CN113462772B - 一种用于检测尼古丁依赖性相关的snp位点的引物组以及应用 - Google Patents
一种用于检测尼古丁依赖性相关的snp位点的引物组以及应用 Download PDFInfo
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Abstract
本发明公开了一种用于检测尼古丁依赖性相关的SNP位点的引物组以及应用，引物组包括26对引物对，引物对的核苷酸序列如SEQ ID NO.1‑52所示；应用于检测尼古丁依赖易感性产品中以及试剂盒中；本发明能够有效提高检测的效率；特异性强，灵敏度高，针对不同个体的适用性较好，整个鉴定流程容易理解，操作简便，结果准确可靠，应用广泛，该方法操作简便，结果准确可靠。
Description
技术领域
本发明涉及生物技术领域，特别涉及一种用于检测尼古丁依赖性相关的SNP位点的引物组以及应用。
背景技术
香烟是最广泛滥用物质之一，香烟内导致成瘾的主要物质为尼古丁，尼古丁依赖（Nicotine Dependence，NP）符合 DSM-Ⅳ中对物质依赖的诊断标准，包括冲动性使用，停药困难以及慢性使用后停药产生戒断症状，其中心理依赖是物质依赖最核心的特征之一。目前，尼古丁依赖已成为全球关注的一个严重问题。据世界卫生组织估计，目前全球每年约有500万人死于由吸烟所引发的包括癌症、心血管疾病以及肺部疾病，预计2030 年每年因吸烟致死的人数将达到1000万。
吸烟与尼古丁依赖的分子遗传学机制：随着候选基因及 GWAS（Genome WideAssociated Study）研究，目前吸烟与尼古丁依赖的分子遗传学研究取得了一定进展。研究发 CHRNB2 基因的单核苷酸多态（single nucleotide polymorphism, SNP）与戒烟能力之间存在强相关性，而位于 CHRNB2 与 CHRNB4 基因的 SNPs 之间的交互作用提示二者影响尼古丁依赖。2007 年研究发现位于 CHRNA5 基因的 rs16969968 是尼古丁依赖的易感因素,另外几个独立研究随后验证该位点和位于 CHRNA3 rs1051730 与尼古丁依赖剂量包括每日吸烟数量（cigarettes per day, CPD）以及尼古丁依赖程度呈高度相关性。GWAS 研究显示位于第 15 号染色体上的 CHRNB5-CHRNA3-CHRNB4 基因束与 CPD 具有相关性，第 8号染色体上的 CHRNB3-CHRNA6 以及第 2 号染色体上的骨骼肌 CHRND-CHRNG基因束均与尼古丁依赖以及吸烟有强相关性。最近来自TGC（the Tobacoo and Genetics Consortium）针对欧美国家人群（n=140，023）的全基因组关联分析及 Meta 分析鉴定了一系列与吸烟行为相关的位点；其中3个位点与每日吸烟量关联，包括位于10q25的rs1329650和rs1028936，以及位于 9q13 的 EGLN2 基因内的rs3733829和位于15q25的CHRNA3 基因的 rs1051730。rs3733829 在 CYP2A6 （Cytochrome P450 2A6）基因 3’端下游 40Kb 处，等位基因 G 的每个拷贝增加约 0.5CPD，CYP2A6 基因是吸烟的一个候选基因，是一种代谢酶可将尼古丁代谢为可替宁，CYP2A6 的某些突变可因减慢尼古丁代谢而减少尼古丁摄人。rs1051730 的A 等位基因与每日吸烟量具有最强关联性， P 值为 2.8×10-7；有 8 个 SNPs 与起动吸烟有关，最强关联性位点为第 11 号染色体上BDNF(Brain-derived neurotrophic factor)基因的 rs6265 的Ｃ等位基因；此外，位于9号染色体上的DBH(Dopamine-hydroxylase)基因附近的 rs3025343 的G 等位基因与吸烟戒断具有关联性。由此可见,遗传学风险因素在尼古丁依赖致病机制中起重要作用。
国内尼古丁依赖的易感基因关联研究尚存在以下问题：1、尚无在同一人群背景中进行尼古丁依赖与多易感基因位点联合研究；2、尚无近年基于大样本量 GWAS 研究鉴定的新易感基因关联研究；3、尚无尼古丁依赖与多易感基因及性格因素关联性的联合研究。
发明内容
我们通过在同一大样本人群背景下同时对若干易感基因位点进行研究，发现与中国汉族人群尼古丁依赖最相关的易感基因SNPs，并设计相应的引物组合，该组合可用于检测中国汉族人群个体的尼古丁依赖易感性，也为开发预防尼古丁依赖的药物提供了依据。
发明的目的在于提供一种用于检测尼古丁依赖性相关的SNP位点的引物组以及应用，解决了背景技术中存在的问题。
本发明是这样实现的，一种用于检测尼古丁依赖性相关的SNP位点的引物组，所述引物组包括26对引物对，所述引物对的核苷酸序列如表1中的SEQ ID NO.1-52所示。
