CN113481296A - 一种早期评估乳腺癌风险的诊断试剂盒 - Google Patents
一种早期评估乳腺癌风险的诊断试剂盒 Download PDFInfo
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Abstract
本发明涉及医学和生物技术领域,尤其为一种早期评估乳腺癌风险的诊断试剂盒,包括一个标准品,一个长链非编码RNA,两个特异基因及内参基因的引物,针对的检测基因及内参基因分别为:LINC00504(GeneID:201853),RFX5(GeneID:5993),VEGFA(GeneID:7422)和β‑actin(GeneID:60);目前对乳腺癌进行发病风险评估多用SNP位点等方法进行分析,存在实用性差且缺乏有效的敏感性和特异性,很少能用于实际的检测和应用,本发明所用检测的基因组合包括了长链非编码RNA一个,乳腺癌相关特异表达基因2个,同时使用标准品及内参基因对检测结果进行有效的校正,通过组合检测分析,能够有效的预测乳腺癌的发病风险,当所检测的3个基因的ct值均小于标准品的ct值时,预警乳腺癌患病为高风险。
Description
技术领域
本发明涉及医学和生物技术领域,具体为早期评估乳腺癌风险的诊断试剂盒。
背景技术
乳腺癌在全世界范围内都是女性最常见的恶心肿瘤之一,而且发病率常年位于女性肿瘤首位,其发病率已占全身各种恶性肿瘤的7%以上,基于乳腺癌具有较强的转移能力,一般患者的预后效果并不理想。随着环境和人口老龄化的加剧,乳腺癌的发病率有明显上升的趋势。早期发现并及时治疗是乳腺癌的防治关键。
长链非编码RNA是一类长度大于200个核苷酸的非编码蛋白的小分子RNA,目前很多研究证明了长链非编码RNA在多种肿瘤中起着关键的作用。本发明试剂盒中提供的长链非编码RNA LINC00504基因引物能特异的检测样品中 LINC00504的丰度,根据目前的研究数据显示,LINC00504的表达丰度跟乳腺癌的风险成正相关性,同时配合转录因子RFX5及特异表达基因VEGFA这两个基因的联合检测,能有效的增加对乳腺癌风险评估的准确率。
与乳腺癌发病直接或间接相关的基因有很多个,因此寻找出一些关联基因的组合检测能大大提高对乳腺癌风险评估的时效性跟准确性,因此建立简单、可靠、快速的关联基因检测的高效检测方法能及时筛查出乳腺癌易感高危人群,从而根据结果尽快采取预防措施,这对提高乳腺癌的早期诊断和治疗有着重要意义。
发明内容
本发明的目的在于提供早期评估乳腺癌风险的诊断试剂盒,解决了目前常见的对乳腺癌进行发病风险评估多用SNP位点等方法进行分析,存在实用性差且缺乏有效的敏感性和特异性,很少能用于实际的检测和应用的问题。
为实现上述目的,本发明提供如下技术方案:早期评估乳腺癌风险的诊断试剂盒,包括一个标准品,一个长链非编码RNA,两个特异基因及内参基因的引物,针对的检测基因及内参基因分别为:LINC00504(GeneID:201853),RFX5 (GeneID:5993),VEGFA(GeneID:7422)和β-actin(GeneID:60),引物能特异的扩增出对应的基因。
优选的,所述LINC00504基因部分序列:
ggggtgtggaaagaggccatgaagaagaaagaggatttgaaagaagagagaggaatgaagaaa gacttctaataaatatgcttgctacgatcaattctgtgtcctcccttttctataaaatatgatccttggcccctata atttcttgactctaaatgtttgaaagagtgaatcagcattatcatggaacaatatgtgctgatgggtatagtctc cccctttgattggtactttgcaagtttcacaaataactaaccacctgagaacttgtctcccttgaactgaattcc attttaattttgttcaccttcttgattctcttctttttgtgtcaaaattgtacttttgcaaagatgatttcaaaagtgtttt ctttctgatttagtgcggccagttttcaacattcggtctgaaattgaggagttgctgtatgaaaagtgaatttatt tgaatggtaaagtttcctgtgaacagaaataaagtattgtttcactctgaaa(SEQ ID NO:9)。
优选的,所述RFX5基因部分序列:
