CN112410426B - 用荧光pcr技术筛查和鉴定pax5重排相关融合基因的引物及探针、组合物和方法 - Google Patents
用荧光pcr技术筛查和鉴定pax5重排相关融合基因的引物及探针、组合物和方法 Download PDFInfo
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Abstract
本发明涉及用荧光PCR技术筛查和鉴定PAX5重排相关融合基因的引物及探针、组合物和方法,筛查及鉴定的相关融合基因包括PAX5‑ETV6、PAX5‑JAK2、PAX5‑ELN、PAX5‑FOXP1、PAX5‑PML、PAX5‑ZNF521、PAX5‑AUTS2、PAX5‑KIDINS220、PAX5‑ESRRB、PAX5‑NOL4L、PAX5‑HIPK1、PAX5‑BRD1、PAX5‑POM121、PAX5‑DACH1、PAX5‑DACH2、PAX5‑NCOR1、PAX5‑GOLGA6A共17种。本发明所述引物、探针、检测组合方式和检测方法方便、经济、快捷,特异性好,灵敏度高,通量大,适于大批量样本的临床结合骨髓检查、染色体核型分析、免疫分型等结果综合评估疾病的发生发展;同时有针对性地进行MRD的监控,预测复发风险。
Description
技术领域
本发明属生命科学和生物技术领域,涉及特别涉及一种采用多重荧光PCR技术筛查PAX5重排相关融合基因的引物及探针、组合物和方法。
背景技术
约47%的儿童及34%的成人B-ALL患者可检出PAX5基因异常,是B-ALL最常见的遗传学异常之一,其中PAX5基因重排的发生率约为14%。涉及PAX5重排的融合基因有20余种之多,包括PAX5-ETV6、PAX5-JAK2、PAX5-ENL、PAX5-PML和PAX5-FOXP1等,这些易位导致PAX5基因异常表达,从而阻滞B淋巴细胞的分化,在B-ALL的发生和发展中起到重要的作用。由于涉及PAX5重排的病例数并不多,对于伴有这类异常的B-ALL预后的分类尚无明确的结论,但仍可以作为ALL筛查的标志物的补充,有助于MRD的监测。
由于PAX5重排涉及的断裂位点多样,涉及的伴侣基因众多,因而产生的融合基因种类也繁多。而目前尚无针对PAX5基因重排的筛查方案。仅某个融合基因而言,一般都通过染色体核型分析发现,或通过FISH的方法和PCR联合电泳的方法进行鉴定及检测。
临床对于融合基因的发现,检测及作为微小残留病灶靶标进行监测都是有强烈需求的。无论这个分子标记物是否与预后相关联,融合基因都是一个不错的监测靶标。染色体核型分析对于各种染色体易位,倒位的变化检出率并不是100%的;该方法受到细胞培养,核***相的个数,结构变化的微小程度,显带的明显程度及技术人员观察判断能力的影响。FISH方法成本高,灵敏度不及PCR;而PCR联合电泳的方法耗时长,过程繁琐,易污染,结果的判断较主观。且PCR反应体系越多,成本越高,不适用于高通量的样本筛查。
发明内容
鉴于目前筛查PAX5重排相关融合基因的方法学不能同时满足经济高效、结果准确且能用于MRD监测等需求,故本发明设计出一套用于多重荧光PCR技术检测PAX5重排常见融合基因的引物、探针,并构思出一种灵敏度高,特异性好,检测通量大,快速且准确的筛查及型别鉴定方案。
一种用荧光PCR技术筛查及鉴定PAX5-ETV6、PAX5-JAK2、PAX5-ELN、PAX5-FOXP1、PAX5-PML、PAX5-ZNF521、PAX5-AUTS2、PAX5-KIDINS220、PAX5-ESRRB、PAX5-NOL4L、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-GOLGA6A这17种相对常见PAX5重排相关融合基因的引物及探针,其特征在于采用多重荧光PCR技术,将存在于PAX5基因的上游引物、及其对应的探针分成两组,配对不同的下游基因上的多条下游引物。其核苷酸序列如下:
PAX5-4F:5'-CGGACAAAAGTACAGCAGCC-3'
PAX5-4P:5'-FAM-AACCAGTCCCAGCTTCCAGTCACAGC-TAMRA-3'
PAX5-5F:5'-CCTCGGTGAGCACGGATT-3'
PAX5-5P:5'-FAM-CATCAGCGGCATCCTGGGCATCAC-TAMRA-3'
PAX5-6F:5'-TTTGAGAGGCAGCACTACTCA-3'
PAX5-6P:5'-FAM-CATCTTCACCACCACAGAGCCCATCA-TAMRA-3'
PAX5-7F:5'-CTGACATCGGGAGCAGTGT-3'
PAX5-7P:5'-FAM-CCAGGCCCGCAGTCCTACCCCAT-TAMRA-3'
(PAX5基因参考序列:NM_016734.3)
ETV6-2R:5'-GAAGCGTAACTCGGCACTG-3'
ETV6-3R:5'-TCAGCCCACTTGAGCCACT-3'
(ETV6基因参考序列:NM_001987.5)
HIPK1-9R:5'-AATCGGTACAGCATTATCCATC-3'
(HIPK1基因参考序列:NM_198268.3)
BRD1-2R:5'-CCTTTCCTCCTCATTTGGTAA-3'
(BRD1基因参考序列:NM_001304808)
POM121-5R:5'-CGGATAGCGTCTTCTAGGTGT-3'
(POM121基因参考序列:NM_001257190.3)
