CN112063712A - 基于高分辨率熔解曲线检测septin9基因甲基化的特异引物对和试剂盒 - Google Patents
基于高分辨率熔解曲线检测septin9基因甲基化的特异引物对和试剂盒 Download PDFInfo
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Abstract
本发明公开了一种基于高分辨率熔解曲线检测septin9基因甲基化的特异引物对和试剂盒。本发明首先保护一种特异引物对,由SEQ ID NO:1所示的单链DNA分子和SEQ ID NO:2所示的单链DNA分子组成。本发明还保护所述特异引物对在制备试剂盒中的应用。所述试剂盒的功能为诊断或辅助诊断结直肠癌、检测septin9基因启动子区甲基化、检测septin9基因启动子区CpG岛甲基化。采用本发明提供的引物对通过甲基化特异性PCR结合HRM曲线分析检测septin9基因甲基化,具有操作简便、特异性好、灵敏度高、成本低廉的优点。本发明可用于结直肠癌患者的早筛或辅助诊断,在健康筛查和临床辅助诊断中具有很好的前景。
Description
技术领域
本发明属于生物技术领域,涉及一种基于高分辨率熔解曲线检测septin9基因甲基化的特异引物对和试剂盒。
背景技术
结直肠癌是常见消化道恶性肿瘤,发病率仅次于胃癌和食道癌。在我国恶性肿瘤的病死率中,结直肠癌患者在男性中占第5位,女性中占第6位。近20年来结直肠癌的发病率在逐渐增加,在我国超过80%患者确诊时已发展到中晚期,早期诊断率仅为10%-15%,国内调查显示,早期术后存活率达90%-95%,而晚期则只有5%。手术治疗可使早期结直肠癌得到根治,而晚期结直肠癌经化学放疗联合分子靶向治疗后平均生存时间仍不足30个月,因此,结直肠癌早期诊断显得非常重要和迫切。
DNA甲基化状态改变与结直肠癌的发生发展关系密切。结直肠癌患者中septin9基因CpG岛高度甲基化。因此,septin9基因CpG岛甲基化可作为结直肠癌潜在的诊断标志物。Septin9基因在结直肠癌发生中起抑癌基因作用,甲基化会抑制该基因正常表达,使其抑癌功能丧失,并最终导致细胞***和癌变。研究发现Septin9基因在结直肠癌患者的肿瘤组织和外周血中均高表达,而在健康人群或其他疾病患者中均低表达,且灵敏度和特异度最高,由此建立了血浆中Septin9基因表达量是结直肠癌最佳候选标志物的基础理念;随后,又通过实验分析结直肠癌患者和健康对照者血浆样本中Septin9基因表达情况发现,在结直肠癌组织中Septin9基因存在高度的甲基化。在结直肠癌患者中Septin9基因CpG岛发生甲基化,正常人Septin9基因CpG岛不发生甲基化。
基因甲基化检测技术大致可以分为两类,特异位点的甲基化检测和全基因组的甲基化分析,后者也称为甲基化图谱分析。甲基化特异性PCR(MS-PCR),具有灵敏度高、不受内切酶的限制、无需特殊仪器、经济实用的优点,并且可用于石蜡包埋样本。对于甲基化特异性PCR来说,最重要的是设计出质量好的引物。亚硫酸氢盐处理结合测序方法,是DNA甲基化分析的金标准,可靠且精确度高,能明确目的片段中每一个CpG位点的甲基化状态,但需要大量的克隆测序,过程较为繁琐。
荧光定量方法(Methylight)优势在于高通量和高灵敏度,且无需在PCR后电泳、杂交等操作,减少污染和操作误差。高分辨率熔解曲线分析(High-resolution melting,HRM)是一种新的分子检测技术,广泛应用于基因突变、基因甲基化水平检测及基因分型检测和HLA配型等领域,具有低成本、高通量、无污染、快捷、操作简便、敏感性和特异性好的特点,灵敏度大于1-5%,且不需要对PCR产物进行后续操作。高分辨率熔解技术与普通熔解曲线原理是一致的,都是通过实时监测升温过程中双链DNA荧光染料与PCR扩增产物的结合情况;二者不同之处主要表现在升温速率和所用的染料。它使用的是一种新型饱和染料(如EVAGreen),解链时染料脱落,荧光信号降低,通过检测荧光信号随温度的变化即可得到扩增产物的熔解曲线。传统熔解曲线技术应用的染料在高浓度时会抑制PCR,故难以达到足够的浓度与PCR产物饱和结合,而HRM应用的饱和染料在相当大的浓度范围内均不影响PCR扩增,故能与PCR产物饱和结合而更准确的反映DNA解链过程。这种染料具有更强的DNA结合能力和很低的抑制作用,同时结合带有高分辨率熔解分析功能的实时荧光定量PCR仪(如Rotor-Gene Q)精确的温度控制能力。此外,HRM熔解仪与常规熔解仪相比,具有更高的数据采集速率、荧光信号敏感度和温度精确度,并确保样品间温度的一致,从而大大提高了数据的精确性。