gccaggaccttggggctgggctggagtgaaggtggctacgagttttccagatttaggagacttc agaaaggtggggcagatagaatggagatggcaaagatctctttgggcatatatgggcctggcgaagta atggaataatttctaattttcggagaaggcaagtgccctcatgccgggatggcagaagatgagcctga tgctaagagccccaagactgggggaagggcccccccaggtggtgctgaggctggggaacctaccaccc ttcttcagaggctccgaggtaccatttccaaggccgtgcagaacaaagtagaggggatcctgcaagat gtacagaaattttctgacaatgacaagctgtatctctaccttcagctcccctcaggacccaccactgg agacaaaagctcagagccaagtacactgagcaatgaggagtacatgtatgcctataggtggatccgca accacctggaagagcacactgacacctg(SEQ ID NO:10)。
优选的,所述VEGFA基因部分序列:
gattccctccacatgctgcacgcgcatctcgcccccaggggcactgcctggaagattcaggagc ctgggcggccttcgcttactctcacctgcttctgagttgcccaggagaccactggcagatgtcccggc gaagagaagagacacattgttggaagaagcagcccatgacagctccccttcctgggactcgccctcat cctcttcctgctccccttcctggggtgcagcctaaaaggacctatgtcctcacaccattgaaaccact agttctgtccccccaggagacctggttgtgtgtgtgtgagtggttgaccttcctccatcccctggtcc ttcccttcccttcccgaggcacagagagacagggcaggatccacgtgcccattgtggaggcagagaaa agagaaagtgttttatatacggtacttatttaatatccctttttaattagaaattaaaacagttaatt taattaaagagtagggttttttttcagt(SEQ ID NO:11)。
优选的,所述β-actin基因部分序列:
gttgctatccaggctgtgctatccctgtacgcctctggccgtaccactggcatcgtgatggact ccggtgacggggtcacccacactgtgcccatctacgaggggtatgccctcccccatgccatcctgcgt ctggacctggctggccgggacctgactgactacctcatgaagatcctcaccgagcgcggctacagctt caccaccacggccgagcgggaaatcgtgcgtgacattaaggagaagctgtgctacgtcgccctggact tcgagcaagagatggccacggctgcttccagctcctccctggagaagagctacgagctgcctgacggc caggtcatcaccattggcaatgagcggttccgctgccctgaggcactcttccagccttccttcctggg catggagtcctgtggcatccacgaaactaccttcaactccatcatgaagtgtgacgtggacatccgca aagacctgtacgccaacacagtgctgtc(SEQ ID NO:12)。
优选的,所述的试剂盒的每一人份包括:
1.逆转录翻译体系:5X逆转录缓冲液4ul,10nm的dNTP 1ul,oligo引物1ul,随机引物1ul,逆转录酶1ul,RNA酶抑制剂0.5ul,无酶水12ul;
2.PCR反应体系:SYBR GREEN 2XPCR混合反应缓冲液10ul,每条引物各1ul,ddH2O7ul;
3.标准品:4ul。
与现有技术相比,本发明的有益效果如下:
目前常见的对乳腺癌进行发病风险评估多用SNP位点等方法进行分析,存在实用性差且缺乏有效的敏感性和特异性,很少能用于实际的检测和应用,本发明所用检测的基因组合包括了长链非编码RNA一个,乳腺癌相关特异表达基因2 个,同时使用标准品及内参基因对检测结果进行有效的校正,通过组合检测分析,能够有效的预测乳腺癌的发病风险,当所检测的3个基因的ct值均小于标准品的ct值时,预警乳腺癌患病为高风险。
附图说明
图1为本发明专利的分析对比示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
通过对1085例乳腺癌患者及291例正常对照的进行长链非编码RNA的表达分析,结果显示LINC00504显著提高乳腺癌的发病风险,数据显示具有显著的差异性。