DACH1-4R:5'-GGTGGGGCATCATCATAAAA-3'
(DACH1基因参考序列:NM_080759.6)
DACH2-4R:5'-CTGAAGTTTCATCGCCTCAG-3'
(DACH2基因参考序列:NM_053281.3)
NCOR1-43R:5'-AACTGCTGGCAGATTAAAGATC-3'
(NCOR1基因参考序列:NM_006311.4)
JAK2-19R:5'-CTTCAAAGGCACCAGAAAAC-3'
(JAK2基因参考序列:NM_004972.4)
FOXP1-7R:5'-AAGGAGCTGTCTTGCCACCT-3'
FOXP1-12R:5'-TGCATACACCATGTCCATAGAG-3'
(FOXP1基因参考序列:NM_032682.6)
ESRRB-5R:5'-TTTGGTGATCTCGCACTCG-3'
(ESRRB基因参考序列:NM_004452.4)
PML-2R:5'-GCAGAAACTGGAACTCCTCCT-3'
(PML基因参考序列:NM_033238.3)
AUTS2-6R:5'-GTTTTTCAGAGCTGGCATCTG-3'
(AUTS2基因参考序列:NM_015570.4)
GOLGA6A-3R:5'-TCCAGGGCTACTGCTAGTTCT-3'
(GOLGA6A基因参考序列:NM_001038640.2)
ELN-2R:5'-CAGGAACTCCACCAGGAATG-3'
(ELN基因参考序列:NM_000501.4)
ZNF521-4R:5'-CCAAGAGCAAGTCGGATCA-3'
(ZNF521基因参考序列:NM_015461.3)
KIDINS220-20R:5'-CCAAGGCTGTTCTGTGAAACT-3'
(KIDINS220基因参考序列:NM_020738.4)
NOL4L-11R:5'-GCCTTTCATGCTGAGGTCC-3'
(NOL4L基因参考序列:NM_001256798.2)。
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ABL的引物和探针,其核苷酸序列如下:
ABL1-F:5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R:5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P:5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
本发明的创新点在于:
1.PAX5基因发生重排时,断裂位点主要集中于内含子4-8,导致PAX5外显子4-8与其他基因发生融合;而其中最常见的融合基因断裂融合位置在PAX5的4内含子,断裂融合位点最多见的为PAX5的5内含子,最少见的为8内含子。为了尽量多地囊括各种PAX5重排产生的融合基因,又使PCR反应尽量减少相互干扰,一管PCR反应中的引物探针尽量单一,本发明选择在PAX5的第4、6和7外显子上各设计一条引物及其对应的探针,作为PAX5重排相关融合基因筛查的共用引物探针。同时将PCR扩增产物的长度尽量控制在250bp以内,以满足荧光PCR技术的检测范围要求。其中第4外显子上的引物探针用于检测4外显子或5外显子融合;第6外显子上的引物探针用于检测第6外显子融合;第7外显子上的引物探针用于检测第7外显子或第8外显子融合。PAX5引物探针设计方案见图1。
2.PAX5基因的断裂位点有5处之多,而与其发生融合的伴侣基因仅已报道的也多达20余种。其中最常见的为PAX5-ETV6。为了尽量减少检测的反应数,提高通量,节约成本,本发明在下游引物设计时需兼顾各种融合方式能检测到,又不宜使一个PCR反应体系中引物过于繁多,相互干扰。下游引物位置设计方案见图1。
3.PAX5重排相关融合基因筛查的组合方案上,本发明兼顾考虑引物二聚体,发夹结构等干扰因素和PAX5基因断裂融合位置的一致性,将筛查分为两组进行:
第一组:筛查PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1和PAX5-ESRRB融合基因。其引物探针序列如下:
PAX5-4F:5'-CGGACAAAAGTACAGCAGCC-3'
PAX5-4P:5'-FAM-AACCAGTCCCAGCTTCCAGTCACAGC-TAMRA-3'
ETV6-2R:5'-GAAGCGTAACTCGGCACTG-3'
ETV6-3R:5'-TCAGCCCACTTGAGCCACT-3'
HIPK1-9R:5'-AATCGGTACAGCATTATCCATC-3'
BRD1-2R:5'-CCTTTCCTCCTCATTTGGTAA-3'
POM121-5R:5'-CGGATAGCGTCTTCTAGGTGT-3'
DACH1-4R:5'-GGTGGGGCATCATCATAAAA-3'
DACH2-4R:5'-CTGAAGTTTCATCGCCTCAG-3'
NCOR1-43R:5'-AACTGCTGGCAGATTAAAGATC-3'
JAK2-19R:5'-CTTCAAAGGCACCAGAAAAC-3'
FOXP1-7R:5'-AAGGAGCTGTCTTGCCACCT-3'
FOXP1-12R:5'-TGCATACACCATGTCCATAGAG-3'
ESRRB-5R:5'-TTTGGTGATCTCGCACTCG-3';
