CN112280866A - 荧光定量pcr技术筛查急性早幼粒细胞白血病相关14种融合基因的方法、引物及探针 - Google Patents
荧光定量pcr技术筛查急性早幼粒细胞白血病相关14种融合基因的方法、引物及探针 Download PDFInfo
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Abstract
本发明涉及使用实时荧光定量PCR技术筛查急性早幼粒细胞白血病相关14种融合基因的引物及探针、组合物和方法,筛查及的相关融合基因包括PML‑RARα、PLZF‑RARα、NuMA‑RARα、STAT5b‑RARα、NPM‑RARα、TBLR1‑RARα、PRKAR1A‑RARα、FNDC3B‑RARα、BCOR‑RARα、FIP1L1‑RARα、IRF2BP2‑RARα、STAT3‑RARα、GTF2I‑RARα、NABP1‑RARα。本发明将实时荧光PCR技术结合采用Tapman探针,将常见或不常见的APL疾病相关的融合基因整合在一起进行筛查,能更全面地辅助APL疾病的诊断、治疗及预后监测,相较于以往的FISH等方法,该方法具有更高精度,且便于结果的判读。而且本发明将引物及探针的合理配比以及优化,将检测条件达到最佳,从而大大提升了效率。
Description
技术领域
本发明属生命科学和生物技术领域,特别涉及针对急性早幼粒细胞相关的融合基因分别设计特异性的引物和探针并使用实时荧光定量PCR技术进行筛查的方法及引物。本发明筛查的APL相关的融合基因包括:PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα、FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα,共计14种融合基因、该方法经济、方便、灵敏度高,适于临床样本的筛查及辅助诊断。
技术背景
急性早幼粒细胞白血病(APL or M3),是一种临床表现相当凶险的急性白血病,患者常以青壮年为主,发病率在急性非淋巴细胞白血病(ANLL)中占10%-15%。而APL中90%以上的分子生物学表现是15号染色体上的PML基因和17号染色体上的维A酸受体α(RARα)基因融合形成了PML/RARα融合基因,编码的PML/RARα融合蛋白。该蛋白阻止细胞分化,从而使骨髓中堆积大量的因分化障碍而停滞在早幼粒阶段的异常细胞。而APL使用全反式维甲酸(ATRA)和砷剂靶向治疗,使APL成为白血病治疗最有效的范例之一。但是仍然有一小部分患者对全反式维甲酸耐药,本发明针对最近的研究成果分别设计特异性的引物探针对包括PML-RARα以及NuMA-RARα、PLZF-RARα、STAT5B-RARα和NPM-RARα、TBLR1-RARα、PRKAR1A-RARα、FNDC38-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1(OBFC2A)–RARα这14种APL相关的常见及少见型别融合基因进行组合,运用RQ-PCR技术进行针对性的筛查,从而能给临床提供更好的辅助诊断和用药指导。
目前APL特异性遗传病变的鉴定可通过常规的核型分析、荧光原位杂交(FISH)、逆转录酶聚合酶链反应(RT-PCR;或实时定量PCR[RQ-PCR]),或基于核酸的同类技术(如逆转录-定量环介导等温扩增[RT-QLAMP])进行。这些方法的特异性都一样,但敏感性不一样,其中细胞遗传学分析最容易出现假阴性,这些方法不能替代RT-PCR或RQ-PCR,后者应始终并行运行,作为唯一允许定义PML/RARΑ同种型的类型并为后续MRD评估定量的技术。因此本发明运用RQ-PCR技术进行针对性的筛查,从而能给临床提供更好的辅助诊断和用药指导。
发明内容
鉴于目前APL的融合基因筛查所涉及的融合基因不是很全面,而综合考虑临床普及问题,本发明采用基于Taqman探针的实时荧光定量PCR技术检测APL相关14种融合基因。本发明设计了检测内参/目的基因的引物、探针序列,采用基于Taqman探针的实时荧光定量PCR技术检测APL相关14种融合基因,通过调整引物、探针浓度及比例,优化PCR的反应体系和反应条件,使扩增效率和速率均达到最佳。
本发明提供了检测样本中APL相关14种融合基因的寡核苷酸,所述寡核苷酸包括检测用上游引物,下游引物及探针,检测内参基因引物及探针
1.上游引物,碱基序列如下(方向为5’-3’):
PML-LF:GGAGCCCCGTCATAGGAAGT
PML-VF:ACCTGGATGGACCGCCTAG
PML-SF:CCGATGGCTTCGACGAGTT
