CN117385025B - 用于检测cyp21a2基因的试剂盒 - Google Patents
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Abstract
本申请涉及生物领域,提供了用于检测CYP21A2基因的试剂盒。用于检测CYP21A2基因的试剂盒包括第一引物组和第二引物组。第一引物组包括SEQ ID NO.2和SEQ ID NO.7。第二引物组包括第一引物池、第二引物池、第三引物池、第四引物池和第五引物池。通过采用本申请的试剂盒,可以基于二代测序结果即可判断突变来源于真基因还是假基因上。另外,除检测点突变外,可同时检测CYP21A2拷贝数,融合基因以及真假基因微转换。
Description
技术领域
本申请涉及基因检测领域,更具体地,涉及用于检测CYP21A2基因的试剂盒。
背景技术
先天性肾上腺皮质增生症(Congenital Adrenal Hyperplasia,CAH)是由基因缺陷引起的肾上腺皮质类固醇激素合成障碍,导致肾上腺皮质增生。这一组常染色体隐性遗传性疾病会导致下丘脑分泌促皮质激素释放激素(CRH)和垂体分泌促肾上腺皮质激素释放激素(ACTH)增加,以代偿性应对类固醇的缺乏。
CAH有多种亚型,其中,最常见的CAH亚型是21-羟化酶缺乏症(21-hydroxylasedeficiency,21-OHD),约占90%~95%的CAH病例。21-OHD是由CYP21A2基因突变所致,CYP21A2基因的突变可以导致21-羟化酶的活性减低或丧失,进而导致类固醇激素的不足或过多合成。因此,对CYP21A2基因的突变进行检测对于CAH的早期诊断和治疗非常重要。但是CYP21A2基因有一个与其相似度非常高且无活性的假基因,即CYP21A1P。这两个基因在结构上非常相似,都包含有3.3kb的长度,并且都拥有10个外显子和9个内含子,同源性高达97%,该特点也是导致分子水平检测该基因困难的主要原因,同时真假基因之间会发生微转换及基因融合,导致CYP21A2基因的变异检测难度进一步增加,进而限制了21-OHD携带者及患者检测的检测难度。目前的检测方法通常都只针对CYP21A2基因进行检测,检测方法有三代测序,一代测序,多重连接依赖性探针扩增(Multiplex Ligation-dependent ProbeAmplification,MLPA)技术,Southern印迹杂交法等。
三代测序单读长的错误率偏高,需重复测序以纠错(增加测序成本),且成本较高通量低,无法进行大规模推广。一代测序通量低,成本高,只能对可疑的突变进行逐一检测,效率低。MLPA检测成本高,操作繁琐,耗时较长,且需要特殊的仪器设备,一般只用于研究使用,不适合筛查方法的推广应用。Southern印迹杂交法操作繁琐,整个流程需要8-10天,且对DNA的用量大,存在潜在的放射性污染。
发明内容
本申请提供了一种二代测序检测CYP21A2基因的试剂盒,通过使用该试剂盒,可以同时对CYP21A2基因的点突变、拷贝数变异以及真假基因发生的微转换及基因融合进行检测并加以区分,能精确定位到突变位于真基因还是假基因上,通过真基因扩增产物与基因组DNA进行混合,可以与其他基因检测位点同时检测,解决了市面上产品只能检测CYP21A2基因的问题,从而增加检测范围,降低检测成本,减少操作复杂度,缩短检测周期。
本申请提供了一种用于检测CYP21A2基因的试剂盒,包括:
第一引物组,所述第一引物组包括SEQ ID NO.2和SEQ ID NO.7;
第二引物组,所述第二引物组包括:
第一引物池,所述第一引物池包括引物对SEQ ID NO.8和SEQ ID NO.16、引物对SEQ ID NO.9和SEQ ID NO.17、引物对SEQ ID NO.10和SEQ ID NO.18、引物对SEQ ID NO.11和SEQ ID NO.19、引物对SEQ ID NO.12和SEQ ID NO.20、引物对SEQ ID NO.13和SEQ IDNO.21、引物对SEQ ID NO.14和SEQ ID NO.22、引物对SEQ ID NO.15和SEQ ID NO.23中的至少一个引物对;
第二引物池,所述第二引物池包括引物对SEQ ID NO.24和SEQ ID NO.33、引物对SEQ ID NO.25和SEQ ID NO.34、引物对SEQ ID NO.26和SEQ ID NO.35、引物对SEQ IDNO.27和SEQ ID NO.36、引物对SEQ ID NO.28和SEQ ID NO.37、引物对SEQ ID NO.29和SEQID NO.38、引物对SEQ ID NO.30和SEQ ID NO.39、引物对SEQ ID NO.31和SEQ ID NO.40、引物对SEQ ID NO.32和SEQ ID NO.41中的至少一个引物对;
第三引物池,所述第三引物池包括引物对SEQ ID NO.42和SEQ ID NO.48、引物对SEQ ID NO.43和SEQ ID NO.49、引物对SEQ ID NO.44和SEQ ID NO.50、引物对SEQ IDNO.45和SEQ ID NO.51、引物对SEQ ID NO.46和SEQ ID NO.52、引物对SEQ ID NO.47和SEQID NO.53中的至少一个引物对;