第二组:筛查PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220、PAX5-NOL4L、PAX5-ESRRB和PAX5-FOXP1融合基因。其引物探针序列如下:
PAX5-6F:5'-TTTGAGAGGCAGCACTACTCA-3'
PAX5-6P:5'-FAM-CATCTTCACCACCACAGAGCCCATCA-TAMRA-3'
PAX5-7F:5'-CTGACATCGGGAGCAGTGT-3'
PAX5-7P:5'-FAM-CCAGGCCCGCAGTCCTACCCCAT-TAMRA-3'
PML-2R:5'-GCAGAAACTGGAACTCCTCCT-3'
AUTS2-6R:5'-GTTTTTCAGAGCTGGCATCTG-3'
GOLGA6A-3R:5'-TCCAGGGCTACTGCTAGTTCT-3'
ELN-2R:5'-CAGGAACTCCACCAGGAATG-3'
ZNF521-4R:5'-CCAAGAGCAAGTCGGATCA-3'
KIDINS220-20R:5'-CCAAGGCTGTTCTGTGAAACT-3'
NOL4L-11R:5'-GCCTTTCATGCTGAGGTCC-3'
ESRRB-5R:5'-TTTGGTGATCTCGCACTCG-3
FOXP1-7R:5'-AAGGAGCTGTCTTGCCACCT-3'
FOXP1-12R:5'-TGCATACACCATGTCCATAGAG-3'。
综合上述因素,最终设计的引物探针及筛查分组方案如上所述。
本发明还提供了鉴定PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1、PAX5-ESRRB、PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220和PAX5-NOL4L这17种融合基因的引物探针,其特征在于公用PAX5基因第4-7外显子中的一组或几组引物探针,配对不同的下游引物进行检测。其引物探针序列如下:
1.PAX5-ETV6融合基因检测引物探针:PAX5-4F,PAX5-4P,ETV6-2R,ETV6-3R;
2.PAX5-HIPK1融合基因检测引物探针:PAX5-5F,PAX5-5P,HIPK1-9R;
3.PAX5-BRD1融合基因检测引物探针:PAX5-5F,PAX5-5P,BRD1-2R;
4.PAX5-POM121融合基因检测引物探针:PAX5-5F,PAX5-5P,POM121-5R;
5.PAX5-DACH1融合基因检测引物探针:PAX5-5F,PAX5-5P,DACH1-4R;
6.PAX5-DACH2融合基因检测引物探针:PAX5-5F,PAX5-5P,DACH2-4R;
7.PAX5-NCOR1融合基因检测引物探针:PAX5-5F,PAX5-5P,NCOR1-43R;
8.PAX5-JAK2融合基因检测引物探针:PAX5-5F,PAX5-5P,JAK2-19R;
9.PAX5-FOXP1融合基因检测引物探针:PAX5-5F,PAX5-5P,PAX5-6F,PAX5-6P,PAX5-7F,PAX5-7P,FOXP1-7R,FOXP1-12R;
10.PAX5-ESRRB融合基因检测引物探针:PAX5-5F,PAX5-5P,PAX5-7F,PAX5-7P,ESRRB-5R;
11.PAX5-PML融合基因检测引物探针:PAX5-6F,PAX5-6P,PML-2R;
12.PAX5-AUTS2融合基因检测引物探针:PAX5-6F,PAX5-6P,AUTS2-6R;
13.PAX5-GOLGA6A融合基因检测引物探针:PAX5-6F,PAX5-6P,GOLGA6A-3R;
14.PAX5-ELN融合基因检测引物探针:PAX5-7F,PAX5-7P,ELN-2R;
15.PAX5-ZNF521融合基因检测引物探针:PAX5-7F,PAX5-7P,ZNF521-4R;
16.PAX5-KIDINS220融合基因检测引物探针:PAX5-7F,PAX5-7P,KIDINS220-20R;
17.PAX5-NOL4L融合基因检测引物探针:PAX5-7F,PAX5-7P,NOL4L-11R。
鉴定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:5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R:5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P:5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
本发明还提供了多重荧光PCR技术筛查及鉴定PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1、PAX5-ESRRB、PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220、PAX5-NOL4L共17种PAX5重排相关融合基因的试剂盒,所述试剂盒包括引物和探针,其核苷酸序列包括:PAX5-4F,PAX5-4P,PAX5-5F,PAX5-5P,PAX5-6F,PAX5-6P,PAX5-7F,PAX5-7P,ETV6-2R,ETV6-3R,HIPK1-9R,BRD1-2R,POM121-5R,DACH1-4R,DACH2-4R,NCOR1-43R,JAK2-19R,FOXP1-7R,FOXP1-12R,ESRRB-5R,PML-2R,AUTS2-6R,GOLGA6A-3R,ELN-2R,ZNF521-4R,KIDINS220-20R,NOL4L-11R。