发明内容
本发明的目的是提供一种基于高分辨率熔解曲线检测septin9基因甲基化的特异引物对和试剂盒。
本发明首先保护一种特异引物对,由SEQ ID NO:1所示的单链DNA分子和SEQ IDNO:2所示的单链DNA分子组成。
所述特异引物对的功能为诊断或辅助诊断结直肠癌。
所述特异引物对的功能为检测septin9基因启动子区甲基化。
所述特异引物对的功能为检测septin9基因启动子区CpG岛甲基化。
本发明还保护所述特异引物对在制备试剂盒中的应用;所述试剂盒的功能为诊断或辅助诊断结直肠癌。
本发明还保护所述特异引物对在制备试剂盒中的应用;所述试剂盒的功能为检测septin9基因启动子区甲基化。
本发明还保护所述特异引物对在制备试剂盒中的应用;所述试剂盒的功能为检测septin9基因启动子区CpG岛甲基化。
本发明还保护一种试剂盒,包括所述特异引物对;所述试剂盒的功能为诊断或辅助诊断结直肠癌。
本发明还保护一种试剂盒,包括所述特异引物对;所述试剂盒的功能为检测septin9基因启动子区甲基化。
本发明还保护一种试剂或试剂盒,包括所述特异引物对;所述试剂盒的功能为检测septin9基因启动子区CpG岛甲基化。
以上任一所述试剂盒还包括PCR扩增试剂和荧光染料。
所述荧光染料具体可为饱和染料EVAGreen。
所述PCR扩增试剂具体包括:TaKaRa Ex Taq、10×Ex Taq Buffer(Mg2+free)、MgCl2、dNTP Mixture。
以上任一所述试剂盒还包括对DNA进行亚硫酸氢盐处理的试剂。
以上任一所述试剂盒还包括记载有如下检测方法的载体:
(1)提取待测样本的总DNA,然后进行亚硫酸氢盐处理;
(2)进行PCR扩增以及高分辨率熔解曲线分析。
PCR扩增的反应程序具体见表2。
高分辨率熔解曲线分析的参数设置具体可为:65℃1s,然后按0.02℃/s的速度从65℃升至95℃(每秒收集25次荧光),最后40℃30s。
以上任一所述试剂盒还包括记载有如下判断标准的载体:在87℃±0.5℃位置出现熔解峰说明样本存在septin9基因甲基化(为候选的结直肠癌患者),未在87℃±0.5℃位置出现熔解峰说明样本不存在septin9基因甲基化(为候选的非结直肠癌患者)。
本发明还保护所述试剂盒的制备方法,包括将各条引物进行独立包装的步骤。
以上任一所述septin9基因为编码人septin9蛋白的基因。
人septin9蛋白具体如序列表的序列4所示。
人cDNA中,septin9基因的开放阅读框具体如序列表的序列3中第309-2048位所示。
septin9基因启动子区CpG岛具体可为序列表的序列3中第34-204位核苷酸。
为了解决现有甲基化检测方法中操作繁琐、存在污染风险、灵敏度低和特异性不好的缺点,本发明提供了一对用于检测septin9甲基化的特异性引物,在septn9基因检测引物中加入了CG位点,选择性的使引物与甲基化序列互补结合,弥补对非甲基化序列倾向性导致的PCR偏倚,有效的增加septin9基因检测的灵敏度,可比甲基化荧光定量PCR具有更好的灵敏度。
采用本发明提供的引物对通过甲基化特异性PCR结合HRM曲线分析检测septin9基因甲基化,具有操作简便、特异性好、灵敏度高、成本低廉的优点。本发明可用于结直肠癌患者的早筛或辅助诊断,在健康筛查和临床辅助诊断中具有很好的前景。
附图说明
图1为实施例3中采用引物对甲时HRM曲线分析的荧光变化曲线。
图2为实施例3中采用引物对甲时的熔解曲线峰图(100%甲基化样本)。
图3为实施例3中采用引物对甲时的熔解曲线峰图(75%甲基化样本)。
图4为实施例3中采用引物对甲时的熔解曲线峰图(50%甲基化样本)。
图5为实施例3中采用引物对甲时的熔解曲线峰图(25%甲基化样本)。
图6为实施例3中采用引物对甲时的熔解曲线峰图(10%甲基化样本)。
图7为实施例3中采用引物对甲时的熔解曲线峰图(5%甲基化样本)。
图8为实施例3中采用引物对甲时的熔解曲线峰图(2%甲基化样本)。
图9为实施例3中采用引物对甲时的熔解曲线峰图(1%甲基化样本)。
图10为实施例3中采用引物对甲时的熔解曲线峰图(0%甲基化样本)。
