CN114107505A - 用于结直肠癌易感基因snp检测的引物、检测方法和试剂盒 - Google Patents
用于结直肠癌易感基因snp检测的引物、检测方法和试剂盒 Download PDFInfo
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- CN114107505A CN114107505A CN202111451110.6A CN202111451110A CN114107505A CN 114107505 A CN114107505 A CN 114107505A CN 202111451110 A CN202111451110 A CN 202111451110A CN 114107505 A CN114107505 A CN 114107505A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
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Abstract
本发明涉及分子生物技术领域，特别涉及用于结直肠癌易感基因SNP检测的引物、检测方法和试剂盒，引物包括与结直肠癌高度相关的9对特异性引物及9个延伸引物。与现有技术相比，本发明通过对3′aQTL与结直肠癌疾病风险关联分析，得到9个与结直肠癌高度相关的SNP位点，并针对9个SNP位点设计9对特异性引物及9个延伸引物，利用9对特异性引物及9个延伸引物对基因样本进行PCR扩增及单碱基延伸后，一次检测可得到9个位点全部的SNP信息，可用于肿瘤的风险预测和早期筛查。
Description
技术领域
本发明涉及分子生物技术领域，特别涉及用于结直肠癌易感基因SNP检测的引物、检测方法和试剂盒。
背景技术
我国大多数群众缺乏癌症早期筛查的意识，由于疾病早期症状不明显，发现明显症状后才到医院进行检查，至被确诊为结直肠癌时，癌细胞已经扩散，错失最佳的治疗时间。中晚期患者所需治疗费用高、疗效差、五年生存率低。如果能够在早期发现结直肠癌，不仅治疗费用降低，还能极大提升治愈几率。目前肠镜检查是结直肠癌检查金标准，但由于其需要侵入检测，普遍接受度低、依从性差，造成肠癌患者的漏检、漏筛。亟需一种肠癌高风险人群划分手段，提升高风险人群肠镜受检比例。
选择性多聚腺苷酸化(APA)是一种重要的前体RNA加工机制，它广泛存在于所有真核生物中。通过在RNA 3’UTR不同位置上添加polyA尾巴，可以选择性地调节3’UTR的长短。由于3′UTR含有多种顺式调控元件，因此，APA可以通过调控3′UTR的长度，影响基因表达与定位调控。非编码区APA数量性状位点 (3′aQTL)是一种特殊的SNP，它以APA作为分子表型，与多种人类复杂疾病相关。通过3′aQTL与结直肠癌疾病风险关联分析，我们筛选到9个与结直肠癌高度相关的SNP位点。
核酸质谱系统基于飞行质谱技术进行四种核苷酸分型，可以一次平行检测 50多个SNP位点，具有高通量、高精度、高自动化、高性价比的检测优势。
发明内容
针对以上述背景技术的不足，本发明提供一种用于结直肠癌易感基因SNP检测的引物、试剂盒及应用，解决了因结直肠癌早期筛查需要侵入检测，普遍接受度低、依从性差，而造成肠癌患者的漏检、漏筛的问题。
一方面，本发明提供用于结直肠癌易感基因SNP检测的引物，关键在于：包括与结直肠癌高度相关的9对特异性引物及9个延伸引物；其中所述9对特异性引物的序列如下：
Seq ID NO.1：F-CTCCTGAGTAGCTGGGATTACAGGT，
Seq ID NO.2：R-ATCACTTCAGGTCAGGAGTTCAAAACCA；
Seq ID NO.3：F-CAGTGTGGTGCAGTGGTTAGAGCAT，
Seq ID NO.4：R-ACTTTATATCCAAGGAGGCCTGACACA；
Seq ID NO.5：F-TGCAGCACTTAACGCAGTGTCCTC，
Seq ID NO.6：R-AATCGATAGTGGGTATGTATAAAGTGCGTA；
Seq ID NO.7：F-TGTGTAACAGACGTCACCTTCAGC，
Seq ID NO.8：R-GGTTTCTCTGTATCCCTATGTAAAGGACT；
Seq ID NO.9：F-GATAGAGAGTTTCTAATCCTTTGGTGA，
Seq ID NO.10：R-CTACATTTGGATATCTGTGAACCTT；
Seq ID NO.11：F-TCTGGTGCTCATTTCCTTCCAGTGGGACA，
Seq ID NO.12：R-ATGCCAGCTGCCATTGTGATGGGA；
Seq ID NO.13：F-TCATGGAAAGAGTATTGATTTATGCC，
