CN112760378A - 检测tpm3-ntrk1融合基因的引物、探针和试剂盒 - Google Patents
检测tpm3-ntrk1融合基因的引物、探针和试剂盒 Download PDFInfo
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Abstract
本发明公开了一种检测TPM3‑NTRK1融合基因的引物、探针和试剂盒,其中引物和探针包括扩增TPM3‑Ex7‑NTRK1‑Ex8、TPM3‑Ex7‑NTRK1‑Ex9融合基因的引物和探针。本发明采用实时荧光定量PCR技术,可快速检测人类TPM3‑NTRK1融合基因情况,特异性好,灵敏度高,操作简单。利用本发明完成的检测结果准确,为了解病情进展和治疗效果提供依据,对肿瘤的研究有重要的参考意义。
Description
技术领域
本发明属于疾病基因检测领域,特别是涉及采用探针实时荧光PCR技术对TPM3-NTRK1的表达量进行检测的试剂盒。
背景技术
恶性肿瘤已成为目前危害人类身体健康最严重的疾病之一,据世界卫生组织估计,全球50亿人口中,每年因恶性肿瘤死亡约500余万人,平均每分钟3500人,每年新发现的恶性肿瘤患者1000万人。肿瘤的防治与康复已成为与人类每一个家庭都密切相关的社会问题。近数年来,随着分子生物学及检测技术的快速发展,驱动基因的研究及相应的靶向药物的研发为恶性肿瘤的治疗带来了革命性的变化,其中Larotrectinib是继Entrectinib之后,FDA加速审批的第2个TRK抑制剂,第1个可用于治疗儿童NTRK融合实体瘤的TRK抑制剂,也是第1个通过“篮子试验”获批的靶向药物,于2018年11月26日在美国上市。Larotrectinib是一种新型口服TRK特异性小分子抑制剂,适用于NTRK融合阳性,无已知获得性耐药突变的无有效替代治疗或其他治疗后进展的转移性或手术切除带来严重后果的成人及儿童实体瘤患者。Larotrectinib可与TRK可逆性结合,下调信号通路,抑制NTRK融合驱动的癌细胞的增殖和扩散,是目前治疗罕见恶性肿瘤的突破性新药。NTRK基因融合已被证实是多种实体瘤的驱动基因,如结直肠癌、甲状腺癌、婴儿型纤维肉瘤等。NTRK基因与其他基因融合后,融合产物为嵌合蛋白发挥非配体依赖性组成型激活作用,引发永久性信号级联反应,驱动癌细胞的生长。
NTRK基因包含NTRK1、NTRK2和NTRK3,他们分别负责编码TRK家族蛋白TRKA、TRKB和TRKC的合成。TRK激酶是一类神经生长因子受体,其家族由高度同源性的原肌球蛋白相关激A(Tropomyosin-relatedkinase A,TrkA)、原肌球蛋白相关激酶B(Tropomyosin-relatedkinase B,TrkB)、原肌球蛋白相关激酶C(Tropomyosin-related kinase C,TrkC)组成。TRK蛋白细胞外结构域与相应配体结合而被激活,在细胞内通过Ras/MEK/ERK,P13K/AKT和PLC-γ途径使受体形成同源二聚体,磷酸化及信号传导,从而影响细胞增殖、分化、代谢、凋亡。TRK信号通路的改变,包括基因融合、蛋白过度表达或单核苷酸改变,已经被发现是许多肿瘤的致病原因,特别是NTRK基因的融合,到目前为止已发现30余种NTRK基因融合类型,如ETV6-NTRK3、NFASCNTRK1、BCAN-NTRK1等。其中,NTRK基因融合最常见的类型为ETV6-NTRK3和TPM3-NTRK1。
NTRK1基因定位于lq21-q22,包含17个外显子、16个内含子,编码酪氨酸激酶受体A(tyrosinekinase receptor A,TRKA),共796个氨基酸,是神经生长因子(nerve growthfactor,NGF)的高亲和力受体。NTRKl蛋白由两部分组成,分别为胞外结构域和胞内酪氨酸激酶结构域(TKD),其中第12~17外显子编码细胞内区域,位于510-781氨基酸残基之间,是参与体内信号转导的关键区域。