表1
本发明的进一步技术方案是：所述引物组用于检测对应的SNP位点，所述SNP位点为：rs1318937、rs17040623、rs279871、rs2349433、rs7747583、rs13225753、rs1451240、rs6474412、rs6996964、rs1028936、rs1329650、rs6265、rs1051730、rs16969968、rs2036527、rs4887077、rs6495308、rs6495309、rs6495314、rs7163369、rs8034191、rs13334632、rs2241617、rs7937、rs3733829和rs4105144。
本发明的进一步技术方案是：rs1318937等位基因型为A/G、rs17040623等位基因型为A/G、rs279871等位基因型为C/T、rs2349433等位基因型为G/A、rs7747583等位基因型为C/A、rs13225753等位基因型为A/G、rs1451240等位基因型为G/A、rs6474412等位基因型为T/C、rs6996964等位基因型为C/T、rs1028936等位基因型为C/A、rs1329650等位基因型为T/G、rs6265等位基因型为T/C、rs1051730等位基因型为G/A、rs16969968等位基因型为G/A，rs2036527等位基因型为G/A、rs4887077等位基因型为C/T、rs6495308等位基因型为C/T、rs6495309等位基因型为C/T、rs6495314等位基因型为A/C、rs7163369等位基因型为A/G、rs8034191等位基因型为T/C、rs13334632等位基因型为T/C、rs2241617等位基因型为C/T、rs7937等位基因型为T/C、rs3733829等位基因型为A/G和rs4105144等位基因型为C/T。
一种用于检测尼古丁依赖性相关的SNP位点的引物组的应用，用于检测尼古丁依赖性相关的SNP位点的引物组在制备检测尼古丁依赖易感性的试剂盒中。
本发明的进一步技术方案是：所述试剂盒还包括Taq DNA聚合酶、10x buffer以及dNTP。
本发明的进一步技术方案是：待测样品来自待测个体的血液、尿、唾液、胃液、头发或活组织检查。
本发明的有益效果：(1)本发明提供的26个SNP位点基于中国人群全基因组测序数据关联分析得到，与中国人群中的尼古丁依赖易感性具有较强的相关性，能够有效提高检测的效率；(2)本发明开发的用于扩增上述SNP位点的引物对，特异性强，灵敏度高，针对不同个体的适用性较好，整个鉴定流程容易理解，操作简便，结果准确可靠，可以广泛应用于中国人群个体中上述SNP位点的鉴定；(3)本发明提供了一种鉴定上述SNP位点等位基因型的试剂盒的应用方法，该方法操作简便，结果准确可靠。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例一：
一种用于检测尼古丁依赖性相关的SNP位点的引物组，所述引物组包括26对引物对，所述引物对的核苷酸序列如SEQ ID NO.1-52所示。
实施例二：
外周血采集及DNA提取：
外周血采血一般采用真空采血管采集，收集好的全血应在5min中内用液氮速冻，保存于-80℃冰箱。加入EDTA抗凝剂后的新鲜血液一般可以在4℃放置12-24小时。务必保证经过速冻后的样本要低温保存，且必须避免反复冻融。
本发明中的外周血DNA提取包括以下步骤：首先向2mL全血中加入5倍体积的1x红细胞裂解液，涡旋并弃上清；然后加入白细胞裂解液0.5mL和20mg/ml蛋白酶K 20μL，37℃水浴裂解白细胞至沉淀溶解；再加入等体积的氯仿待分层后转移上清至新EP管中，加入1/10体积的3M醋酸铵和等体积的冰乙醇沉淀DNA，用1mL的体积浓度为75％乙醇洗涤后自然干燥，加入TE溶解即可得到DNA。为确保后续血液样本的核酸提取质量合格，每个样本应提取1μg以上的全基因组DNA，浓度>12.5ng/μL，OD260/280在1.8～2.0之间。DNA质量检测包括完整性、总量及浓度的检测，可采用Qubit或AGE仪器完成。DNA样品含量检测可采用以下两种方法的一种：荧光定量或琼脂糖凝胶电泳检测。荧光定量主要对样品浓度进行精确定量(如QubitFluorometer)，琼脂糖凝胶电泳使用质量浓度为1％的琼脂糖凝胶在150V电压下电泳约40min(缓冲液为1xTAE)，检测样品完整性及是否存在RNA、蛋白和次生代谢物污染。根据DNA总量和浓度对血样给出等级判定。优：DNA总量≥2μg、浓度≥12.5ng/μL且样品没有降解或者轻微降解。良：DNA总量介于1～2μg、浓度≥12.5ng/μL且样品没有降解或者轻微降解。尽量选用质量检测为优的DNA样品进行后续的工作。