(PML基因参考序列:NM_033238.3)
PLZF-3F:TGAGAATGCACTTACTGGCTCAT
PLZF-4F:TGGAGACACACAGGCAGACC
(PLZF基因参考序列:NM_001018011.3)
NuMA-F:AGTTCTGCTCGTCGTTCCC
(NuMA1基因参考序列:NM_006185.4)
STAT5b-13F:GCCTGTCTGTGTCCTGGTC
STAT5b-15F:TGGGACCTTCCTCCTGAGA
(STAT5b基因参考序列:NM_012448.4)
NPM1-4F:GGCTTTGAAATAACACCACCA
(NPM1基因参考序列:NM_001355006.2)
TBLR1-5F:GGGAGGAGAATGGAGCACA
(TBLR1基因参考序列:NM_001321193.3)
FNDC3B-24F:ACCTTCAGCACAACCAAAAGT
(FNDC3B基因参考序列:NM_001135095.2)
PRKAR1A-2F:TGCACTGCTCGACCTGAGA
PRKAR1A-3F:GGCACTCGTACAGACTCAAGG
(PRKAR1A基因参考序列:NM_001276289.2)
BCOR-F:CATGAAAATGACCCACAGTGA
(BCOR基因参考序列:NM_001123383.1)
FIP1L1-13F:ACTGCTCCACCTCTGATTCCA
FIP1L1-15F:TGGACATTCCTCTGGTTATGAT
(FIP1L1基因参考序列:NM_001134937.2)
IRF2BP2-1F:CCCTTCGAGAGCAAGTTTAAG
IRF2BP2-2F:TGCTTCCCTTGCTCCAGAC
(IRF2BP2基因参考序列:NM_182972.3)
STAT3-21F:AGGCATTCGGAAAGTATTGTC
STAT3-23F:GGAAATAATGGTGAAGGTGCT
(STAT3基因参考序列:NM_001369512.1)
GTF2I-5F:CAAAGCAGGGATTTCATTCA
GTF2I-6F:TTTAGAGCCAAAGAAGCATGTAG
(GTF2I基因参考序列:NM_032999.4)
NABP1-5F:TGGGTACAGGTACATTTGGACC
(NABP1基因参考序列:NM_001031716.5)
2.下游引物及探针碱基序列如下:
RARα-R:AGGGCTGGGCACTATCTCTTC
RARα-P:FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA
(RARα基因参考序列:NM_000964.4)
上述上游引物皆与该RARα基因的引物和探针结合从而测得融合基因的表达水平。
3.内参基因为ABL1基因,该基因的引物及探针碱基序列如下(方向为5’-3’):
ABL1-F:GATACGAAGGGAGGGTGTACCA
ABL1-R:CTCGGCCAGGGTGTTGAA
ABL1-P:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
本发明涉及相同基因的融合基因共用下游引物及探针,配对针对不同的伙伴基因上游引物进行检测,其引物探针序列如下:
用于PML-RARα融合基因检测引物和探针的核苷酸序列是:PML-SF,PML-VF,PML-LF,RARA-R,RARA-P;
用于PLZF-RARα融合基因检测引物和探针的核苷酸序列是:PLZF-3F,PLZF-4F,RARA-P,RARA-R;
用于NuMA-RARα融合基因检测引物和探针的核苷酸序列是:NuMA-F,RARA-R,RARA-P;
用于STAT5b-RARα融合基因检测引物和探针的核苷酸序列是:STAT5b-13F,STAT5b-15F,RARA-R,RARA-P;
用于NPM-RARα融合基因检测引物和探针的核苷酸序列是:NPM1-4F,RARA-R,RARA-P;
用于TBLR1-RARα融合基因检测引物和探针的核苷酸序列是:TBLR1-5F,RARA-R,RARA-P;
用于PRKAR1A-RARα融合基因检测引物和探针的核苷酸序列是:PRKAR1A-2F,PRKAR1A-3F,RARA-R,RARA-P;
用于FNDC3B-RARα融合基因检测引物和探针的核苷酸序列是:FNDC3B-24F,RARA-R,RARA-P;
用于BCOR-RARα融合基因检测引物和探针的核苷酸序列是:BCOR-F,RARA-R,RARA-P;