Seq ID NO.14：R-TAAACCTGTGTTGTATTTAATCAAGTT；
Seq ID NO.15：F-AGTGAGCTGTGATCATGCCAAGGTACT，
Seq ID NO.16：R-TGAGCCCTGGCCCACAGAACGAGA；
Seq ID NO.17：F-ATAGGAAATCACAAGGGTATTGACTGG，
Seq ID NO.18：R-TTTCATGGACATGTATTAGTTCCCCAA；
延伸引物序列如下：
Seq ID NO.19：CTAATTTTTGTATTTTCAGTAGAG；
Seq ID NO.20：GGCTGTGGAGGCAGATGGATCTCAGGCCAGCC；
Seq ID NO.21：GCAGTGTCCTCATACTCTTTTATTTCTTCTTGG；
Seq ID NO.22：CTCTCTGTCAAACAGGCATTCTTCCCCCC；
Seq ID NO.23：TGAAAAGTAAAAGAAGGTTAAAATA；
Seq ID NO.24：TCCCTTGCTCCCTCAGTACCCTGTCTCTCA；
Seq ID NO.25：CTCTAATCAGTATCGGTGAAGTCACC；
Seq ID NO.26：AAACCTGTCACCTTTTTTTTGGGGGGGG；
Seq ID NO.27：ATGAAATCTTTACAATTTATGCTTAGAGA。
另一方面，本发明提供一种结直肠癌易感基因SNP检测方法，关键在于包括以下步骤：
S1.提取体液中游离的基因组；体液包括血液，淋巴液，精液和尿液；
S2.将基因组作为模板，加入如权利要求1所述的9对特异性引物，进行多重PCR扩增；
S3.将扩增产物中加入虾碱性磷酸酶进行多余引物及dNTP消化；
S4.将消化产物中加入权利要求1所述的9个延伸引物进行单碱基延伸；
S5.将单碱基延伸产物进行核酸质谱检测。
优选的，所述S1具体为：采用QiagenDNA提取试剂盒从口腔拭子中提取 cfDNA。
优选的，所述S2的扩增程序为：98℃/45s，1个循环；98℃/15s，60℃/30 s，72℃/30s，40个循环；72℃/3min，1个循环；4℃/hold。
优选的，所述S2中具体为：将提取后的10ng基因组DNA作为模板，加入如权利要求1所述的9对特异性引物及试剂形成扩增体系如下：
| 组分 | 单个反应加入量(μL) |
| KAPA HiFi HotStartReadyMix(2X) | 2.5 |
| 10×引物mix | 0.5 |
| 基因组DNA(10ng/μL) | 1 |
| 无核酶水 | 1 |
优选的，所述S3中具体为：将扩增产物中加入虾碱性磷酸酶及试剂形成消化体系如下：
优选的，所述S3的消化程序为：37℃/60min；80℃/15min，4℃/hold。
优选的，所述S4中具体为：将消化产物中加入权利要求1所述的9个延伸引物及试剂形成单碱基延伸体系如下：
| 组分 | 单个反应加入量(μL) |
| 上步消化体系 | 7 |
| 无核酶水 | 0.3 |
| 10×iPLEX缓冲液 | 1 |
| iPLEXddNTP | 0.2 |
| 延伸引物mix | 1 |
| iPEX酶(无核酶水稀释10倍) | 0.5 |
优选的，所述S4中延伸程序为：95℃/60s，1个循环；95℃/10s，55℃/10s， 80℃/5s，40个循环；72℃/3min，1个循环；4℃/hold。
另一方面，本发明提供一种用于结直肠癌易感基因SNP检测的试剂盒，关键在于：包括以上方案的特异性引物、延伸引物及试剂。
与现有技术相比，本发明具有以下有益效果：
1.本发明通过对3′aQTL与结直肠癌疾病风险关联分析，得到9个与结直肠癌高度相关的SNP位点，包括rs12065110,rs10737238,rs665211，rs4858894, rs34747192,rs9804992，rs6604676,rs8103493,rs4652760，针对9个SNP位点设计9对特异性引物及9个延伸引物；
利用9对特异性引物及9个延伸引物对基因样本进行PCR扩增及单碱基延伸后在核酸质谱系统进行上述9个SNP位点的平行检测，一次检测可得到9个位点全部的SNP信息，可用于肿瘤的早期筛查、早期诊断、疗效评价和追踪。
具体实施方式
为使本领域技术人员更好的理解本发明的技术方案，下面结合具体实施方式对本发明作详细说明。
实施例1用于结直肠癌易感基因SNP检测的试剂盒
包括Qiagen游离DNA提取试剂盒(Cat:56304)、虾碱性磷酸酶(SAP，thermo, Cat:783901000UN)、Agena单碱基延伸试剂盒及9对特异性引物及9个延伸引物；其中所述9对特异性引物的序列如下：
Seq ID NO.1：F-CTCCTGAGTAGCTGGGATTACAGGT，
