CN106636306A - 一种用于结直肠癌中相关基因启动子甲基化的检测方法 - Google Patents
一种用于结直肠癌中相关基因启动子甲基化的检测方法 Download PDFInfo
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Abstract
本发明涉及一种用于结直肠癌中相关基因启动子甲基化的检测方法,本发明涉及与三个大肠癌相关基因FBN1、SNCA、SPG20,包括这三个基因甲基化互补的特异性探针序列,通过对特异性序列的扩增和检测,可以快速高效确定甲基化位点,只需少量粪便样本完成大肠癌检测,具有良好的临床应用价值。
Description
技术领域
本发明涉及用于结直肠癌相关基因启动子超甲基化的新型标记物,本发明结直肠癌相关基因超甲基化状态检测技术领域,特别是涉及一种甲基化荧光PCR定性检测大肠癌技术。
背景技术
随着人民生活水平的提高,大肠癌(包括结直肠癌)已成为我国常见恶性肿瘤之一。截至2012年,我国大肠癌死亡率已跃居各肿瘤第五位,发病率也以每年4.2%的速率递增。大肠癌及相关疾病已成为危害我国人民健康的重要因素。
由于大肠癌早期症状不明显,仅有不适、消化不良、大便潜血等等,难以引起注意,加上人们长期以来缺乏体检意识,约有34%的患者入院即诊断为中晚期,并常伴有全身转移,大大降低了治疗效果。因此,迫切需要一种能够达成早期诊断。准确率高并且无痛苦的大肠癌检测技术。
目前,市场上已有数种粪便检测试剂盒,可对粪便隐血及其中少量蛋白进行检测,但由于粪便样本数量的限制等因素,无法检测出粪便中少量DNA等分子标记物,而这类分子标记物已被证实具有高参考价值,特别是对于大肠癌等消化道肿瘤的早期诊断具有重要意义。肠镜是一种侵入性检查,可能有并发症的发生,给病人带来的痛苦让患者望而生畏。因此,改进现有技术以提高检测能力,对于提高大肠癌的早期诊断率至关重要。
发明内容
为了克服上述现有技术的不足,本发明提供了一种快速检测大肠癌相关基因的甲基化荧光PCR技术,包括相关引物和探针组合物。与传统检测方法相比,本方案针对性更强,能够有效避免假阳性,大大提高大肠癌早期诊断率,同时具有容易操作、经济高效的特性。
为达到以上目的,本发明采用以下技术方案:
a 提取粪便中DNA;
b 对提取DNA进行甲基化修饰;
c 采用PCR引物及探针对步骤b中所述的修饰后DNA进行荧光定性检测;
d 根据荧光PCR中手机的荧光信号形成的扩增曲线,分析提取DNA中中是否存在大肠癌早期DNA
所述的荧光PCR定性检测中,采用引物及探针序列如下:
FBN1
上游引物GAGTTATAGTTGGGATAGTTGCGAGC
下游引物 AACGACGACTCCGACTCCC
探针
6FAM-CGCTACAACCACTACTCGA-MGB
SNCA
上游引物 GCGTTTTGGGCGTTTTTTTAC
下游引物 CGCTATAAACCGACGACGC
探针
6FAM-CGCTAACCTATCGTCGAA-MGB
SPG20
上游引物 GCGCGTCGTGGAACGT
下游引物 CTACGCTCGCCGAAAACC
探针
6FAM-CGCGCTTACCGTAACAA-MGB
所属的试剂盒组成包括
本发明的原理是基本原理在于:样本经亚硫氢酸盐处理后,甲基化的胞嘧啶(C)保持不变,但非甲基化的胞嘧啶被转化成脲嘧啶,因此在利用该处理产物作为模板的PCR产物中,甲基化的胞嘧啶还是胞嘧啶,但非甲基化胞嘧啶变成了脲嘧啶(胸腺嘧啶),此时检测到的胞嘧啶(C)即是样品中本身的甲基化位点;
本发明的创新之处在于通过荧光PCR技术,检测粪便样本中大肠癌早期相关基因。相对于传统技术,本方法中基因具有特异性,准确性高达95%,操作简便,经济便捷,具有良好的临床实用价值。
附图说明
图1 荧光PCR定性检测FBN1扩增图
图2荧光PCR定性检测SNCA扩增图