用于FIP1L1-RARα融合基因检测引物和探针的核苷酸序列是:FIP1L1-13F,FIP1L1-15F,RARA-R,RARA-P;
用于IRF2BP2-RARα融合基因检测引物和探针的核苷酸序列是:IRF2BP2-1F,IRF2BP2-2F,RARA-R,RARA-P;
用于STAT3-RARα融合基因检测引物和探针的核苷酸序列是:STAT3-21F,STAT3-23F,RARA-R,RARA-P;
用于GTF2I-RARα融合基因检测引物和探针的核苷酸序列是:GTF2I-5F,GTF2I-6F,RARA-R,RARA-P;
用于NABP1-RARα融合基因检测引物和探针的核苷酸序列是:NABP1-5F,RARA-R,RARA-P。
筛查上述融合基因的引物探针还包括扩增作为内参的管家基因ABL1的引物和探针,其核苷酸序列如下:
ABL1-F:GATACGAAGGGAGGGTGTACCA
ABL1-R:CTCGGCCAGGGTGTTGAA
ABL1-P:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
本发明还提供实时荧光定量PCR技术筛查PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα、FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα共14种急性早幼粒细胞白血病相关融合基因的组合物,所述组合物包括组合物1和组合物2,其中:组合物1包括筛查PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα融合基因的引物和探针,所述引物和探针的核苷酸序列是:RARA-R,RARA-P,PML-LF,PML-VF,PML-SF,PLZF-3F,PLZF-4F,NuMA-F,STAT5b-13F,STAT5b-15F,NPM1-4F,TBLR1-5F,PRKAR1A-2F,PRKAR1A-3F;组合物2包括筛查FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα融合基因的引物和探针,所述引物和探针的核苷酸序列是:RARA-R,RARA-P,FNDC3B-24F,BCOR-F,FIP1L1-13F,FIP1L1-15F,IRF2BP2-1F,IRF2BP2-2F,STAT3-21F,STAT3-23F,GTF2I-5F,GTF2I-6F,NABP1-5F。
本发明还提供实时荧光定量PCR技术筛查PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα、FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα共14种急性早幼粒细胞白血病相关融合基因的引物和探针在制备检测14种急性早幼粒细胞白血病相关融合基因的试剂盒中的应用。
本发明还提供了上述引物探针的筛查及鉴定PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα、FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα这14种融合基因的方法:
⑴采用红细胞裂解液裂解红细胞的方法提取全血样本中的有核细胞。10×红细胞裂解液配方为:NH4Cl 82g,NaHCO38.4g,EDTA-Na2 3.72g,加ddH2O定容至1000ml。
⑵采用TRIzol RNA提取方法提取有核细胞中的总RNA,并将RNA反转录成cDNA。
⑶以步骤2中的cDNA为模版,进行APL相关融合基因的初步筛查。筛查分第一组和第二组2个体系进行。反应条件为:95℃预变性1min;95℃10s,58℃50s,共40个循环;58℃时采集荧光。
⑷根据步骤3中的检测结果,判断是第一和第二组中的哪一组为阳性。在内参ABL1基因扩增正常(CT值≤32)的情况下,各融合基因CT值<36判定为阳性。若第一组阳性,则需鉴定是PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα融合基因中的哪一个为阳性;若第二组阳性,则需鉴定是FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα融合基因中的哪一个为阳性。
本发明将实时荧光PCR技术结合采用Tapman探针,将常见或不常见的APL疾病相关的融合基因整合在一起进行筛查,能更全面地辅助APL疾病的诊断、治疗及预后监测,相较于以往的FISH等方法,该方法具有更高精度,且便于结果的判读。而且本发明将引物及探针的合理配比以及优化,将检测条件达到最佳,从而大大提升了效率。