Seq ID NO.2：R-ATCACTTCAGGTCAGGAGTTCAAAACCA；
Seq ID NO.3：F-CAGTGTGGTGCAGTGGTTAGAGCAT，
Seq ID NO.4：R-ACTTTATATCCAAGGAGGCCTGACACA；
Seq ID NO.5：F-TGCAGCACTTAACGCAGTGTCCTC，
Seq ID NO.6：R-AATCGATAGTGGGTATGTATAAAGTGCGTA；
Seq ID NO.7：F-TGTGTAACAGACGTCACCTTCAGC，
Seq ID NO.8：R-GGTTTCTCTGTATCCCTATGTAAAGGACT；
Seq ID NO.9：F-GATAGAGAGTTTCTAATCCTTTGGTGA，
Seq ID NO.10：R-CTACATTTGGATATCTGTGAACCTT；
Seq ID NO.11：F-TCTGGTGCTCATTTCCTTCCAGTGGGACA，
Seq ID NO.12：R-ATGCCAGCTGCCATTGTGATGGGA；
Seq ID NO.13：F-TCATGGAAAGAGTATTGATTTATGCC，
Seq ID NO.14：R-TAAACCTGTGTTGTATTTAATCAAGTT；
Seq ID NO.15：F-AGTGAGCTGTGATCATGCCAAGGTACT，
Seq ID NO.16：R-TGAGCCCTGGCCCACAGAACGAGA；
Seq ID NO.17：F-ATAGGAAATCACAAGGGTATTGACTGG，
Seq ID NO.18：R-TTTCATGGACATGTATTAGTTCCCCAA；
延伸引物序列如下：
Seq ID NO.19：CTAATTTTTGTATTTTCAGTAGAG；
Seq ID NO.20：GGCTGTGGAGGCAGATGGATCTCAGGCCAGCC；
Seq ID NO.21：GCAGTGTCCTCATACTCTTTTATTTCTTCTTGG；
Seq ID NO.22：CTCTCTGTCAAACAGGCATTCTTCCCCCC；
Seq ID NO.23：TGAAAAGTAAAAGAAGGTTAAAATA；
Seq ID NO.24：TCCCTTGCTCCCTCAGTACCCTGTCTCTCA；
Seq ID NO.25：CTCTAATCAGTATCGGTGAAGTCACC；
Seq ID NO.26：AAACCTGTCACCTTTTTTTTGGGGGGGG；
Seq ID NO.27：ATGAAATCTTTACAATTTATGCTTAGAGA。
实施例2检测结直肠癌易感基因SNP的方法
1.使用Qiagen游离DNA提取试剂盒从口腔拭子中提取人基因组；
2.将提取后的10ng基因组DNA作为模板，加入9对特异性引物及试剂形成扩增体系如下：
| 组分 | 单个反应加入量(μL) |
| KAPA HiFi HotStartReadyMix(2X) | 2.5 |
| 10×引物mix | 0.5 |
| 基因组DNA(10ng/μL) | 1 |
| 无核酶水 | 1 |
设置如下程序在PCR仪中进行多重PCR扩增：
3.将扩增产物中加入虾碱性磷酸酶及试剂形成消化体系如下：
| 组分 | 单个反应加入量(μL) |
| 上步PCR体系 | 5 |
| SAP(1U/μL) | 0.5 |
| 10×SAP缓冲液 | 0.7 |
| 无核酶水 | 0.8 |
设置如下程序进行多余引物及dNTP消化：37℃/60min；80℃/15min，4℃/hold。
4.将消化产物中加入9个延伸引物及试剂形成单碱基延伸体系如下：
| 组分 | 单个反应加入量(μL) |
| 上步消化体系 | 7 |
| 无核酶水 | 0.3 |
| 10×iPLEX缓冲液 | 1 |
| iPLEXddNTP | 0.2 |
| 延伸引物mix | 1 |
| iPEX酶(无核酶水稀释10倍) | 0.5 |
设置如下程序进行单碱基延伸：
5.将单碱基延伸产物加入20μL无核酶水、6mg树脂，充分混匀后，分离纯化，通过Agena核酸质谱仪对纯化的样品进行易感基因SNP检测。
对8例结直肠患者的口腔拭子样本同时采用本发明进行易感基因SNP检测，得到检测结果如下表所示：
从上表结果可以看出每个SNP位点可以有效检出，且SNP频率与既往研究基本一致。
最后需要说明，上述描述仅为本发明的优选实施例，本领域的技术人员在本发明的启示下，在不违背本发明宗旨及权利要求的前提下，可以做出多种类似的表示，这样的变换均落入本发明的保护范围之内。
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1 5 10 15