TPM3,原肌球蛋白家族中一员,位于1q22→1q23和由1个外显子组成。在肌肉组织中,TPM3介导的肌球蛋白与Ca离子反应,并且参与细胞骨架微丝的稳定性。TPM3基因可以和其他基因形成融合基因可以导致肿瘤的发展。比如,酪氨酸蛋白激酶受体(TRK),血小板生长因子受体β(PDGFRB),间变性淋巴瘤激酶(ALK),神经营养酪氨酸激酶受体I型(NTRK1)。
目前,检测NTRK基因融合的方法主要有NGS、免疫组织化学(immunohistochemistry,IHC)、逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)和荧光原位杂交法(fluorescence in situhybrid-ization,FISH)。免疫组化方法尽管原理简单,但是试验过程过于繁琐,需要的试剂种类繁多,且试验结果需要经验丰富的专家来判读,判读结果存在较大的主观性,一定程度上限制了该法的应用;荧光原位杂交技术(FISH)检测结果需要经验丰富的专家来判读,结果判读存在较大的主观性,而且FISH只能定性,而不能用于微小残留的检测;相对于二代测序后期数据处理复杂,实时荧光定量PCR检测结果直观,容易断断,同时具有操作简单,成本低廉等特点。因此本研究采用实时荧光定量PCR技术,具有特异性好、灵敏度高、线性关系好、操作简单、自动化程度高、防污染、有较大的线性范围等优点,可以对TMP3-NTRK1融合基因的定量检测微小残留病灶。
发明内容
本发明的目的是提供一种检测TPM3-NTRK1融合基因的引物、探针和检测试剂盒,采用实时荧光PCR技术可快速检测人类TPM3-NTRK1融合基因情况,特异性好,灵敏度高,操作简单,检测结果准确。
本发明提供了检测TPM3-NTRK1融合基因的引物和探针。
进一步地,所述引物和探针还包括扩增内参基因的引物和探针。
本发明还提供了一种检测TPM3-NTRK1融合基因的试剂盒,包括:RNA抽提试剂、逆转录试剂和PCR反应液,所述PCR反应液包括用于扩增TPM3-NTRK1融合基因的引物和探针。
进一步地,所述试剂盒还包括扩增内参基因的引物和探针。
进一步地,所述试剂盒还包括阴性对照和阳性对照。
本发明的有益效果:
(1)上游引物同时针对TPM3基因的第7外显子进行设计,下游引物通过NTRK1的第8、第9外显子进行设计;探针共用了位于TPM3第7外显子上的一段核酸序列,可降低引物及探针的合成成本。
(2)在引物设计上,尽量避开引物3'端使用A碱基及重复3个以上碱基,降低引物的错配几率;同时在筛选引物和探针时,尽量避免两条引物间及引物与探针间的互补配对,如此可提高引物及探针的特异性,提高引物的扩增效率。
(3)本发明将实时荧光PCR技术结合采用Taqman探针,利用双标准曲线的方法,分别构建内参基因ACTIN和TPM3-NTRK1目的基因的定量标准曲线,检测受测者体内TPM3-NTRK1的表达水平。相比于以往的FISH和△△CT法,该试剂盒经测试特异性好、灵敏度高、操作简便、自动化程度高、防污染、有较大的线性范围等优点,可以对TMP3-NTRK1融合基因的定量检测微小残留病灶。
附图说明
图1为实施例3中TPM3-ex7-NTRK1-ex8融合基因标准曲线实验结果;
图2为实施例3中TPM3-ex7-NTRK1-ex9融合基因标准曲线实验结果;
图3为实施例3中内参基因标准曲线实验结果;
图4为实施例4中44例样本T7N8(TPM3-ex7-NTRK1-ex8)扩增曲线图;
图5为实施例4中44例样本T7N9(TPM3-ex7-NTRK1-ex9)扩增曲线图。
具体实施方式
实施例1
本发明用于辅助临床上广谱抗肿瘤药TRK抑制剂Larotrectinib的用药制定的方法。主要包括以下试剂:RNA提取试剂、RNA反转录试剂、检测体系PCR反应液。
其中检测体系PCR反应液包括:ReverTra Ace qPCR RT Kit;THUNDERBIRD ProbeqPCR Mix(2×)、ACTIN内参基因及TPM3-NTRK1目的基因的引物和探针均为10μM;
检测融合基因TPM3-NTRK1和内参基因ACTIN的引物和探针,碱基序列分别为:
阳性对照品:含TPM3-NTRK1的溶液;
阴性对照品:不含TPM3-NTRK1的溶液。
实施例2
本发明的操作流程:
(1)抽提血液中的总RNA:在洁净的1.5ml的离心管中加入1ml红细胞裂解液,取抗凝血0.5ml混匀。室温静置10min;1500rpm离心5min,弃上清,收集底部的细胞;再次加入0.5ml红细胞裂解液,1500rpm离心5min,弃上清,收集底部的细胞;向细胞中加入1mlTRIzol,反复吹打直至沉淀完全溶解,室温静止5min;加入0.2ml氯仿,震荡均匀;14000rpm4℃离心10min,吸取上清层转移至另一新的离心管中;加入等体积的异丙醇,上下充分混匀,室温静置10min;14000rpm 4℃离心10min,弃上清,加入75%乙醇1ml,轻轻上下颠倒洗涤管壁;14000rpm 4℃离心5min,弃乙醇;室温干燥10-15min,加入20ulRNase-free水溶解沉淀。
(2)参考Rever Tra Ace qPCR RT Kit试剂盒说明书,将RNA反转为cDNA。
(3)试剂配置:按检测人份数配置检测体系PCR反应液各XμL,每人份23μL分装:
X=23μL反应液×(8份内参基因(标准曲线)+8份目的基因(标准曲线)+n份标本+1份阳性对照+1份阴性对照+1份空白对照);
n为检测标本数。
(4)加样:加入检测体系PCR反应液中2μl cDNA;阳性对照和阴性对照直接加2μl阳性对照品和阴性对照品;空白对照加2μl生理盐水或不加任何物质。
(5)扩增:检测在实时荧光PCR仪上进行,可用仪器包括ABI7300,7500(美国Applied Biosystems公司)等。
实时荧光PCR扩增反应条件:
阶段 | 温度 | 时间 | 循环数 |
Stage 1 | 95℃ | 1min | |
Stage 2Step1 | 95℃ | 15s | |
Stage 2Step2 | 58℃ | 35sec | 40 |
(6)结果判断:将阈值线调整至背景信号及阴性扩增线以上,系统根据标准曲线和CT值自动计算出拷贝数。
1)内参阳性时,检测结果才认为有效;
2)阳性判断标准:Ct<36,为阳性;35≤Ct≤38,为疑似阳性,需要再次验证;Ct>38,为阴性。
实施例3
构建TPM3-NTRK1的2种融合形式的阳性质粒pUC57/TPM3-ex7-NTRK1-ex8、pUC57/TPM3-ex7-NTRK1-ex9及内参基因的阳性质粒pUC57/actin作为阳性对照,稀释浓度分别为107、106、105、104、103、102copies/μL进行PCR扩增,绘制标准曲线,结果如图1、图2、图3所示,通过计算,内参基因ACTIN的扩增效率为100.98%,TPM3-ex7-NTRK1-ex8融合基因的扩增效率为100.98%,TPM3-ex7-NTRK1-ex9融合基因的扩增效率为99%。
实施例4
选取临床特检乳腺癌及甲状腺结节样本44例,按照实施例2提取样本RNA,并配制试剂进行检测,每份样品加入检测体系PCR反应液中2μl,每例样本重复2次。同时做阳性,阴性,空白对照,内参基因,目的基因的标准曲线各一份。对于内参基因Actin,阳性质粒起线,阴性对照未起线,而全部样本的内参基因Actin全都起线,此结果说明提取的RNA及反转录得到的cDNA可以使用。对于融合基因,阳性质粒起线,阴性对照未起线,而全部样本中的融合基因均未起线,结果如图4、图5所示,即在所有被检测的样本中未发现TPM3-NTRK1基因融合现象。
序列表
<110> 合肥艾迪康医学检验实验室有限公司
<120> 检测TPM3-NTRK1融合基因的引物、探针和试剂盒
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tactgataaa ctcaaggagg caga 24
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cccgtgctga gtttgctgag agatc 25
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctccaggaac tcagtgaaga tga 23
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gggtctccag atgtgctgtt ag 22