图11为实施例3中采用引物对甲时的熔解曲线峰图(各个样本的叠加图)。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。如无特殊说明,实施例中的DNA分子中,A均代表腺嘌呤脱氧核糖核苷酸,T均代表胸腺嘧啶脱氧核糖核苷酸,C均代表胞嘧啶脱氧核糖核苷酸,G均代表鸟嘌呤脱氧核糖核苷酸。pMD20-T载体:宝日医生物技术(北京)有限公司,货号6028。
实施例1、引物的设计和制备以及标准品质粒的制备
一、引物的设计和制备
基于septin9基因CpG岛设计多个引物对,通过预实验分别验证多个引物对的效果,初步筛选得到3个引物对。
引物对甲由引物F1和引物R1组成。
F1(SEQ ID NO:1):5’-GTTAGTGCGAGATAGGGAGGTC-3’;
R1(SEQ ID NO:2):5’-CAATACAAAACTAACCGCCG-3’。
引物对乙由引物F2和引物R2组成。
F2:5’-AGAAATTTTAGGATTTGGGCG-3’;
R2:5’-GACGTTCTCCACCTACTTAAACG-3’。
引物对丙由引物F3和引物R3组成。
F3:5’-GTGTTGGGTTATAAGACGTTAGAATC-3’;
R3:5’-TAACAAAAACAATAAACACCTCGAA-3’。
分别合成上述各条引物。
二、标准品质粒的制备
标准品质粒Ⅰ(非甲基化质粒)的制备:将序列表的序列3所示的DNA分子(C均代表胞嘧啶脱氧核糖核苷酸)***pMD20-T载体Hind III和Kpn I酶切位点之间,得到标准品质粒Ⅰ。
标准品质粒Ⅱ(甲基化质粒)的制备:将序列表的序列3所示的DNA分子(三个特定区间中的所有CG中的C均代表5-甲基胞嘧啶脱氧核糖核苷酸,其它C均代表胞嘧啶脱氧核糖核苷酸;三个特异区间为:序列3中的第34-204位、第469-654位和第696-828位)***pMD20-T载体Hind III和Kpn I酶切位点之间,得到标准品质粒Ⅱ。
实施例2、通过甲基化特异性PCR结合HRM曲线分析检测甲基化的方法的建立
1、亚硫酸氢盐处理
取20μl样本溶液,进行亚硫酸氢盐处理,得到10μl产物溶液,即为模板溶液。进行亚硫酸氢盐处理采用的试剂盒为:EZ-96DNA Methylation-Gold MagPrep(ZYMO,货号D5042);按试剂盒说明书进行操作。
2、进行PCR扩增以及高分辨率熔解曲线分析
反应体系见表1。
扩增反应程序见表2。
扩增后进行高分辨率熔解(high-resolution melting,HRM)曲线分析。HRM曲线分析的参数设置为:65℃1s,然后按0.02℃/s的速度从65℃升至95℃(每秒收集25次荧光),最后40℃30s。
表1
组分 | 加入体积 |
TaKaRa Ex Taq | 0.4μL |
10×Ex Taq Buffer(Mg<sup>2+</sup>free) | 3μL |
25mM MgCl<sub>2</sub>溶液 | 3μL |
引物溶液 | 2μL |
dNTP Mixture | 3μL |
饱和染料EVAGreen | 1.5μL |
模板溶液 | 5μL |
DNase-free water | 补足至30μL |
TaKaRa Ex Taq:Takara,货号RR01AM,产品规格5U/μl;产品网址:https://www.takarabiomed.com.cn/ProductShow.aspx?m=20141215102854153148&productID=20141223192454673006。
10×Ex Taq Buffer(Mg2+free)和25mM MgCl2溶液:Takara,货号9152AM;产品网址:https://www.takarabiomed.com.cn/ProductShow.aspx?m=20141215102854153148&productID=20141223195903453090。
引物溶液提供的有效成分为引物F和引物R。引物溶液中,引物F和引物R的浓度均为10μM。
dNTP Mixture提供的有效成分为dATP、dTTP、dCTP和dGTP。dNTP Mixture:生工生物工程(上海)股份有限公司,货号B500056-0005;产品网址:https://www.sangon.com/productDetail?productInfo.code=B500056。
饱和染料EVAGreen:北京博蕾德科技发展有限公司(厂家美国Biotium);产品网址:http://www.bjbiolead.com/biolead2013-ParentList-859509/。