附图说明
图1是本发明检测区域、引物设计区域示意图。
图2是使用本发明引物测序方法对第一个待检血液样本进行筛查的荧光扩增图(阴性样本)
图3是使用本发明引物测序方法对第二个待检血液样本进行筛查的荧光扩增图(APL-2组阳性)
图4是使用本发明引物测序方法对第二个待检血液样本进行分型的荧光扩增图(FIP1L1-RARα阳性)
图5是使用本发明引物测序方法对第二个待检血液样本进行筛查的Sanger测序图(FIP1L1的15外显子与RARα的3外显子融合)
图6是使用本发明引物测序方法对第二个待检血液样本进行筛查的核型分析结果图(t(4;17)(q12;q21))
图7是使用本发明引物测序方法对第三个待检血液样本进行筛查的荧光扩增图(APL-1组阳性)
图8是使用本发明引物测序方法对第三个待检血液样本进行分型的荧光扩增图(PML-RARα的S型阳性)
图9是使用本发明引物测序方法对第三个待检血液样本的染色体核型分析结果图(t(15;17)(q21;q21))
图10是使用本发明引物测序方法对第四个待检血液样本进行筛查的荧光扩增图(APL-1组中的TBLR1-RARα阳性)
图11是使用本发明引物测序方法对第五个待检血液样本进行筛查的荧光扩增图(APL-1组中的STAT5B-RARα阳性)
图12是使用本发明引物测序方法对第五个待检血液样本的染色体核型分析结果图(t(17;17)(q21;q21))
具体实施方式
实施例1:
本发明所使用的试剂及引物如下:
1.红细胞裂解液:包括16μmol/L氯化铵、1mmol/L碳酸氢钾和12.5μmol/L EDTA;
2.样本RNA提取液:TRIzol、氯仿、异丙醇、75%乙醇和RNase-free水;RNA逆转录试剂:ReverTra Ace qPCR RT;Kit(TOYOBO公司);
3.检测体系PCR反应液:THUNDERBIRD Probe qPCR Mix(2×);检测用上游引物,下游引物和探针;内参基因上游引物,下游引物和探针浓度皆为10μM,其中引物探针序列如下(方向为5’-3’):
PML-LF:GGAGCCCCGTCATAGGAAGT
PML-VF:ACCTGGATGGACCGCCTAG
PML-SF:CCGATGGCTTCGACGAGTT
PLZF-3F:TGAGAATGCACTTACTGGCTCAT
PLZF-4F:TGGAGACACACAGGCAGACC
NuMA-F:AGTTCTGCTCGTCGTTCCC
STAT5b-13F:GCCTGTCTGTGTCCTGGTC
STAT5b-15F:TGGGACCTTCCTCCTGAGA
NPM1-4F:GGCTTTGAAATAACACCACCA
TBLR1-5F:GGGAGGAGAATGGAGCACA
FNDC3B-24F:ACCTTCAGCACAACCAAAAGT
PRKAR1A-2F:TGCACTGCTCGACCTGAGA
PRKAR1A-3F:GGCACTCGTACAGACTCAAGG
BCOR-F:CATGAAAATGACCCACAGTGA
FIP1L1-13F:ACTGCTCCACCTCTGATTCCA
FIP1L1-15F:TGGACATTCCTCTGGTTATGAT
IRF2BP2-1F:CCCTTCGAGAGCAAGTTTAAG
IRF2BP2-2F:TGCTTCCCTTGCTCCAGAC
STAT3-21F:AGGCATTCGGAAAGTATTGTC
STAT3-23F:GGAAATAATGGTGAAGGTGCT
GTF2I-5F:CAAAGCAGGGATTTCATTCA
GTF2I-6F:TTTAGAGCCAAAGAAGCATGTAG
NABP1-5F:TGGGTACAGGTACATTTGGACC
RARα-R:AGGGCTGGGCACTATCTCTTC
RARα-P:FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA
ABL1-F:GATACGAAGGGAGGGTGTACCA
ABL1-R:CTCGGCCAGGGTGTTGAA
ABL1-P:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA
实施例2:
1.血液RNA的提取:
在1.5ml离心管中加入1ml 1×红细胞裂解液,取抗凝全血0.5ml混匀,室温静置5min,4000rpm离心3min。吸弃上清,再加1ml 1×红细胞裂解液重悬细胞,吹打混匀。4000rpm离心3min。吸弃上清,收集底部细胞;向细胞团块中加入l ml Trizol Reagent。反复吹吸直至裂解液中无明显细胞团块,,室温静置5min。加入氯仿200μl,漩涡混匀。待充分乳化至粉红色(无分相现象)后,在冰上静置10min。14,000rpm,4℃离心10min。吸取上清液450μl转移至另一新的离心管中。向上清中加入等体积的预冷异丙醇,上下颠倒离心管充分混匀后,在冰上静置10min。14,000rpm,4℃离心10min。倒弃上清,加入l ml 75%的DEPC-乙醇,轻轻颠倒洗涤离心管,洗涤沉淀。14,000rpm,4℃离心5min。弃75%乙醇,加入l ml无水乙醇,轻轻颠倒洗涤离心管,洗涤沉淀。14,000rpm,4℃离心5min。弃无水乙醇,室温干燥5min、加入RNase-free水溶解沉淀,震荡混匀,即为所提取RNA。
2.将RNA逆转录为cDNA
参考TOYOBO公司的Rever Tra Ace qPCR RT Kit试剂盒说明书,将(1)中获得的RNA逆转录为cDNA。
实施例3:
初筛荧光PCR扩增:
1.本发明检测区域、引物设计区域示意图如图1所示,根据表1所示进行试剂配制,整个筛查试剂分为3个反应管:第1和第2反应管为14种融合基因的筛查检测管,第3的反应管为内参基因ABL1检测管(APL-1+APL-2+ABL总共三个反应管):
表1:
2.加样:在分装好的检测体系PCR反应液中加入实例2中的cDNA 2μl中;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,最终反应体系为25ul。
3.检测:其扩增程序如下:95℃1min;95℃1min,54℃30s,60℃30s,6个循环;95℃10s,58℃30s,☆58℃40s,40个循环。
备注:☆收集荧光信号
实施例4:
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。
内参基因ABL1基因扩增正常(CT值≤32)的情况下,检测结果才认为有效;
阳性判断标准:Ct<36,为阳性;36≤Ct≤38,为疑似阳性,需要再次验证;Ct>38,为阴性。
实施例5:
第一个待检全血样本检测:对第一个待检全血样本按实施例2-4所述进行RNA提取、逆转录,荧光扩增及结果分析,得到的荧光图谱如图2,其内参基因ABL1为阳性,而其他两组没有扩增线,则判读为阴性
实施例6:
第二个待检全血样本检测:对第二个待检全血样本按实施例2-4所述进行RNA提取、逆转录,荧光扩增及结果分析,得到的荧光图谱如图3,内参基因ABL1及APL-2组皆阳性,则对APL-2组进行分别鉴定,试剂配制如表2所示:
1.试剂配制:
表2:
2.加样:在分装好的检测体系PCR反应液中加入实例2中的cDNA 2μl中;最终反应体系为25ul。
3.检测:其扩增程序如下:95℃1min;95℃1min,54℃30s,60℃30s,6个循环;95℃10s,58℃30s,☆58℃40s,40个循环。
备注:☆收集荧光信号
4.按实例4分析结果,得到的荧光谱图如图4,其中有内参基因ABL1及FIP1L1-RARα的检测孔阳性,则可确定为FIP1L1-RARΑ阳性,进一步地我们对该样本进行了sanger测序,得到的测序图谱如图5,为FIP1L1的第15外显子与RARα的3外显子融合形成,同时该样本检测了染色体核型分析的项目,其结果为(t(4;17)(q12;q21)如图6,三种方法均验证了第二个样本为FIP1L1-RARα阳性。
实施例7:
第三个待检全血样本检测:对第三个待检全血样本按实施例2-4所述进行RNA提取、逆转录,荧光扩增及结果分析,得到的荧光图谱如图7,第三个待检全血样本检测:对第三个待检全血样本按实施例2-4所述进行RNA提取、逆转录,荧光扩增及结果分析,得到的荧光图谱如图3,内参基因ABL1及APL-1组皆阳性,则对APL-1组进行分析鉴定,试剂配制如表3所示:1.试剂配制:
表3:
2.加样:在分装好的检测体系PCR反应液中加入实例2中的cDNA 2μl中;最终反应体系为25ul。
3.检测:其扩增程序如下:95℃1min;95℃1min,54℃30s,60℃30s,6个循环;95℃10s,58℃30s,☆58℃40s,40个循环。
备注:☆收集荧光信号
4.按实例4分析结果,得到的荧光谱图如图8,其中有内参基因ABL及PML-RARα的S型的检测孔阳性,另外染色体核型分析结果如图9,为t(15;17)(q21;q21),则符合PML-RARαS型阳性。
实施例8:
第四个待检全血样本检测:对第四个待检全血样本按实施例2-4所述进行RNA提取、逆转录,荧光扩增及结果分析,同实施例7:APL-1及内参基因ABL1阳性,对APL-1进行分型同实施例7中的实际配制、加样、检测,分析结果。得到的荧光图谱如图10,确定为TBLR1-RARα阳性,符合临床。
实施例9:
第五个待检全血样本检测:对第五个待检全血样本按实施例2-4所述进行RNA提取、逆转录,荧光扩增及结果分析,同实施例7:APL-1及内参基因ABL1阳性,对APL-1进行分型同实施例7中的实际配制、加样、检测,分析结果。得到的荧光图谱如图11,结合临床及染色体核型分析为t(17;17)(q21;q21)如图12,则第五个样本STAT5B-RARα阳性符合。
序列表
<110> 福州艾迪康医学检验所有限公司
<120> 荧光定量PCR技术筛查急性早幼粒细胞白血病相关14种融合基因的方法、引物及探针
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<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gcctgtctgt gtcctggtc 19
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tgggaccttc ctcctgaga 19
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggctttgaaa taacaccacc a 21
<210> 10
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gggaggagaa tggagcaca 19
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
accttcagca caaccaaaag t 21
<210> 12
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgcactgctc gacctgaga 19
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggcactcgta cagactcaag g 21
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
catgaaaatg acccacagtg a 21
<210> 15
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
actgctccac ctctgattcc a 21
<210> 16
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tggacattcc tctggttatg at 22
<210> 17
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cccttcgaga gcaagtttaa g 21
<210> 18
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tgcttccctt gctccagac 19
<210> 19
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aggcattcgg aaagtattgt c 21
<210> 20
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ggaaataatg gtgaaggtgc t 21
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
caaagcaggg atttcattca 20
<210> 22
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tttagagcca aagaagcatg tag 23
<210> 23
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tgggtacagg tacatttgga cc 22
<210> 24
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
agggctgggc actatctctt c 21
<210> 25