早期评估乳腺癌风险的诊断试剂盒,包括一个标准品,一个长链非编码RNA,两个特异基因及内参基因的引物,针对的检测基因及内参基因分别为: LINC00504(GeneID:201853),RFX5(GeneID:5993),VEGFA(GeneID:7422) 和β-actin(GeneID:60),引物能特异的扩增出对应的基因。
本实施例中,LINC00504基因部分序列:
ggggtgtggaaagaggccatgaagaagaaagaggatttgaaagaagagagaggaatgaagaaa gacttctaataaatatgcttgctacgatcaattctgtgtcctcccttttctataaaatatgatccttggcccctata atttcttgactctaaatgtttgaaagagtgaatcagcattatcatggaacaatatgtgctgatgggtatagtctc cccctttgattggtactttgcaagtttcacaaataactaaccacctgagaacttgtctcccttgaactgaattcc attttaattttgttcaccttcttgattctcttctttttgtgtcaaaattgtacttttgcaaagatgatttcaaaagtgtttt ctttctgatttagtgcggccagttttcaacattcggtctgaaattgaggagttgctgtatgaaaagtgaatttatt tgaatggtaaagtttcctgtgaacagaaataaagtattgtttcactctgaaa(SEQ ID NO:9)。
本实施例中,RFX5基因部分序列:
gccaggaccttggggctgggctggagtgaaggtggctacgagttttccagatttaggagacttc agaaaggtggggcagatagaatggagatggcaaagatctctttgggcatatatgggcctggcgaagta atggaataatttctaattttcggagaaggcaagtgccctcatgccgggatggcagaagatgagcctga tgctaagagccccaagactgggggaagggcccccccaggtggtgctgaggctggggaacctaccaccc ttcttcagaggctccgaggtaccatttccaaggccgtgcagaacaaagtagaggggatcctgcaagat gtacagaaattttctgacaatgacaagctgtatctctaccttcagctcccctcaggacccaccactgg agacaaaagctcagagccaagtacactgagcaatgaggagtacatgtatgcctataggtggatccgca accacctggaagagcacactgacacctg(SEQ ID NO:10)。
本实施例中,VEGFA基因部分序列:
gattccctccacatgctgcacgcgcatctcgcccccaggggcactgcctggaagattcaggagc ctgggcggccttcgcttactctcacctgcttctgagttgcccaggagaccactggcagatgtcccggc gaagagaagagacacattgttggaagaagcagcccatgacagctccccttcctgggactcgccctcat cctcttcctgctccccttcctggggtgcagcctaaaaggacctatgtcctcacaccattgaaaccact agttctgtccccccaggagacctggttgtgtgtgtgtgagtggttgaccttcctccatcccctggtcc ttcccttcccttcccgaggcacagagagacagggcaggatccacgtgcccattgtggaggcagagaaa agagaaagtgttttatatacggtacttatttaatatccctttttaattagaaattaaaacagttaatt taattaaagagtagggttttttttcagt(SEQ ID NO:11)。
本实施例中,β-actin基因部分序列:
gttgctatccaggctgtgctatccctgtacgcctctggccgtaccactggcatcgtgatggact ccggtgacggggtcacccacactgtgcccatctacgaggggtatgccctcccccatgccatcctgcgt ctggacctggctggccgggacctgactgactacctcatgaagatcctcaccgagcgcggctacagctt caccaccacggccgagcgggaaatcgtgcgtgacattaaggagaagctgtgctacgtcgccctggact tcgagcaagagatggccacggctgcttccagctcctccctggagaagagctacgagctgcctgacggc caggtcatcaccattggcaatgagcggttccgctgccctgaggcactcttccagccttccttcctggg catggagtcctgtggcatccacgaaactaccttcaactccatcatgaagtgtgacgtggacatccgca aagacctgtacgccaacacagtgctgtc(SEQ ID NO:12)。