表2
3、结果判断
质控标准:甲基化质粒溶液作为样本溶液依次进行步骤1和步骤2,熔解曲线正常,仅在87℃±0.5℃位置出现熔解峰;非甲基化质粒溶液作为样本溶液依次进行步骤1和步骤2,熔解曲线正常,仅在82℃±0.5℃位置出现熔解峰。
结果判断标准:样本溶液(即待测生物样本进行基因组DNA提取得到的溶液)依次进行步骤1和步骤2,在87℃±0.5℃位置出现熔解峰说明样本存在septin9基因甲基化(为候选的结直肠癌患者),未在87℃±0.5℃位置出现熔解峰说明样本不存在septin9基因甲基化(为候选的非结直肠癌患者)。
实施例3、灵敏度检测
用甲基化质粒和TE缓冲液制备样本溶液,又称100%甲基化样本。用甲基化质粒、非甲基化质粒和TE缓冲液制备样本溶液(甲基化质粒占总质粒的质量百分比为75%),又称75%甲基化样本。用甲基化质粒、非甲基化质粒和TE缓冲液制备样本溶液(甲基化质粒占总质粒的质量百分比为50%),又称50%甲基化样本。用甲基化质粒、非甲基化质粒和TE缓冲液制备样本溶液(甲基化质粒占总质粒的质量百分比为25%),又称25%甲基化样本。用甲基化质粒、非甲基化质粒和TE缓冲液制备样本溶液(甲基化质粒占总质粒的质量百分比为10%),又称10%甲基化样本。用甲基化质粒、非甲基化质粒和TE缓冲液制备样本溶液(甲基化质粒占总质粒的质量百分比为5%),又称5%甲基化样本。用甲基化质粒、非甲基化质粒和TE缓冲液制备样本溶液(甲基化质粒占总质粒的质量百分比为2%),又称2%甲基化样本。用甲基化质粒、非甲基化质粒和TE缓冲液制备样本溶液(甲基化质粒占总质粒的质量百分比为1%),又称1%甲基化样本。用非甲基化质粒和TE缓冲液制备样本溶液,又称0%甲基化样本。每5μL样本溶液中,DNA含量为100ng。
依次按照实施例2的步骤1和步骤2进行检测。分别采用实施例1的三个引物对。
2%甲基化样本至100%甲基化样本,采用三个引物对均可以检测到甲基化发生。1%甲基化样本,只有引物对甲可以检测到甲基化发生,引物对乙和引物对丙均未检测到甲基化发生。0%甲基化样本,采用三个引物对均未检测到甲基化发生。即,引物对甲检测甲基化的灵敏度为1%,引物对乙和引物对丙检测甲基化的灵敏度为2%,引物对甲的灵敏度为另外两个引物对的2倍。
采用引物对甲时HRM曲线分析的荧光变化曲线见图1。采用引物对甲时的熔解曲线峰图见图2至图11。图2至图10依次对应100%甲基化样本至0%甲基化样本,图11为图2至图10的叠加图。
实施例4、重复性检测
用甲基化质粒、非甲基化质粒和TE缓冲液制备样本溶液。每5μL样本溶液中,DNA含量为100ng。样本溶液1中,甲基化质粒占总质粒的质量百分比为10%。样本溶液2中,甲基化质粒占总质粒的质量百分比为1%。
依次按照实施例2的步骤1和步骤2进行检测。采用实施例1的引物对甲。
进行20次重复试验。结果见表3。对两种浓度的混合模板重复检测20次,结果均能正常检出甲基化,表明具有很好的重复性。
表3
实施例5、临床样本检测
从医院获取10位志愿者的结直肠组织石蜡切片。其中临床样本2和临床样本8均获自医院确诊的结直肠癌患者。剩余的8个临床样本均获自医院确诊的非结直肠癌患者。
取石蜡切片,提取总DNA,作为样本溶液。
依次按照实施例2的步骤1和步骤2进行检测。采用实施例1的引物对甲。
结果见表4。临床样本2和临床样本8能够检出Septin9基因甲基化。
表4
临床样本编号 | 临床样本1 | 临床样本2 | 临床样本3 | 临床样本4 | 临床样本5 |
结果 | 未甲基化 | 甲基化 | 未甲基化 | 未甲基化 | 未甲基化 |
临床样本编号 | 临床样本6 | 临床样本7 | 临床样本8 | 临床样本9 | 临床样本10 |
结果 | 未甲基化 | 未甲基化 | 甲基化 | 未甲基化 | 未甲基化 |
SEQUENCE LISTING
<110> 北京康美天鸿生物科技有限公司
<120> 基于高分辨率熔解曲线检测septin9基因甲基化的特异引物对和试剂盒
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<213> Homo sapiens
<400> 3
ccccaggcct ggccttgaca ggcgggcgga gcagccagtg cgagacaggg aggccggtgc 60