Thr Gly Ala Ala Gly Thr Cys Ala Cys Cys
20 25
<210> 26
<211> 28
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 26
Ala Ala Ala Cys Cys Thr Gly Thr Cys Ala Cys Cys Thr Thr Thr Thr
1 5 10 15
Thr Thr Thr Thr Gly Gly Gly Gly Gly Gly Gly Gly
20 25
<210> 27
<211> 29
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 27
Ala Thr Gly Ala Ala Ala Thr Cys Thr Thr Thr Ala Cys Ala Ala Thr
1 5 10 15
Thr Thr Ala Thr Gly Cys Thr Thr Ala Gly Ala Gly Ala
20 25
Claims (10)
1.用于结直肠癌易感基因SNP检测的引物，其特征在于：包括与结直肠癌高度相关的9对特异性引物及9个延伸引物；其中所述9对特异性引物的序列如下：
Sea ID NO.1：F-CTCCTGAGTAGCTGGGATTACAGGT，
Seq ID NO.2：R-ATCACTTCAGGTCAGGAGTTCAAAACCA；
Seq ID NO.3：F-CAGTGTGGTGCAGTGGTTAGAGCAT，
Seq ID NO.4：R-ACTTTATATCCAAGGAGGCCTGACACA；
Sea ID NO.5：F-TGCAGCACTTAACGCAGTGTCCTC，
Seq ID NO.6：R-AATCGATAGTGGGTATGTATAAAGTGCGTA；
Seq ID NO.7：F-TGTGTAACAGACGTCACCTTCAGC，
Seq ID NO.8：R-GGTTTCTCTGTATCCCTATGTAAAGGACT；
Sea ID NO.9：F-GATAGAGAGTTTCTAATCCTTTGGTGA，
Seq ID NO.10：R-CTACATTTGGATATCTGTGAACCTT；
Seq ID NO.11：F-TCTGGTGCTCATTTCCTTCCAGTGGGACA，
Sea ID NO.12：R-ATGCCAGCTGCCATTGTGATGGGA；
Sea ID NO.13：F-TCATGGAAAGAGTATTGATTTATGCC，
Seq ID NO.14：R-TAAACCTGTGTTGTATTTAATCAAGTT；
Seq ID NO.15：F-AGTGAGCTGTGATCATGCCAAGGTACT，
Sea ID NO.16：R-TGAGCCCTGGCCCACAGAACGAGA；
Sea ID NO.17：F-ATAGGAAATCACAAGGGTATTGACTGG，
Seq ID NO.18：R-TTTCATGGACATGTATTAGTTCCCCAA；
延伸引物序列如下：
Sea ID NO.19：CTAATTTTTGTATTTTCAGTAGAG；
Seq ID NO.20：GGCTGTGGAGGCAGATGGATCTCAGGCCAGCC；
Seq ID NO.21：GCAGTGTCCTCATACTCTTTTATTTCTTCTTGG；
Seq ID NO.22：CTCTCTGTCAAACAGGCATTCTTCCCCCC；
Seq ID NO.23：TGAAAAGTAAAAGAAGGTTAAAATA；
Seq ID NO.24：TCCCTTGCTCCCTCAGTACCCTGTCTCTCA；
Seq ID NO.25：CTCTAATCAGTATCGGTGAAGTCACC；
Seq ID NO.26：AAACCTGTCACCTTTTTTTTGGGGGGGG；
Sea ID NO.27：ATGAAATCTTTACAATTTATGCTTAGAGA。
2.一种结直肠癌易感基因SNP检测方法，其特征在于包括以下步骤：
S1.提取体液中游离的基因组；
S2.将基因组作为模板，加入如权利要求1所述的9对特异性引物，进行多重PCR扩增；
S3.将扩增产物中加入虾碱性磷酸酶进行多余引物及dNTP消化；
S4.将消化产物中加入权利要求1所述的9个延伸引物进行单碱基延伸；
S5.将单碱基延伸产物进行核酸质谱检测。
3.根据权利要求2所述结直肠癌易感基因SNP检测方法，其特征在于所述S1具体为：采用QiagenDNA提取试剂盒从口腔拭子中提取cfDNA。
4.根据权利要求2所述结直肠癌易感基因SNP检测方法，其特征在于所述S2的扩增程序为：98℃/45s，1个循环；98℃/15s，60℃/30s，72℃/30s，40个循环；72℃/3min，1个循环；4℃/hold。
