CN113999915A - 用于食管癌早期筛查的生物标志物组合、试剂盒及应用 - Google Patents
用于食管癌早期筛查的生物标志物组合、试剂盒及应用 Download PDFInfo
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Abstract
本发明涉及分子生物技术领域,特别涉及用于食管癌早期筛查的生物标志物组合、试剂盒及应用,生物标志物组合包括与食管癌人群早期甲基化位点相关的4对靶标特异引物、4个特异性引物探针及对应的内参引物、内参引物探针。与现有技术相比,本发明通过筛选中国人群食管癌早期基因组超甲基化区域,针对这些区域选择含有CCGC,CCGG,GCGC,ACGT,GCGG中至少两个位点的区间作为靶点设计若干对PCR引物,最终筛选出4对可以在甲基化敏感内切酶的辅助下可以区分肿瘤组织与正常组织的PCR效率高,特异性强,稳定性好的引物及对应探针序列,提高食道癌早癌检出率、改善食道癌治疗效果、降低食道癌死亡率。
Description
技术领域
本发明涉及分子生物技术领域,特别涉及用于食管癌早期筛查的生物标志物组合、试剂盒及应用。
背景技术
食管癌是常见的恶性肿瘤之一,常发生在消化系统里,是一种原发于食管上皮的恶性肿瘤。此病在我国的发病率和死亡率居于前列,是高发癌症。诱发食管癌的因素有很多,例如吸烟、饮酒、遗传等因素。研究发现,食管癌早期患者的五年生存率可达到95%以上,但多数食管癌患者的早期症状不典型,若出现进行件吞咽困难一般已属中晚期,而中晚期患者预后差,五年生存率较低,总生存率不超过15%,因此建立食管癌预警和早期筛查的方法,对食管癌的防治非常重要,早发现、早诊断、早治疗、早手术是防控食管癌的有效手段。
目前用于食道癌的检测和筛查方法有影像学检测技术、组织活检、肿瘤血清标识物检测等。这此方法不仅设备成本高,操作技术要求强,检测成本高;并且侵入性强,不适于早癌的筛查;且灵敏度很低,无法满足临床检测需要。因此有必要开发灵敏、特异的新型食道癌标识物和检测技术,提高食道癌早癌检出率、改善食道癌治疗效果、降低食道癌死亡率。
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食管癌早期筛查的生物标志物组合、试剂盒及应用,解决了食道癌早期检测敏感度低,特异性差的问题。
9.本发明采用的技术方案如下:用于食管癌早期筛查的生物标志物组合,关键在于:包括与食管癌人群早期甲基化位点相关的4对靶标特异引物、4个特异性引物探针及对应的内参引物、内参引物探针;其中所述4对靶标特异引物的序列如下:
Seq ID NO.1:F-CCCGGCGCGATTGCAAAGTTTTCGT,
Seq ID NO.2:R-CCGCCCCGTCGCCGAGTCC;
Seq ID NO.3:F-GTCAGCAGCACCCCCGAGCCTT,
Seq ID NO.4:R-CGCACTCTTGGGGCGGGGTCCTATCTC;
Seq ID NO.5:F-GGTACTGTGAAGGGTCCGGGTC,
Seq ID NO.6:R-AAGTCGGGGTCGAAAGTCCTC;
Seq ID NO.7:F-ACGTTTGCACGCAGGTTCA,
Seq ID NO.8:R-CCGGGATCGTGTCCGTAAGC;
内参引物的序列如下:
Seq ID NO.9:F-GGTGCCAGATTTTCTCCATGTCGTC,
Seq ID NO.10:R-ACGAGGCCCAGAGCAAGAGA;
所述特异性引物探针序列如下:
Seq ID NO.11:FAM-CCCCTCTGGCCCGGAGTTGCGGCTGA-BHQ1,
Seq ID NO.12:FAM-CCTGCGCCGTCCCCTCCGCCCGTA-BHQ1;
Seq ID NO.13:FAM-CCAAAGCCCGAGTCCGCCCTG-BHQ1;
Seq ID NO.14:FAM-AAGATAAAAGGGAAAGCGCCTCCACGAA-BHQ1;
内参引物探针序列如下:
Seq ID NO.15:VIC-TACCCCATCGAGCACGGCATCGTC-BHQ1。
用于食管癌早期筛查的试剂盒,关键在于:包括用于食管癌早期筛查的生物标志物组合及甲基化敏感内切酶体系。
优选的,所述甲基化敏感内切酶体系包含HinP1I,HpaII,AciI,HpyCH4IV。
试剂盒在制备食管癌早期筛查试剂中的应用,关键在于筛查方法包括以下步骤:
S1.提取血浆中游离的cfDNA;
S2.将cfDNA与甲基化敏感内切酶体系在37℃下孵育;
S3.将孵育产物作为模板,加入如权利要求3所述的试剂盒中的4对靶标特异引物及内参引物,进行多重PCR扩增;
S4.将扩增产物作为模板,加入如权利要求3所述的试剂盒中的4对靶标特异引物、4个特异性引物探针、内参引物探针及内参引物,进行qPCR检测反应;