还包括扩增作为内参的管家基因ABL的引物和探针,其核苷酸序列如下:
ABL1-F:5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R:5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P:5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
本发明还提供了使用上述引物及探针筛查及鉴定PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1、PAX5-ESRRB、PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220和PAX5-NOL4L这17种PAX5重排相关融合基因的方法,其特征包括以下步骤:
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PAX5重排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PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1和PAX5-ESRRB融合基因中的哪一个为阳性;若第二组阳性,则需鉴定是PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220、PAX5-NOL4L、PAX5-ESRRB和PAX5-FOXP1融合基因中的哪一个为阳性。
据目前报道显示,这17种PAX5相关融合基因在ALL中的发生率约为4-6%,在与临床预后的相关性上还没有具有统计学意义上的结论,但仍然不失为监测MRD的有效标志物。若通过常规染色体核型分析发现,会漏检一些隐性的染色体变化,其灵敏度也不能满足临床的要求;若通过FISH的方法进行PAX5分离探针的检测,则无法分辨出其伴侣基因,不利于之后的随访和MRD监测;若通过PCR联合电泳的方法,则判断主观,灵敏度也满足不了监测MRD的要求。本发明利用三对上游引物探针覆盖PAX5基因已报道的所有断裂位点,对应多种伴侣基因的断裂方式,先进行筛查后进行鉴定,将筛查检测的反应体系缩减到2管,既节约了成本,又增加了检测的通量,为大样本的筛查工作提供便利。同时将这种组合方式与荧光PCR技术相结合,避免普通PCR的开盖反应,提高了结果的准确性,避免污染的发生;也提高了结果的易判断性,使检测结果更加客观易读。该方法利于临床结合骨髓检查、染色体核型分析、免疫分型等结果综合评估疾病的发生发展,同时有针对性地进行MRD的监控,预测复发风险。
附图说明
图1是本发明引物、探针设计区域示意图。
图2是使用本发明引物探针及方法对一个待检样本进行筛查的荧光扩增曲线图。
图3是使用本发明引物探针及方法对一个已知阳性质控物进行筛查及具体融合基因鉴定的荧光扩增曲线图。
具体实施方式
实施例1:
全血RNA的提取:在1.5ml离心管中加入1ml 1×红细胞裂解液,取待检全血样本0.5ml,颠倒混匀。4000rpm离心3min,吸弃上清,加红细胞裂解液洗涤一次,得所需细胞;加lml Total RNA Isolation Reagent,反复吹吸直至无明显细胞团块,加入氯仿200μl,漩涡混匀30s,冰上静置10min。14,000rpm,4℃离心10min。用移液器吸取上清液450μl转移至另一离心管中,加入等体积的预冷异丙醇,颠倒混匀后在冰上静置10min。14,000rpm,4℃离心10min。然后用75%的乙醇和无水乙醇分别洗涤离心一次。室温干燥5min,加入50μl DEPC-H2O溶解。
实施例2:
逆转录:取实施例1中RNA溶液4ul(浓度约200ng/ul)加1ul Primer mix(ReverTraAce qPCR RT Kit)和3ulDEPC-H2O混匀,70℃预变性5min;冰上骤冷1min后加入5*RTbuffer4ul(ReverTra Ace qPCR RT Kit),Enzyme Mix 1ul(ReverTra Ace qPCR RT Kit),并加DEPC-H20 7ul至总体积为20ul。37℃60min反应后98℃5min灭活,所得即为待检样本的cDNA。
实施例3:
多重荧光PCR筛查:根据下表1所示各材料及用量配置筛查试剂。筛查试剂共含3个反应管:第一第二管为筛查检测组;第三管为ABL1内参基因检测组,用于判断RNA提取质量是否符合要求。加入实施例2中所得cDNA各2ul。按如下程序进行检测:95℃预变性60s;95℃10s,58℃50s,共40个循环;58℃时采集荧光。所得结果如图2所示:筛查显示结果为阴性。
表1
实施例4:
对PAX5-ETV6阳性质控物按实施例3所述进行多重荧光PCR筛查,所得结果如图3-A所示:显示第一组阳性。对第一组的各个融合基因进行分别鉴定,试剂配置如表2所示,并加入PAX5-ETV6阳性质控物各2ul。检测程序同实施例3。所得结果如图3-B所示:融合基因鉴定为PAX5-ETV6阳性。结果与特异性质控物型别相符合。