工作原理:早期评估乳腺癌风险的诊断试剂盒在血液样品中使用时,先抽取患者静脉血液1ml,使用氯仿法抽提总RNA,然后对RNA进行定量分析,使用 50nm总RNA作为起始量进行下一步操作,其次在使用试剂盒提供的逆转录试剂进行逆转录反应,体系为20ul,反应条件如下:42℃/60分钟,75℃/5分钟,然后再使用试剂盒提供的PCR试剂进行PCR反应,取上述逆转录反应产物1ul 作为模板,分别单独扩增LINC00504,RXF5,VEGFA及β-actin,所使用的引物分别如下:(1)LINC00504正向引物:5`-GCTACGATCAATTCTGTGTCCTC-3`(SEQ IDNO:1),反向引物:5`-CCCATCAGCACATATTGTTCC-3`(SEQ ID NO:2);(2) RFX5正向引物:5`-GATGAGCCTGATGCTAAGAGC-3`(SEQ ID NO:3),反向引物: 5`-CCCTCTACTTTGTTCTGCACG-3`(SEQ ID NO:4);(3)VEGFA正向引物:5`- AGGAGACCACTGGCAGATGT-3`(SEQ ID NO:5),反向引物:5`- CAATGGTGTGAGGACATAGG-3`(SEQ ID NO:6);(4)β-actin正向引物:5`-TCGTGCGTGACATTAAGGAG-3`(SEQ ID NO:7),反向引物:5`- AACCGCTCATTGCCAATGGT-3`(SEQID NO:8),同时进行标准品的PCR反应,取试剂盒内提供的标准品1ul作为模板,分别单独扩增LINC00504,RXF5,VEGFA 及β-actin,所使用的引物分别如下:(5)1)LINC00504正向引物:5`-GCTACGATCAATTCTGTGTCCTC-3`(SEQ ID NO:1),反向引物: 5`-CCCATCAGCACATATTGTTCC-3`(SEQ ID NO:2);(6)RFX5正向引物: 5`-GATGAGCCTGATGCTAAGAGC-3`(SEQ ID NO:3),反向引物: 5`-CCCTCTACTTTGTTCTGCACG-3`(SEQ ID NO:4);(7)VEGFA正向引物:5`- AGGAGACCACTGGCAGATGT-3`(SEQ ID NO:5),反向引物:5`- CAATGGTGTGAGGACATAGG-3`(SEQ ID NO:6);(8)β-actin正向引物:5`-TCGTGCGTGACATTAAGGAG-3`(SEQ ID NO:7),反向引物:5`- AACCGCTCATTGCCAATGGT-3`(SEQID NO:8),PCR扩增的反应体系为:SYBR GREEN 2XPCR混合反应缓冲液10ul,每条引物各1ul,ddH2O 7ul,逆转录产物1ul或标准品1ul,而PCR反应条件为:94℃/5分钟变性酶激活,94℃/30秒变性,60℃ /30秒退火,72℃/30秒延伸,共40个循环,使用仪器为实时定量PCR仪器,最后进行结果分析,PCR下机数据采用直接读取ct值的方法,首先通过内参基因进行校正,校正后的数据跟标准品比较,当LINC00504,RFX5及VEGFA基因的ct值同时小于标准品ct值时,预警乳腺癌为高风险;
当早期评估乳腺癌风险的诊断试剂盒在活检组织中使用时,先进行活检取出的组织样品使用氯仿法抽提总RNA,对RNA进行定量分析,使用100nm总RNA作为起始量进行下一步操作,让母后使用试剂盒提供的逆转录试剂进行逆转录反应,体系为20ul,反应条件如下:42℃/60分钟,75℃/5分钟,然后再使用试剂盒提供的PCR试剂进行PCR反应,取上述逆转录反应产物1ul作为模板,分别单独扩增LINC00504,RXF5,VEGFA及β-actin,所使用的引物分别如下:(1) LINC00504正向引物:5`-GCTACGATCAATTCTGTGTCCTC-3`(SEQ ID NO:1),反向引物:5`-CCCATCAGCACATATTGTTCC-3`(SEQ ID NO:2);(2)RFX5正向引物:5`-GATGAGCCTGATGCTAAGAGC-3`(SEQ ID NO:3),反向引物: 