gggtgcggga acctgatccg cccgggaggc gggggcgggg cgggggcgca gcgcgcgggg 120
aggggccggc gcccgccttc ctcccccatt cattcagctg agccaggggg cctaggggct 180
cctccggcgg ctagctctgc actgcaggag cgcgggcgcg gcgccccagc cagcgcgcag 240
ggcccgggcc ccgccggggg cgcttcctcg ccgctgccct ccgcgcgacc cgctgcccac 300
cagccatcat gtcggacccc gcggtcaacg cgcagctgga tgggatcatt tcggacttcg 360
aagccttgaa aagatctttt gaggtcgagg aggtcgagac acccaactcc accccacccc 420
ggagggtcca gactccccta ctccgagcca ctgtggccag ctccacccag aaattccagg 480
acctgggcgt gaagaactca gaaccctcgg cccgccatgt ggactcccta agccaacgct 540
cccccaaggc gtccctgcgg agggtggagc tctcgggccc caaggcggcc gagccggtgt 600
cccggcgcac tgagctgtcc attgacatct cgtccaagca ggtggagaac gccggggcca 660
tcggcccgtc ccggttcggg ctcaagaggg ccgaggtgtt gggccacaag acgccagaac 720
cggcccctcg gaggacggag atcaccatcg tcaaacccca ggagtcagcc caccggagga 780
tggagccccc tgcctccaag gtccccgagg tgcccactgc ccctgccacc gacgcagccc 840
ccaagagggt ggagatccag atgcccaagc ctgctgaggc gcccaccgcc cccagcccag 900
cccagacctt ggagaattca gagcctgccc ctgtgtctca gctgcagagc aggctggagc 960
ccaagcccca gccccctgtg gctgaggcta caccccggag ccaggaggcc actgaggcgg 1020
ctcccagctg cgttggcgac atggccgaca cccccagaga tgccgggctc aagcaggcgc 1080
ctgcatcacg gaacgagaag gccccggtgg acttcggcta cgtggggatt gactccatcc 1140
tggagcagat gcgccggaag gccatgaagc agggcttcga gttcaacatc atggtggtcg 1200
ggcagagcgg cttgggtaaa tccaccttaa tcaacaccct cttcaaatcc aaaatcagcc 1260
ggaagtcggt gcagcccacc tcagaggagc gcatccccaa gaccatcgag atcaagtcca 1320
tcacgcacga tattgaggag aaaggcgtcc ggatgaagct gacagtgatt gacacaccag 1380
ggttcgggga ccacatcaac aacgagaact gctggcagcc catcatgaag ttcatcaatg 1440
accagtacga gaaatacctg caggaggagg tcaacatcaa ccgcaagaag cgcatcccgg 1500
acacccgcgt ccactgctgc ctctacttca tccccgccac cggccactcc ctcaggcccc 1560
tggacatcga gtttatgaaa cgcctgagca aggtggtcaa catcgtccct gtcatcgcca 1620
aggcggacac actcaccctg gaggagaggg tccacttcaa acagcggatc accgcagacc 1680