S5.根据4对靶标特异引物与内参引物的Ct值之差ΔCt,评估食管癌风险;所述S5的评估方式为:
ΔCt=4对引物特异扩增Ct值-内参引物扩增Ct值;
ΔCt>5.03食管癌低风险;
ΔCt≤5.03食管癌高风险。
优选的,所述S1具体为:采用Qiagen血浆游离DNA提取试剂盒提取血液中的cfDNA。
优选的,所述S2的孵育条件为:37℃孵育16h,在80℃酶失活20min。
优选的,所述S3的扩增程序为:98℃/45s,8个循环;98℃/15s,55℃/30s,72℃/30s,8个循环;72℃/1min,1个循环;4℃/hold。
根据权利要求4所述的应用,其特征在于所述S4的qPCR检测反应程序为:95℃/3min,1个循环;98℃/15s,60℃/60s,45个循环。
与现有技术相比,本发明具有以下有益效果:
1.本发明通过对比食管癌(包括鳞癌和腺癌)与癌变甲基化数据,筛选中国人群食管癌早期基因组超甲基化区域,并与白细胞基因组对应区域进行比对,筛选在食管癌中呈现超甲基化状态,而在白细胞中呈现低甲基化状态的区域。针对这些区域选择含有CCGC,CCGG,GCGC,ACGT,GCGG中至少两个位点的区间作为靶点设计若干对PCR引物,最终筛选出4对可以在甲基化敏感内切酶的辅助下可以区分肿瘤组织与正常组织的PCR效率高,特异性强,稳定性好的引物及对应探针序列;
2.利用甲基化敏感内切酶不能切割甲基化位点的特性,降解非肿瘤来源cfDNA,特异扩增肿瘤来源ctDNA极大提升检查灵敏度,克服了单个DNA甲基化信号低的问题,提高了检测的灵敏度及特异性,提高食道癌早癌检出率、改善食道癌治疗效果、降低食道癌死亡率;
3.基于DNA甲基化的检测简单易行、判读客观,避免了人为判读结果的主观性,提高了准确率;可以区分食道癌患者与健康人群,用于肿瘤的早期筛查、早期诊断、疗效评价和追踪以及预后评估。
附图说明
图1为基于本发明的风险判定图;
图2为基于本发明的分类性能AUC曲线图;
图3为基于本发明的风险模型测试图。
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,下面结合附图和具体实施方式对本发明作详细说明。
实施例1用于食管癌早期筛查的试剂盒
包括Qiagen血浆游离DNA提取试剂盒、包含HinP1I,HpaII,AciI,HpyCH4IV的甲基化敏感内切酶体系、4对靶标特异引物及内参引物、4对特异性引物探针、内参引物探针及内参引物探针;
其中其中所述4对靶标特异引物的序列如下:
Seq ID NO.1:F-CCCGGCGCGATTGCAAAGTTTTCGT,
Seq ID NO.2:R-CCGCCCCGTCGCCGAGTCC;
Seq ID NO.3:F-GTCAGCAGCACCCCCGAGCCTT,
Seq ID NO.4:R-CGCACTCTTGGGGCGGGGTCCTATCTC;
Seq ID NO.5:F-GGTACTGTGAAGGGTCCGGGTC,
Seq ID NO.6:R-AAGTCGGGGTCGAAAGTCCTC;
Seq ID NO.7:F-ACGTTTGCACGCAGGTTCA,
Seq ID NO.8:R-CCGGGATCGTGTCCGTAAGC;
内参引物的序列如下:
Seq ID NO.9:F-GGTGCCAGATTTTCTCCATGTCGTC,
Seq ID NO.10:R-ACGAGGCCCAGAGCAAGAGA;
所述特异性引物探针序列如下:
Seq ID NO.11:FAM-CCCCTCTGGCCCGGAGTTGCGGCTGA-BHQ1,
Seq ID NO.12:FAM-CCTGCGCCGTCCCCTCCGCCCGTA-BHQ1;
Seq ID NO.13:FAM-CCAAAGCCCGAGTCCGCCCTG-BHQ1;
Seq ID NO.14:FAM-AAGATAAAAGGGAAAGCGCCTCCACGAA-BHQ1;
内参引物探针序列如下:
Seq ID NO.15:VIC-TACCCCATCGAGCACGGCATCGTC-BHQ1。
实施例2检测ctDNA甲基化水平变化的方法
1.取5mL血浆,采用Qiagen血浆游离DNA提取试剂盒(Cat:55204)提取游离cfDNA;
2.取20ng cfDNA与20μHinP1I,HpaII,AciI,HpyCH4IV四种甲基化敏感内切酶体系(终浓度10U/μL),于37℃孵育16h,80℃酶失活20min,体系如下表1所示;
表1
将孵育完成的全部产物作为模板,4对靶标特异引物(每种50nM)加1个内参引物(10nM)配置体系(见表3),设置以下表4设定程序在普通PCR仪中进行扩增程序;
表3
表4
4.取上步扩增产物1μL做为模板,加入4对靶标特异引物(每种0.25μM),4个特异性引物探针(每种0.1μM)加1个内参引物(0.05μM)及内参引物探针(0.02μM)构成混合体系(见表5),设置以下表6设定程序在ABI 7500荧光PCR仪中进行qPCR反应,每轮循环结束前采集FAM及VIC通道信号;