5`-CCCTCTACTTTGTTCTGCACG-3`(SEQ ID NO:4);(3)VEGFA正向引物:5`- AGGAGACCACTGGCAGATGT-3`(SEQ ID NO:5),反向引物:5`- CAATGGTGTGAGGACATAGG-3`(SEQ ID NO:6);(4)β-actin正向引物:5`-TCGTGCGTGACATTAAGGAG-3`(SEQ ID NO:7),反向引物:5`- AACCGCTCATTGCCAATGGT-3`(SEQID NO:8),同时进行标准品的PCR反应,取试剂盒内提供的标准品1ul作为模板,分别单独扩增LINC00504,RXF5,VEGFA 及β-actin,所使用的引物分别如下:(5)1)LINC00504正向引物:5`-GCTACGATCAATTCTGTGTCCTC-3`(SEQ ID NO:1),反向引物: 5`-CCCATCAGCACATATTGTTCC-3`(SEQ ID NO:2);(6)RFX5正向引物: 5`-GATGAGCCTGATGCTAAGAGC-3`(SEQ ID NO:3),反向引物: 5`-CCCTCTACTTTGTTCTGCACG-3`(SEQ ID NO:4);(7)VEGFA正向引物:5`- AGGAGACCACTGGCAGATGT-3`(SEQ ID NO:5),反向引物:5`- CAATGGTGTGAGGACATAGG-3`(SEQ ID NO:6);(8)β-actin正向引物:5`-TCGTGCGTGACATTAAGGAG-3`(SEQ ID NO:7),反向引物:5`- AACCGCTCATTGCCAATGGT-3`(SEQID NO:8),PCR扩增的反应体系为:SYBR GREEN 2XPCR混合反应缓冲液10ul,每条引物各1ul,ddH2O 7ul,逆转录产物1ul或标准品1ul,而PCR反应条件为:94℃/5分钟变性酶激活,94℃/30秒变性,60℃ /30秒退火,72℃/30秒延伸,共40个循环,使用仪器为实时定量PCR仪器,最后进行结果分析,PCR下机数据采用直接读取ct值的方法,首先通过内参基因进行校正,校正后的数据跟标准品比较,当LINC00504,RFX5及VEGFA基因的ct值同时小于标准品ct值时,预警乳腺癌为高风险。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 广东省人民医院
<120> 一种早期评估乳腺癌风险的诊断试剂盒
<141> 2021-05-12
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 500
<212> DNA
<213> 人工序列
<400> 1
ggggtgtgga aagaggccat gaagaagaaa gaggatttga aagaagagag aggaatgaag 60
aaagacttct aataaatatg cttgctacga tcaattctgt gtcctccctt ttctataaaa 120
tatgatcctt ggcccctata atttcttgac tctaaatgtt tgaaagagtg aatcagcatt 180
atcatggaac aatatgtgct gatgggtata gtctccccct ttgattggta ctttgcaagt 240
ttcacaaata actaaccacc tgagaacttg tctcccttga actgaattcc attttaattt 300
tgttcacctt cttgattctc ttctttttgt gtcaaaattg tacttttgca aagatgattt 360
caaaagtgtt ttctttctga tttagtgcgg ccagttttca acattcggtc tgaaattgag 420
gagttgctgt atgaaaagtg aatttatttg aatggtaaag tttcctgtga acagaaataa 480
agtattgttt cactctgaaa 500
<210> 2
<211> 500
<212> DNA
<213> 人工序列
<400> 2
gccaggacct tggggctggg ctggagtgaa ggtggctacg agttttccag atttaggaga 60
cttcagaaag gtggggcaga tagaatggag atggcaaaga tctctttggg catatatggg 120
cctggcgaag taatggaata atttctaatt ttcggagaag gcaagtgccc tcatgccggg 180
atggcagaag atgagcctga tgctaagagc cccaagactg ggggaagggc ccccccaggt 240
