ ID NO.23-24所示，
用于检测基因ANKK1位点rs1800497的引物对PG13如SEQ ID NO.25-26所示，
用于检测基因NAT2位点rs1801280的引物对PG14如SEQ ID NO.27-28所示，
用于检测基因UGT2B15位点rs1902023的引物对PG15如SEQ ID NO.29-30所示，
用于检测基因UGT1A4位点rs2011425的引物对PG16如SEQ ID NO.31-32所示，
用于检测基因EPHX1位点rs2234922的引物对PG17如SEQ ID NO.33-34所示，
用于检测基因CYP2B6位点rs2279343的引物对PG18如SEQ ID NO.35-36所示，
用于检测基因ADORA2A位点rs2298383的引物对PG19如SEQ ID NO.37-38所示，
用于检测基因SCN2A位点rs2304016的引物对PG20如SEQ ID NO.39-40所示，
用于检测基因SACM1L位点rs2742435的引物对PG21如SEQ ID NO.41-42所示，
用于检测基因POLG位点rs3087374的引物对PG22如SEQ ID NO.43-44所示，
用于检测基因SCN1A位点rs3812718的引物对PG23如SEQ ID NO.45-46所示，
用于检测基因BMP5位点rs41271330的引物对PG24如SEQ ID NO.47-48所示，
用于检测基因CYP1A2位点rs4646425的引物对PG25如SEQ ID NO.49-50所示，
用于检测基因CYP1A2位点rs762551的引物对PG26如SEQ ID NO.51-52所示，
用于检测基因FKBP5位点rs4713916的引物对PG27如SEQ ID NO.53-54所示，
用于检测基因MC4R位点rs489693的引物对PG28如SEQ ID NO.55-56所示，
用于检测基因GNB3位点rs5443的引物对PG29如SEQ ID NO.57-58所示，
用于检测基因HTR1A位点rs6295的引物对PG30如SEQ ID NO.59-60所示，
用于检测基因OPRD1位点rs678849的引物对PG31如SEQ ID NO.61-62所示，
用于检测基因RGS4位点rs951439的引物对PG32如SEQ ID NO.63-64所示。
2.如权利要求1所述一种精神类个体化用药基因检测的引物组合物，其特征在于：所述用于二代测序的芯片接头序列分别为：AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT和CAAGCAGAAGACGGCATACGAGATTACCGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC。
3.如权利要求1所述一种精神类个体化用药基因检测的引物组合物，其特征在于：所述引物的摩尔比为PG1：PG2：PG3：PG4：PG5：PG6：PG7：PG8：PG9：PG10：PG11：PG12：PG13：PG14：PG15：PG16：PG17：PG18：PG19：PG20：PG21：PG22：PG23：PG24：PG25：PG26：PG27：PG28：PG29：PG30：PG31：PG32＝0.1～0.2:0.1～0.2:0.15～0.2:0.3～0.5:0.15～0.2:0.1～0.2:0.1～0.2:0.15～0.2:0.2～0.3:0.4～0.5:0.25～0.3:0.1～0.3:0.1～0.2:0.1～0.2:0.1～0.2:0.3～0.5:0.1～0.2:0.1～0.2:0.25～0.3:0.3～0.4:0.1～0.3:0.3～0.4:0.1～0.2:0.3～0.4:0.2～0.25:0.1～0.2:0.1～0.2:0.1～0.2:0.1～0.2:0.15～0.2:0.2～0.3:0.1～0.3。
4.如权利要求3所述一种精神类个体化用药基因检测的引物组合物，其特征在于：所述引物的摩尔比为PG1：PG2：PG3：PG4：PG5：PG6：PG7：PG8：PG9：PG10：PG11：PG12：PG13：PG14：PG15：PG16：PG17：PG18：PG19：PG20：PG21：PG22：PG23：PG24：PG25：PG26：PG27：PG28：PG29：PG30：PG31：PG32＝0.15:0.15:0.175:0.4:0.175:0.15:0.15:0.175:0.25:0.45:0.275:0.2:0.15:0.15:0.15:0.4:0.15:0.15:0.275:0.35:0.2:0.35:0.15:0.35:0.215:0.15:0.15:0.15:0.15:0.175:0.25:0.2。
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Denomination of invention: A gene detection method for individualized medication of psychosis Effective date of registration: 20220727 Granted publication date: 20210720 Pledgee: Chongqing Rural Commercial Bank Co.,Ltd. Beibei sub branch Pledgor: Chongqing puluotong Life Technology Group Co.,Ltd. Registration number: Y2022500000048 |
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