单核苷酸多态性检测：
利用本发明中根据基因多态性区域特征设计的引物对，以上述DNA样品为模板进行PCR扩增得到包含多态性区域的DNA片段。具体如下：
PCR反应20μL体系：Taq DNA聚合酶1μL，正向引物10pmol/μL 0.6μL，反向引物10pmol/μL 0.6μL，模板DNA 2μL，10x buffer 2μL，dNTP 2.5mM 1.8mL，ddH2O 12.0μL，总计20μL。
PCR反应条件如下：94℃预变性3min；94℃变性45s，59℃退火45s，72℃延伸45s，共50个循环；72℃最后延伸5min，4℃保存。
PCR扩增完成后用琼脂糖凝胶电泳检测，切胶回收产物，随后用sanger测序得到样本中上述单核苷酸多态性位点的基因型。
实施例三：
本发明提供了一系列与尼古丁依赖风险相关的单核苷酸多态性位点(SNP)。本发明在1496个中国人群样本中进行基因分型和表型关联分析，得到了26个与中国汉族人群尼古丁依赖相关的SNP。所述SNP位点的物理位置是根据人参考基因组GRCh37/hg19确定的，SNP的编号的来源为dbSNP151。所述的SNP在人基因组的染色体及位置如表2所示。
表2中国人群尼古丁依赖相关的26个SNPs位点所在染色体及位置
| 单核苷酸多态位点 | 染色体 | 基因 | 位置(hg19) |
| rs1318937 | 3 | SH3BP5 | chr3:15,295,364-15,295,364 |
| rs17040623 | 3 | NR2C2 | chr3:15,028,478-15,028,478 |
| rs279871 | 4 | GABRA2 | chr4:46,305,733-46,305,733 |
| rs2349433 | 6 | RGS17 | chr6:153,383,755-153,383,755 |
| rs7747583 | 6 | RGS17 | chr6:153,371,768-153,371,768 |
| rs13225753 | 7 | CACNA2D1 | chr7:82,158,523-82,158,523 |
| rs1451240 | 8 | CHRNB3 | chr8:42,546,751-42,546,751 |
| rs6474412 | 8 | CHRNB3 | chr8:42,550,498-42,550,498 |
| rs6996964 | 8 | CSGALNACT1 | chr8:19,623,911-19,623,911 |
| rs1028936 | 10 | HECTD2-AS1 | chr10:93,349,797-93,349,797 |
| rs1329650 | 10 | HECTD2-AS1 | chr10:93,348,120-93,348,120 |
| rs6265 | 11 | BDNF | chr11:27,679,916-27,679,916 |
| rs1051730 | 15 | CHRNA3 | chr15:78,894,339-78,894,339 |
| rs16969968 | 15 | CHRNA5 | chr15:78,882,925-78,882,925 |
| rs2036527 | 15 | PSMA4//CHRNA5 | chr15:78,851,615-78,851,615 |
| rs4887077 | 15 | CHRNB4 | chr15:78,978,364-78,978,364 |
| rs6495308 | 15 | CHRNA3 | chr15:78,907,656-78,907,656 |
| rs6495309 | 15 | CHRNB4 | chr15:78,915,245-78,915,245 |
| rs6495314 | 15 | CHRNB4 | chr15:78,960,529-78,960,529 |
| rs7163369 | 15 | SLCO3A1 | chr15:92,533,914-92,533,914 |
| rs8034191 | 15 | HYKK | chr15:78,806,023-78,806,023 |
| rs13334632 | 16 | ZCCHC14 | chr16:87,490,819-87,490,819 |
| rs2241617 | 16 | ZCCHC14 | chr16:87,441,048-87,441,048 |
| rs7937 | 19 | RAB4B | chr19:41,302,706-41,302,706 |
| rs3733829 | 19 | EGLN2 | chr19:41,310,571-41,310,571 |