表2
实施例5:
根据实施例3和4所得的PAX5重排相关融合基因检测结果,总结如表3所示
表3
序列表
<110> 福州艾迪康医学检验所有限公司
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ctgaagtttc atcgcctcag 20
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gatacgaagg gagggtgtac ca 22
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ctcggccagg gtgttgaa 18
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Claims (7)
1.用多重荧光PCR技术筛查及鉴定PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1、PAX5-ESRRB、PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220、PAX5-NOL4L共17种PAX5重排相关融合基因的引物和探针,其特征在于,所述引物和探针的核苷酸序列如下:
PAX5-4F:5'-CGGACAAAAGTACAGCAGCC-3'
PAX5-4P:5'-FAM-AACCAGTCCCAGCTTCCAGTCACAGC-TAMRA-3'
PAX5-5F:5'-CCTCGGTGAGCACGGATT-3'
PAX5-5P:5'-FAM-CATCAGCGGCATCCTGGGCATCAC-TAMRA-3'
PAX5-6F:5'-TTTGAGAGGCAGCACTACTCA-3'
PAX5-6P:5'-FAM-CATCTTCACCACCACAGAGCCCATCA-TAMRA-3'
PAX5-7F:5'-CTGACATCGGGAGCAGTGT-3'
PAX5-7P:5'-FAM-CCAGGCCCGCAGTCCTACCCCAT-TAMRA-3'
ETV6-2R:5'-GAAGCGTAACTCGGCACTG-3'
ETV6-3R:5'-TCAGCCCACTTGAGCCACT-3'
HIPK1-9R:5'-AATCGGTACAGCATTATCCATC-3'
BRD1-2R:5'-CCTTTCCTCCTCATTTGGTAA-3'
POM121-5R:5'-CGGATAGCGTCTTCTAGGTGT-3'
DACH1-4R:5'-GGTGGGGCATCATCATAAAA-3'
DACH2-4R:5'-CTGAAGTTTCATCGCCTCAG-3'
NCOR1-43R:5'-AACTGCTGGCAGATTAAAGATC-3'
JAK2-19R:5'-CTTCAAAGGCACCAGAAAAC-3'
FOXP1-7R:5'-AAGGAGCTGTCTTGCCACCT-3'
FOXP1-12R:5'-TGCATACACCATGTCCATAGAG-3'
ESRRB-5R:5'-TTTGGTGATCTCGCACTCG-3'
PML-2R:5'-GCAGAAACTGGAACTCCTCCT-3'
AUTS2-6R:5'-GTTTTTCAGAGCTGGCATCTG-3'
GOLGA6A-3R:5'-TCCAGGGCTACTGCTAGTTCT-3'
ELN-2R:5'-CAGGAACTCCACCAGGAATG-3'
ZNF521-4R:5'-CCAAGAGCAAGTCGGATCA-3'
KIDINS220-20R:5'-CCAAGGCTGTTCTGTGAAACT-3'
NOL4L-11R:5'-GCCTTTCATGCTGAGGTCC-3'。
2.根据权利要求1所述的引物和探针,其特征在于,
用于PAX5-ETV6融合基因检测引物和探针的核苷酸序列是:PAX5-4F,PAX5-4P,ETV6-2R,ETV6-3R;
用于PAX5-HIPK1融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,HIPK1-9R;
用于PAX5-BRD1融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,BRD1-2R;
用于PAX5-POM121融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,POM121-5R;
用于PAX5-DACH1融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,DACH1-4R;
用于PAX5-DACH2融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,DACH2-4R;
用于PAX5-NCOR1融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,NCOR1-43R;
用于PAX5-JAK2融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,JAK2-19R;
用于PAX5-FOXP1融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,PAX5-6F,PAX5-6P,PAX5-7F,PAX5-7P,FOXP1-7R,FOXP1-12R;