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ccattgagac ccagagcagc agttc 25
<210> 26
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gatacgaagg gagggtgtac ca 22
<210> 27
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ctcggccagg gtgttgaa 18
<210> 28
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tgcttctgat ggcaagctct acgtctcct 29
Claims (7)
1.实时荧光定量PCR技术筛查PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα、FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα共14种急性早幼粒细胞白血病相关融合基因的引物及探针,其特征在于,所述引物和探针的核苷酸序列如下:
PML-LF:GGAGCCCCGTCATAGGAAGT
PML-VF:ACCTGGATGGACCGCCTAG
PML-SF:CCGATGGCTTCGACGAGTT
PLZF-3F:TGAGAATGCACTTACTGGCTCAT
PLZF-4F:TGGAGACACACAGGCAGACC
NuMA-F:AGTTCTGCTCGTCGTTCCC
STAT5b-13F:GCCTGTCTGTGTCCTGGTC
STAT5b-15F:TGGGACCTTCCTCCTGAGA
NPM1-4F:GGCTTTGAAATAACACCACCA
TBLR1-5F:GGGAGGAGAATGGAGCACA
FNDC3B-24F:ACCTTCAGCACAACCAAAAGT
PRKAR1A-2F:TGCACTGCTCGACCTGAGA
PRKAR1A-3F:GGCACTCGTACAGACTCAAGG
BCOR-F:CATGAAAATGACCCACAGTGA
FIP1L1-13F:ACTGCTCCACCTCTGATTCCA
FIP1L1-15F:TGGACATTCCTCTGGTTATGAT
IRF2BP2-1F:CCCTTCGAGAGCAAGTTTAAG
IRF2BP2-2F:TGCTTCCCTTGCTCCAGAC
STAT3-21F:AGGCATTCGGAAAGTATTGTC
STAT3-23F:GGAAATAATGGTGAAGGTGCT
GTF2I-5F:CAAAGCAGGGATTTCATTCA
GTF2I-6F:TTTAGAGCCAAAGAAGCATGTAG
NABP1-5F:TGGGTACAGGTACATTTGGACC
RARA-R:AGGGCTGGGCACTATCTCTTC
RARA-P:FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA。
2.根据权利要求1所述的引物和探针,其特征在于,
用于PML-RARα融合基因检测引物和探针的核苷酸序列是:PML-SF,PML-VF,PML-LF,RARA-R,RARA-P;
用于PLZF-RARα融合基因检测引物和探针的核苷酸序列是:PLZF-3F,PLZF-4F,RARA-R,RARA-P;
用于NuMA-RARα融合基因检测引物和探针的核苷酸序列是:NuMA-F,RARA-R,RARA-P;
用于STAT5b-RARα融合基因检测引物和探针的核苷酸序列是:STAT5b-13F,STAT5b-15F,RARA-R,RARA-P;
用于NPM-RARα融合基因检测引物和探针的核苷酸序列是:NPM1-4F,RARA-R,RARA-P;
用于TBLR1-RARα融合基因检测引物和探针的核苷酸序列是:TBLR1-5F,RARA-R,RARA-P;
用于PRKAR1A-RARα融合基因检测引物和探针的核苷酸序列是:PRKAR1A-2F,PRKAR1A-3F,RARA-R,RARA-P;
用于FNDC3B-RARα融合基因检测引物和探针的核苷酸序列是:FNDC3B-24F,RARA-R,RARA-P;
用于BCOR-RARα融合基因检测引物和探针的核苷酸序列是:BCOR-F,RARA-R,RARA-P;
用于FIP1L1-RARα融合基因检测引物和探针的核苷酸序列是:FIP1L1-13F,FIP1L1-15F,RARA-R,RARA-P;