| rs4105144 | 19 | CYP2A6 | chr19:41,358,624-41,358,624 |
所述引物组用于检测对应的SNP位点，所述SNP位点为：rs1318937、rs17040623、rs279871、rs2349433、rs7747583、rs13225753、rs1451240、rs6474412、rs6996964、rs1028936、rs1329650、rs6265、rs1051730、rs16969968、rs2036527、rs4887077、rs6495308、rs6495309、rs6495314、rs7163369、rs8034191、rs13334632、rs2241617、rs7937、rs3733829和rs4105144。
rs1318937等位基因型为A、rs17040623等位基因型为A、rs279871等位基因型为C、rs2349433等位基因型为G、rs7747583等位基因型为C、rs13225753等位基因型为A、rs1451240等位基因型为G、rs6474412等位基因型为T、rs6996964等位基因型为C、rs1028936等位基因型为C、rs1329650等位基因型为T、rs6265等位基因型为C、rs1051730等位基因型为G、rs16969968等位基因型为G，rs2036527等位基因型为G、rs4887077等位基因型为C、rs6495308等位基因型为C、rs6495309等位基因型为C、rs6495314等位基因型为A、rs7163369等位基因型为A、rs8034191等位基因型为T、rs13334632等位基因型为T、rs2241617等位基因型为C、rs7937等位基因型为T、rs3733829等位基因型为A和rs4105144等位基因型为C。
实施例四：
一种用于检测尼古丁依赖性相关的SNP位点的引物组的应用，用于检测尼古丁依赖性相关的SNP位点的引物组在制备检测尼古丁依赖易感性的试剂盒中。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
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Claims (5)
1.一种用于检测尼古丁依赖性相关的SNP位点的引物组，其特征在于：所述引物组包括26对引物对，所述引物对的核苷酸序列如SEQ ID NO.1-52所示。
2.根据权利要求1所述的一种用于检测尼古丁依赖性相关的SNP位点的引物组，其特征在于：所述引物组用于检测对应的SNP位点，所述SNP位点为：rs1318937、rs17040623、rs279871、rs2349433、rs7747583、rs13225753、rs1451240、rs6474412、rs6996964、rs1028936、rs1329650、rs6265、rs1051730、rs16969968、rs2036527、rs4887077、rs6495308、rs6495309、rs6495314、rs7163369、rs8034191、rs13334632、rs2241617、rs7937、rs3733829和rs4105144。
3.根据权利要求2所述的一种用于检测尼古丁依赖性相关的SNP位点的引物组，其特征在于：rs1318937等位基因型为A、rs17040623等位基因型为A、rs279871等位基因型为C、rs2349433等位基因型为G、rs7747583等位基因型为C、rs13225753等位基因型为A、rs1451240等位基因型为G、rs6474412等位基因型为T、rs6996964等位基因型为C、rs1028936等位基因型为C、rs1329650等位基因型为T、rs6265等位基因型为C、rs1051730等位基因型为G、rs16969968等位基因型为G，rs2036527等位基因型为G、rs4887077等位基因型为C、rs6495308等位基因型为C、rs6495309等位基因型为C、rs6495314等位基因型为A、rs7163369等位基因型为A、rs8034191等位基因型为T、rs13334632等位基因型为T、rs2241617等位基因型为C、rs7937等位基因型为T、rs3733829等位基因型为A和rs4105144等位基因型为C。
4.根据权利要求1-3中任意一项所述一种用于检测尼古丁依赖性相关的SNP位点的引物组在制备检测尼古丁依赖易感性的试剂盒中的应用。
5.根据权利要求4所述一种用于检测尼古丁依赖性相关的SNP位点的引物组在制备检测尼古丁依赖易感性的试剂盒中的应用，其特征在于：所述试剂盒还包括Taq DNA聚合酶、10x buffer以及dNTP。
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