图3荧光PCR定性检测SPG20扩增图
图中:包含四个样本,阳性对照A,阳性病例B,阴性对照C,阴性病D。
具体实施方式
反应体系的建立与优化
PCR反应体系
引物和探针浓度的优化
DNA甲基化方法:
1、将约2ugDNA于1.5mlEP管中使用DDW稀释至50ul;
2、加5.5ul新鲜配制的3M NaOH;
3、42℃水浴30min; 水浴期间配制;
4、10mM对苯二酚(氢醌),加30ul至上述水浴后混合液中;(溶液变成淡黄色)
5、加3.6M亚硫酸氢钠520ul至上述水浴后溶液中;
6、EP管外裹以铝箔纸,避光,轻柔颠倒混匀溶液;
7、加200 ul石蜡油,防止水分蒸发,限制氧化;
8、50℃避光水浴16h。
Claims (5)
1.本专利涉及一种荧光PCR定性检测基因FBN1的甲基化引物及探针序列,其特征在于,所述的序列为
上游引物GAGTTATAGTTGGGATAGTTGCGAGC
下游引物
AACGACGACTCCGACTCCC
探针
6FAM-CGCTACAACCACTACTCGA-MGB。
2. 本专利涉及一种荧光PCR定性检测基因SNCA的甲基化引物及探针序列,其特征在于,所述的序列为
上游引物
GCGTTTTGGGCGTTTTTTTAC
下游引物
CGCTATAAACCGACGACGC
探针
6FAM-CGCTAACCTATCGTCGAA-MGB。
3.本专利涉及一种荧光PCR定性检测基因SPG20的甲基化引物及探针序列,其特征在于,所述的序列为
上游引物
GCGCGTCGTGGAACGT
下游引物
CTACGCTCGCCGAAAACC
探针
6FAM-CGCGCTTACCGTAACAA-MGB。
4.根据权利要求1-3所述的三种甲基化引物及探针序列,其特征在于,其应用对象为经亚硫酸氢钠甲基化处理的人类DNA。
5.根据权利要求4所述的人类DNA,其特征在于DNA来自于粪便样本。
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Cited By (3)
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CN111534590A (zh) * | 2020-04-30 | 2020-08-14 | 福建西陇生物技术有限公司 | 一种结直肠癌多基因甲基化联合检测试剂盒及其应用 |
CN112538529A (zh) * | 2019-11-14 | 2021-03-23 | 广州市基准医疗有限责任公司 | 血液中结直肠癌的分子标记物及其检测试剂盒和方法 |
CN114085904A (zh) * | 2020-08-24 | 2022-02-25 | 广州达健生物科技有限公司 | 结直肠癌基因甲基化检测引物探针组合及其试剂盒与应用 |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112538529A (zh) * | 2019-11-14 | 2021-03-23 | 广州市基准医疗有限责任公司 | 血液中结直肠癌的分子标记物及其检测试剂盒和方法 |
CN111534590A (zh) * | 2020-04-30 | 2020-08-14 | 福建西陇生物技术有限公司 | 一种结直肠癌多基因甲基化联合检测试剂盒及其应用 |
CN114085904A (zh) * | 2020-08-24 | 2022-02-25 | 广州达健生物科技有限公司 | 结直肠癌基因甲基化检测引物探针组合及其试剂盒与应用 |
CN114085904B (zh) * | 2020-08-24 | 2024-02-23 | 广州达健生物科技有限公司 | 结直肠癌基因甲基化检测引物探针组合及其试剂盒与应用 |
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