用于IRF2BP2-RARα融合基因检测引物和探针的核苷酸序列是:IRF2BP2-1F,IRF2BP2-2F,RARA-R,RARA-P;
用于STAT3-RARα融合基因检测引物和探针的核苷酸序列是:STAT3-21F,STAT3-23F,RARA-R,RARA-P;
用于GTF2I-RARα融合基因检测引物和探针的核苷酸序列是:GTF2I-5F,GTF2I-6F,RARA-R,RARA-P;
用于NABP1-RARα融合基因检测引物和探针的核苷酸序列是:NABP1-5F,RARA-R,RARA-P。
3.根据权利要求1或2所述的引物和探针,其特征在于,还包括扩增作为内参的管家基因ABL1的引物和探针,其核苷酸序列如下:
ABL1-F:GATACGAAGGGAGGGTGTACCA
ABL1-R:CTCGGCCAGGGTGTTGAA
ABL1-P:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
4.实时荧光定量PCR技术筛查PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα、FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα共14种急性早幼粒细胞白血病相关融合基因的组合物,其特征在于,所述组合物包括组合物1和组合物2,其中:
组合物1包括筛查PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα融合基因的引物和探针,所述引物和探针的核苷酸序列是:RARA-R,RARA-P,PML-LF,PML-VF,PML-SF,PLZF-3F,PLZF-4F,NuMA-F,STAT5b-13F,STAT5b-15F,NPM1-4F,TBLR1-5F,PRKAR1A-2F,PRKAR1A-3F;
组合物2包括筛查FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα融合基因的引物和探针,所述引物和探针的核苷酸序列是:RARA-R,RARA-P,FNDC3B-24F,BCOR-F,FIP1L1-13F,FIP1L1-15F,IRF2BP2-1F,IRF2BP2-2F,STAT3-21F,STAT3-23F,GTF2I-5F,GTF2I-6F,NABP1-5F。
5.根据权利要求4所述的组合物,其特征在于,还包括扩增作为内参的管家基因ABL1的引物和探针,其核苷酸序列如下:
ABL1-F:GATACGAAGGGAGGGTGTACCA
ABL1-R:CTCGGCCAGGGTGTTGAA
ABL1-P:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
6.权利要求1-5中任意一项所述的引物和探针在制备检测14种急性早幼粒细胞白血病相关融合基因的试剂盒中的应用。
7.实时荧光定量PCR技术筛查PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα、FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα共14种急性早幼粒细胞白血病相关融合基因的方法,其特征在于,包括以下步骤:
⑴采用红细胞裂解液裂解红细胞的方法提取全血样本中的有核细胞。10×红细胞裂解液配方为:NH4Cl 82g,NaHCO38.4g,EDTA-Na2 3.72g,加ddH2O定容至1000ml。
⑵采用TRIzol RNA提取方法提取有核细胞中的总RNA,并将RNA反转录成cDNA。
⑶以步骤2中的cDNA为模版,进行APL相关融合基因的初步筛查。筛查分第一组和第二组2个体系进行。反应条件为:95℃预变性1min;95℃10s,58℃50s,共40个循环;58℃时采集荧光。
⑷根据步骤3中的检测结果,判断是第一和第二组中的哪一组为阳性。在内参ABL1基因扩增正常(CT值≤32)的情况下,各融合基因CT值<36判定为阳性。若第一组阳性,则需鉴定是PML-RARα、PLZF-RARα、NuMA-RARα、STAT5b-RARα、NPM-RARα、TBLR1-RARα、PRKAR1A-RARα融合基因中的哪一个为阳性;若第二组阳性,则需鉴定是FNDC3B-RARα、BCOR-RARα、FIP1L1-RARα、IRF2BP2-RARα、STAT3-RARα、GTF2I-RARα、NABP1-RARα融合基因中的哪一个为阳性。
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