5.根据权利要求2所述结直肠癌易感基因SNP检测方法，其特征在于所述S2中具体为：将提取后的10ng基因组DNA作为模板，加入如权利要求1所述的9对特异性引物及试剂形成扩增体系如下：
6.根据权利要求2所述结直肠癌易感基因SNP检测方法，其特征在于所述S3中具体为：将扩增产物中加入虾碱性磷酸酶及试剂形成消化体系如下：
7.根据权利要求2所述结直肠癌易感基因SNP检测方法，其特征在于所述S3的消化程序为：37℃/60min；80℃/15min，4℃/hold。
8.根据权利要求2所述结直肠癌易感基因SNP检测方法，其特征在于所述S4中具体为：将消化产物中加入权利要求1所述的9个延伸引物及试剂形成单碱基延伸体系如下：
9.根据权利要求2所述结直肠癌易感基因SNP检测方法，其特征在于所述S4中延伸程序为：95℃/60s，1个循环；95℃/10s，55℃/10s，80℃/5s，40个循环；72℃/3min，1个循环；4℃/hold。
10.一种用于结直肠癌易感基因SNP检测的试剂盒，其特征在于：包括权利要求2-9中的特异性引物、延伸引物及试剂。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006104370A1 (en) * | 2005-04-01 | 2006-10-05 | Samsung Electronics Co., Ltd. | Multiple snp for diagnosing colorectal cancer, microarray and kit comprising the same, and method of diagnosing colorectal cancer using the same |
| WO2012049279A1 (en) * | 2010-10-14 | 2012-04-19 | Universitaet Des Saarlandes | MEANS AND METHODS APPLYING SINGLE NUCLEOTIDE PRIMER EXTENSION WITH ION PAIR-, REVERSED-PHASE HPLC (SIRPH) FOR THE DIAGNOSIS OF SNPs |
| CN104232768A (zh) * | 2014-09-05 | 2014-12-24 | 北京大学人民医院 | 结直肠癌易感性诊断试剂盒和snp在其制备中的应用 |
| CN110283908A (zh) * | 2019-06-04 | 2019-09-27 | 华中科技大学 | 一种结直肠癌辅助诊断snp标志物及其应用 |
| CN111154871A (zh) * | 2019-11-22 | 2020-05-15 | 上海鉴研生物技术有限公司 | 一种基于核酸质谱技术检测结直肠癌相关基因突变的方法 |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006104370A1 (en) * | 2005-04-01 | 2006-10-05 | Samsung Electronics Co., Ltd. | Multiple snp for diagnosing colorectal cancer, microarray and kit comprising the same, and method of diagnosing colorectal cancer using the same |
| WO2012049279A1 (en) * | 2010-10-14 | 2012-04-19 | Universitaet Des Saarlandes | MEANS AND METHODS APPLYING SINGLE NUCLEOTIDE PRIMER EXTENSION WITH ION PAIR-, REVERSED-PHASE HPLC (SIRPH) FOR THE DIAGNOSIS OF SNPs |
| CN104232768A (zh) * | 2014-09-05 | 2014-12-24 | 北京大学人民医院 | 结直肠癌易感性诊断试剂盒和snp在其制备中的应用 |
| CN110283908A (zh) * | 2019-06-04 | 2019-09-27 | 华中科技大学 | 一种结直肠癌辅助诊断snp标志物及其应用 |
| CN111154871A (zh) * | 2019-11-22 | 2020-05-15 | 上海鉴研生物技术有限公司 | 一种基于核酸质谱技术检测结直肠癌相关基因突变的方法 |
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