tgctgtccaa cggcatcgac gtgtaccccc agaaggaatt tgatgaggac tcggaggacc 1740
ggctggtgaa cgagaagttc cgggagatga tcccatttgc tgtggtgggc agtgaccacg 1800
agtaccaggt caacggcaag aggatccttg ggaggaagac caagtggggt accatcgaag 1860
ttgaaaacac cacacactgt gagtttgcct acctgcggga ccttctcatc aggacgcaca 1920
tgcagaacat caaggacatc accagcagca tccacttcga ggcgtaccgt gtgaagcgcc 1980
tcaacgaggg cagcagcgcc atggccaacg gcatggagga gaaggagcca gaagccccgg 2040
agatgtag 2048
<210> 4
<211> 579
<212> PRT
<213> Homo sapiens
<400> 4
Met Ser Asp Pro Ala Val Asn Ala Gln Leu Asp Gly Ile Ile Ser Asp
1 5 10 15
Phe Glu Ala Leu Lys Arg Ser Phe Glu Val Glu Glu Val Glu Thr Pro
20 25 30
Asn Ser Thr Pro Pro Arg Arg Val Gln Thr Pro Leu Leu Arg Ala Thr
35 40 45
Val Ala Ser Ser Thr Gln Lys Phe Gln Asp Leu Gly Val Lys Asn Ser
50 55 60
Glu Pro Ser Ala Arg His Val Asp Ser Leu Ser Gln Arg Ser Pro Lys
65 70 75 80
Ala Ser Leu Arg Arg Val Glu Leu Ser Gly Pro Lys Ala Ala Glu Pro
85 90 95
Val Ser Arg Arg Thr Glu Leu Ser Ile Asp Ile Ser Ser Lys Gln Val
100 105 110
Glu Asn Ala Gly Ala Ile Gly Pro Ser Arg Phe Gly Leu Lys Arg Ala
115 120 125
Glu Val Leu Gly His Lys Thr Pro Glu Pro Ala Pro Arg Arg Thr Glu
130 135 140
Ile Thr Ile Val Lys Pro Gln Glu Ser Ala His Arg Arg Met Glu Pro
145 150 155 160
Pro Ala Ser Lys Val Pro Glu Val Pro Thr Ala Pro Ala Thr Asp Ala
165 170 175
Ala Pro Lys Arg Val Glu Ile Gln Met Pro Lys Pro Ala Glu Ala Pro
180 185 190
Thr Ala Pro Ser Pro Ala Gln Thr Leu Glu Asn Ser Glu Pro Ala Pro
195 200 205
Val Ser Gln Leu Gln Ser Arg Leu Glu Pro Lys Pro Gln Pro Pro Val
210 215 220
Ala Glu Ala Thr Pro Arg Ser Gln Glu Ala Thr Glu Ala Ala Pro Ser
225 230 235 240
Cys Val Gly Asp Met Ala Asp Thr Pro Arg Asp Ala Gly Leu Lys Gln
245 250 255
Ala Pro Ala Ser Arg Asn Glu Lys Ala Pro Val Asp Phe Gly Tyr Val
260 265 270
Gly Ile Asp Ser Ile Leu Glu Gln Met Arg Arg Lys Ala Met Lys Gln
275 280 285
Gly Phe Glu Phe Asn Ile Met Val Val Gly Gln Ser Gly Leu Gly Lys