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tgagcgaggc tacagctt 18
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tccttgatgt cgcgcacgat tt 22
<210> 7
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
accaccacgg ccgagcgg 18
Claims (7)
1.检测TPM3-NTRK1融合基因的引物和探针,其特征在于,碱基序列分别为:
TPM3-ex7-F:TACTGATAAACTCAAGGAGGCAGA
TPM3-ex7-P:CCCGTGCTGAGTTTGCTGAGAGATC
NTRK1-ex8-R:CTCCAGGAACTCAGTGAAGATGA
NTRK1-ex9-R:GGGTCTCCAGATGTGCTGTTAG。
2.如权利要求1所述的引物和探针,其特征在于,还包括扩增内参基因的引物和探针,其碱基序列分别为:
actin-F:TGAGCGAGGCTACAGCTT
actin-R:TCCTTGATGTCGCGCACGATTT
actin-probe:FAM-ACCACCACGGCCGAGCGG-TAMRA。
3.检测检测人类TPM3-NTRK1融合基因的方法,包括如下步骤:
(1)提取样品中的组织RNA;
(2)将(1)中提取出的组织RNA逆转录为cDNA;
(3)试剂配置;
(4)加样:检测体系PCR反应液加入cDNA;
(5)扩增:利用实时荧光PCR仪进行扩增反应;
(6)结果判断,将阈值线调整至背景信号及阴性扩增线以上,系统根据标准曲线和CT值自动计算出拷贝数;
1)内参阳性时,检测结果才认为有效;
2)阳性判断标准:Ct<36,为阳性;35≤Ct≤38,为疑似阳性,需要再次验证;Ct>38,为阴性;
其特征在于,引物和探针的碱基序列分别为:
TPM3-ex7-F:TACTGATAAACTCAAGGAGGCAGA
TPM3-ex7-P:CCCGTGCTGAGTTTGCTGAGAGATC
NTRK1-ex8-R:CTCCAGGAACTCAGTGAAGATGA
NTRK1-ex9-R:GGGTCTCCAGATGTGCTGTTAG。
4.如权利要求3所述的方法,其特征在于,还包括扩增内参基因的引物和探针,其碱基序列分别为:
actin-F:TGAGCGAGGCTACAGCTT
actin-R:TCCTTGATGTCGCGCACGATTT
actin-probe:FAM-ACCACCACGGCCGAGCGG-TAMRA。
5.用于检测人类TPM3-NTRK1融合基因的试剂盒,其特征在于,包括权利要求1和2之一所述的引物和探针。
6.根据权利要求5所述的检测人类TPM3-NTRK1融合基因的试剂盒,其特征在于还包括阳性对照、阴性对照和空白对照。
7.根据权利要求4所述的检测人类TPM3-NTRK1融合基因的试剂盒,其特征在于,所述试剂盒包括:
(i)RNA抽提试剂;
(ii)逆转录试剂;
(iii)检测体系PCR扩增反应液:包括扩增TPM3-NTRK1融合基因的引物和探针,其碱基序列分别为:
TPM3-ex7-F:TACTGATAAACTCAAGGAGGCAGA
TPM3-ex7-P:CCCGTGCTGAGTTTGCTGAGAGATC
NTRK1-ex8-R:CTCCAGGAACTCAGTGAAGATGA
NTRK1-ex9-R:GGGTCTCCAGATGTGCTGTTAG;
(iv)检测体系PCR扩增反应液:还包括扩增内参基因的引物和探针,其碱基序列分别为:
actin-F:TGAGCGAGGCTACAGCTT
actin-R:TCCTTGATGTCGCGCACGATTT
actin-probe:FAM-ACCACCACGGCCGAGCGG-TAMRA;
(v)阳性对照品和阴性对照品。
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