用于PAX5-ESRRB融合基因检测引物和探针的核苷酸序列是:PAX5-5F,PAX5-5P,PAX5-7F,PAX5-7P,ESRRB-5R;
用于PAX5-PML融合基因检测引物和探针的核苷酸序列是:PAX5-6F,PAX5-6P,PML-2R;
用于PAX5-AUTS2融合基因检测引物和探针的核苷酸序列是:PAX5-6F,PAX5-6P,AUTS2-6R;用于PAX5-GOLGA6A融合基因检测引物和探针的核苷酸序列是:PAX5-6F,PAX5-6P,GOLGA6A-3R;
用于PAX5-ELN融合基因检测引物和探针的核苷酸序列是:PAX5-7F,PAX5-7P,ELN-2R;
用于PAX5-ZNF521融合基因检测引物和探针的核苷酸序列是:PAX5-7F,PAX5-7P,ZNF521-4R;
用于PAX5-KIDINS220融合基因检测引物和探针的核苷酸序列是:PAX5-7F,PAX5-7P,KIDINS220-20R;
用于PAX5-NOL4L融合基因检测引物和探针的核苷酸序列是:PAX5-7F,PAX5-7P,NOL4L-11R。
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ABL的引物和探针,其核苷酸序列如下:
ABL1-F:5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R:5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P:5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
4.用多重荧光PCR技术筛查及鉴定PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1、PAX5-ESRRB、PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220、PAX5-NOL4L共17种PAX5重排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PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1和PAX5-ESRRB融合基因的引物和探针,所述引物和探针的核苷酸序列是:PAX5-4F,PAX5-4P,ETV6-2R,ETV6-3R,HIPK1-9R,BRD1-2R,POM121-5R,DACH1-4R,DACH2-4R,NCOR1-43R,JAK2-19R,FOXP1-7R,FOXP1-12R,ESRRB-5R;
组合物2包括筛查PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220、PAX5-NOL4L、PAX5-ESRRB和PAX5-FOXP1融合基因的引物和探针,所述引物和探针的核苷酸序列是:PAX5-6F,PAX5-6P,PAX5-7F,PAX5-7P,PML-2R,AUTS2-6R,GOLGA6A-3R,ELN-2R,ZNF521-4R,KIDINS220-20R,NOL4L-11R,ESRRB-5R,FOXP1-7R,FOXP1-12R。
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ABL的引物和探针,其核苷酸序列如下:
ABL1-F:5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R:5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P:5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
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17种PAX5相关融合基因的试剂盒中的应用。
7.筛查及鉴定PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1、PAX5-ESRRB、PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220和PAX5-NOL4L共17种PAX5重排相关融合基因在试剂盒中的应用,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PAX5重排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PAX5-ETV6、PAX5-HIPK1、PAX5-BRD1、PAX5-POM121、PAX5-DACH1、PAX5-DACH2、PAX5-NCOR1、PAX5-JAK2、PAX5-FOXP1和PAX5-ESRRB融合基因中的哪一个为阳性;若第二组阳性,则需鉴定是PAX5-PML、PAX5-AUTS2、PAX5-GOLGA6A、PAX5-ELN、PAX5-ZNF521、PAX5-KIDINS220、PAX5-NOL4L、PAX5-ESRRB和PAX5-FOXP1融合基因中的哪一个为阳性。
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