290 295 300
Ser Thr Leu Ile Asn Thr Leu Phe Lys Ser Lys Ile Ser Arg Lys Ser
305 310 315 320
Val Gln Pro Thr Ser Glu Glu Arg Ile Pro Lys Thr Ile Glu Ile Lys
325 330 335
Ser Ile Thr His Asp Ile Glu Glu Lys Gly Val Arg Met Lys Leu Thr
340 345 350
Val Ile Asp Thr Pro Gly Phe Gly Asp His Ile Asn Asn Glu Asn Cys
355 360 365
Trp Gln Pro Ile Met Lys Phe Ile Asn Asp Gln Tyr Glu Lys Tyr Leu
370 375 380
Gln Glu Glu Val Asn Ile Asn Arg Lys Lys Arg Ile Pro Asp Thr Arg
385 390 395 400
Val His Cys Cys Leu Tyr Phe Ile Pro Ala Thr Gly His Ser Leu Arg
405 410 415
Pro Leu Asp Ile Glu Phe Met Lys Arg Leu Ser Lys Val Val Asn Ile
420 425 430
Val Pro Val Ile Ala Lys Ala Asp Thr Leu Thr Leu Glu Glu Arg Val
435 440 445
His Phe Lys Gln Arg Ile Thr Ala Asp Leu Leu Ser Asn Gly Ile Asp
450 455 460
Val Tyr Pro Gln Lys Glu Phe Asp Glu Asp Ser Glu Asp Arg Leu Val
465 470 475 480
Asn Glu Lys Phe Arg Glu Met Ile Pro Phe Ala Val Val Gly Ser Asp
485 490 495
His Glu Tyr Gln Val Asn Gly Lys Arg Ile Leu Gly Arg Lys Thr Lys
500 505 510
Trp Gly Thr Ile Glu Val Glu Asn Thr Thr His Cys Glu Phe Ala Tyr
515 520 525
Leu Arg Asp Leu Leu Ile Arg Thr His Met Gln Asn Ile Lys Asp Ile
530 535 540
Thr Ser Ser Ile His Phe Glu Ala Tyr Arg Val Lys Arg Leu Asn Glu
545 550 555 560
Gly Ser Ser Ala Met Ala Asn Gly Met Glu Glu Lys Glu Pro Glu Ala
565 570 575
Pro Glu Met
Claims (10)
1.特异引物对,由SEQ ID NO:1所示的单链DNA分子和SEQ ID NO:2所示的单链DNA分子组成。
2.如权利要求1所述的特异引物对,其特征在于:所述特异引物对的功能为诊断或辅助诊断结直肠癌。
3.如权利要求1所述的特异引物对,其特征在于:所述特异引物对的功能为检测septin9基因启动子区甲基化。
4.权利要求1所述特异引物对在制备试剂盒中的应用;所述试剂盒的功能为诊断或辅助诊断结直肠癌。
5.权利要求1所述特异引物对在制备试剂盒中的应用;所述试剂盒的功能为检测septin9基因启动子区甲基化。
6.权利要求1所述特异引物对在制备试剂盒中的应用;所述试剂盒的功能为检测septin9基因启动子区CpG岛甲基化。
7.一种试剂盒,包括权利要求1所述特异引物对;所述试剂盒的功能为诊断或辅助诊断结直肠癌。
8.一种试剂盒,包括权利要求1所述特异引物对;所述试剂盒的功能为检测septin9基因启动子区甲基化。
9.如权利要求7或8所述的试剂盒,其特征在于:所述试剂盒还具有PCR扩增试剂和荧光染料。
10.权利要求7或8所述试剂盒的制备方法,包括将各条引物进行独立包装的步骤。
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