ggtgctgagg ctggggaacc taccaccctt cttcagaggc tccgaggtac catttccaag 300
gccgtgcaga acaaagtaga ggggatcctg caagatgtac agaaattttc tgacaatgac 360
aagctgtatc tctaccttca gctcccctca ggacccacca ctggagacaa aagctcagag 420
ccaagtacac tgagcaatga ggagtacatg tatgcctata ggtggatccg caaccacctg 480
gaagagcaca ctgacacctg 500
<210> 3
<211> 500
<212> DNA
<213> 人工序列
<400> 3
gattccctcc acatgctgca cgcgcatctc gcccccaggg gcactgcctg gaagattcag 60
gagcctgggc ggccttcgct tactctcacc tgcttctgag ttgcccagga gaccactggc 120
agatgtcccg gcgaagagaa gagacacatt gttggaagaa gcagcccatg acagctcccc 180
ttcctgggac tcgccctcat cctcttcctg ctccccttcc tggggtgcag cctaaaagga 240
cctatgtcct cacaccattg aaaccactag ttctgtcccc ccaggagacc tggttgtgtg 300
tgtgtgagtg gttgaccttc ctccatcccc tggtccttcc cttcccttcc cgaggcacag 360
agagacaggg caggatccac gtgcccattg tggaggcaga gaaaagagaa agtgttttat 420
atacggtact tatttaatat ccctttttaa ttagaaatta aaacagttaa tttaattaaa 480
gagtagggtt ttttttcagt 500
<210> 4
<211> 500
<212> DNA
<213> 人工序列
<400> 4
gttgctatcc aggctgtgct atccctgtac gcctctggcc gtaccactgg catcgtgatg 60
gactccggtg acggggtcac ccacactgtg cccatctacg aggggtatgc cctcccccat 120
gccatcctgc gtctggacct ggctggccgg gacctgactg actacctcat gaagatcctc 180
accgagcgcg gctacagctt caccaccacg gccgagcggg aaatcgtgcg tgacattaag 240
gagaagctgt gctacgtcgc cctggacttc gagcaagaga tggccacggc tgcttccagc 300
tcctccctgg agaagagcta cgagctgcct gacggccagg tcatcaccat tggcaatgag 360
cggttccgct gccctgaggc actcttccag ccttccttcc tgggcatgga gtcctgtggc 420
atccacgaaa ctaccttcaa ctccatcatg aagtgtgacg tggacatccg caaagacctg 480
tacgccaaca cagtgctgtc 500
Claims (6)
1.一种早期评估乳腺癌风险的诊断试剂盒,其特征在于:包括一个标准品,一个长链非编码RNA,两个特异基因及内参基因的引物,针对的检测基因及内参基因分别为:LINC00504(GeneID:201853),RFX5(GeneID:5993),VEGFA(GeneID:7422)和β-actin(GeneID:60),引物能特异的扩增出对应的基因。
6.根据权利要求1所述的一种早期评估乳腺癌风险的诊断试剂盒,其特征在于:所述的试剂盒的每一人份包括:
1.逆转录翻译体系:5X逆转录缓冲液4ul,10nm的dNTP 1ul,oligo引物1ul,随机引物1ul,逆转录酶1ul,RNA酶抑制剂0.5ul,无酶水12ul;
2.PCR反应体系:SYBR GREEN 2XPCR混合反应缓冲液10ul,每条引物各1ul,ddH20 7ul;
3.标准品:4ul。
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