表5
表6
根据内参引物与4对靶标特异引物的Ct值之差ΔCt,如图1所示,评估食管癌风险,ΔCt=4对引物特异扩增Ct值-内参引物扩增Ct值;
ΔCt>5.03食管癌低风险
ΔCt≤5.03食管癌高风险,建议定期CT检测。
对35例健康者和45例Ⅰ-Ⅲ期食道癌患者的血液样本同时采用本发明评估食道癌风险性能,样本信息和测试结果如下表7所示:
表7
将表7中的数据导入到Graphpadprism 6.0软件中,自动生成ROC曲线,并统计曲线下面积AUC值(图2),横坐标为1-特异度,纵坐标为敏感度,软件统计结果以ΔCt≤5.03为阈值,分类器敏感度63.04%,特异度94.30%;
以ΔCt≤5.03为阈值,构建风险模型,将对35例健康者和45例Ⅰ-Ⅲ期食道癌患者进行食道癌风险等级评估(图3)。
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。
序列表
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Claims (8)
1.用于食管癌早期筛查的生物标志物组合,其特征在于:包括与食管癌人群早期甲基化位点相关的4对靶标特异引物、4个特异性引物探针及对应的内参引物、内参引物探针;其中所述4对靶标特异引物的序列如下:
Seq ID NO.1:F-CCCGGCGCGATTGCAAAGTTTTCGT,
Seq ID NO.2:R-CCGCCCCGTCGCCGAGTCC;
Seq ID NO.3:F-GTCAGCAGCACCCCCGAGCCTT,
Seq ID NO.4:R-CGCACTCTTGGGGCGGGGTCCTATCTC;
Seq ID NO.5:F-GGTACTGTGAAGGGTCCGGGTC,
Seq ID NO.6:R-AAGTCGGGGTCGAAAGTCCTC;
Seq ID NO.7:F-ACGTTTGCACGCAGGTTCA,
Seq ID NO.8:R-CCGGGATCGTGTCCGTAAGC;
内参引物的序列如下:
Seq ID NO.9:F-GGTGCCAGATTTTCTCCATGTCGTC,
Seq ID NO.10:R-ACGAGGCCCAGAGCAAGAGA;
所述特异性引物探针序列如下:
Seq ID NO.11:FAM-CCCCTCTGGCCCGGAGTTGCGGCTGA-BHQ1,
Seq ID NO.12:FAM-CCTGCGCCGTCCCCTCCGCCCGTA-BHQ1;
Seq ID NO.13:FAM-CCAAAGCCCGAGTCCGCCCTG-BHQ1;
Seq ID NO.14:FAM-AAGATAAAAGGGAAAGCGCCTCCACGAA-BHQ1;
内参引物探针序列如下:
Seq ID NO.15:VIC-TACCCCATCGAGCACGGCATCGTC-BHQ1。
2.用于食管癌早期筛查的试剂盒,其特征在于:包括权利要求1所述的用于食管癌早期筛查的生物标志物组合及甲基化敏感内切酶体系。
3.根据权利要求2所述用于食管癌早期筛查的试剂盒,其特征在于:所述甲基化敏感内切酶体系包含HinP1I,HpaII,AciI,HpyCH4IV。
4.根据权利要求3所述的试剂盒在制备食管癌早期筛查试剂中的应用,其特征在于筛查方法包括以下步骤:
S1.提取血浆中游离的cfDNA;
S2.将cfDNA与甲基化敏感内切酶体系在37℃下孵育;
S3.将孵育产物作为模板,加入如权利要求3所述的试剂盒中的4对靶标特异引物及内参引物,进行多重PCR扩增;
S4.将扩增产物作为模板,加入如权利要求3所述的试剂盒中的4对靶标特异引物、4个特异性引物探针、内参引物探针及内参引物,进行qPCR检测反应;
S5.根据4对靶标特异引物与内参引物的Ct值之差ΔCt,评估食管癌风险;所述S5的评估方式为:
ΔCt=4对引物特异扩增Ct值-内参引物扩增Ct值;
ΔCt>5.03食管癌低风险;
ΔCt≤5.03食管癌高风险。
5.根据权利要求4所述的应用,其特征在于所述S1具体为:采用Qiagen血浆游离DNA提取试剂盒提取血液中的cfDNA。
6.根据权利要求4所述的应用,其特征在于所述S2的孵育条件为:37℃孵育16h,在80℃酶失活20min。
7.根据权利要求4所述的应用,其特征在于所述S3的扩增程序为:98℃/45s,8个循环;98℃/15s,55℃/30s,72℃/30s,8个循环;72℃/1min,1个循环;4℃/hold。
8.根据权利要求4所述的应用,其特征在于所述S4的qPCR检测反应程序为:95℃/3min,1个循环;98℃/15s,60℃/60s,45个循环。
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