第四引物池,所述第四引物池包括引物对SEQ ID NO.54和SEQ ID NO.63、引物对SEQ ID NO.55和SEQ ID NO.64、引物对SEQ ID NO.56和SEQ ID NO.65、引物对SEQ IDNO.57和SEQ ID NO.66、引物对SEQ ID NO.58和SEQ ID NO.67、引物对SEQ ID NO.59和SEQID NO.68、引物对SEQ ID NO.60和SEQ ID NO.69、引物对SEQ ID NO.61和SEQ ID NO.70、引物对SEQ ID NO.62和SEQ ID NO.71中的至少一个引物对;
第五引物池,所述第五引物池包括引物对SEQ ID NO.72和SEQ ID NO.81、引物对SEQ ID NO.73和SEQ ID NO.82、引物对SEQ ID NO.74和SEQ ID NO.83、引物对SEQ IDNO.75和SEQ ID NO.84、引物对SEQ ID NO.76和SEQ ID NO.85、引物对SEQ ID NO.77和SEQID NO.86、引物对SEQ ID NO.78和SEQ ID NO.87、引物对SEQ ID NO.79和SEQ ID NO.88、引物对SEQ ID NO.80和SEQ ID NO.89中的至少一个引物对。
在一些实施例中,所述第一引物组还包括SEQ ID NO.3-6。
在一些实施例中,所述第一引物池、所述第二引物池、所述第三引物池、所述第四引物池和所述第五引物池分别放置在不同的容器中。
在一些实施例中,所述第一引物池包括SEQ ID NO.8-23,所述第二引物池包括SEQID NO.24-41,所述第三引物池包括SEQ ID NO.42-53,所述第四引物池包括SEQ ID NO.54-71,所述第五引物池包括SEQ ID NO.72-89。
在一些实施例中,所述试剂盒还包括第三引物组,所述第三引物组包括SEQ IDNO. 90-97。
本申请采用了特异性的预扩增CYP21A2基因与二代测序的方法,通过预扩增产物与基因组DNA按照一定比例进行混合,从而基于二代测序结果即可判断突变来源于真基因还是假基因上。另外,除检测点突变外,可同时检测CYP21A2拷贝数,融合基因以及真假基因微转换。在本申请中,对预扩增产物按照一定拷贝数投入到样本gDNA中,投入过高则会导致测序成本增加,过低则无法区分突变来源。另外,通过CYP21A2基因与其他基因相结合的方式,可以同时检测多种疾病类型。
附图说明
图1示出了根据本申请的实施例的CYP21A2拷贝数的检测。
图2示出了根据本申请的实施例的样本S7的真假基因微转换及融合基因的检测。
图3示出了根据本申请的实施例的样本S8的真假基因微转换及融合基因的检测。
图4示出了根据本申请的实施例的样本S9的真假基因微转换及融合基因的检测。
图5示出了根据本申请的实施例的样本S10的真假基因微转换及融合基因的检测。
图6示出了根据本申请的实施例的样本S11的真假基因微转换及融合基因的检测。
图7示出了根据本申请的实施例的样本S12的真假基因微转换及融合基因的检测。
图8示出了根据本申请的实施例的样本S13的真假基因微转换及融合基因的检测。
图9示出了根据本申请的实施例的样本S14的真假基因微转换及融合基因的检测。
具体实施方式
下面的实施例可以使本领域技术人员更全面地理解本申请,但不以任何方式限制本申请。在本申请中,除非特别说明,采用的试剂均为常规商购试剂。
本申请基于引物设计与特异的CYP21A2基因预扩增,与基因组DNA按照一定比例混合后进行多重PCR的扩增子二代测序,结合数据分析,同时检测CYP21A2基因、CYP21A2/CYP21A1P融合基因、其他遗传病相关基因,本申请成本低、操作简单、检测周期短。
本申请的方法包含主要4个步骤,分别为引物设计、真基因预扩增、扩增子测序和数据分析。
引物设计:
特异扩增CYP21A2基因全长的引物序列为:
SEQ ID NO.1 | GGGTCGGTGGGAGGGTACCTGAAG |
SEQ ID NO.2 | CACCTCTCTCGCACCCCAGTATGACT |
其中上游引物序列3’端包含CYP21A2基因和CYP21A1P基因的差异位点,下游引物不包含两基因的差异位点。
进一步地,为了覆盖c.-113G>A突变,将含差异位点的上游引物重新设计:
SEQ ID NO.3 | CCCTTCCTTGCTTCTTGATGGGTGATCA |
SEQ ID NO.4 | CCTTGCTTCTTGATGGGTGATCA |
SEQ ID NO.5 | ATTGCCTGCACAGTTGATGTGG |
SEQ ID NO.6 | ATTGCCTGCACAGTTGATGTGGAAC |
SEQ ID NO.7 | TTTCCCTTCCTTGCTTCTTGATGGGTGATCA |
优选地,特异扩增CYP21A2基因全长的引物序列为:上游引物:SEQ ID NO.7:TTTCCCTTCCTTGCTTCTTGATGGGTGATCA;下游引物:SEQ ID NO.2:CACCTCTCTCGCACCCCAGTATGACT。
二代测序引物为覆盖CYP21A2基因全部外显子区域,二代测序引物不包含差异位点,因此用于同时扩增CYP21A2基因和CYP21A1P基因。为了提高单个位点/区域内覆盖度的准确性,设计了多对引物进行扩增,同时为了避免相同区域内相近的引物之间的互相干扰,将距离相近的引物对放在不同的引物池中进行平行扩增,本申请采用5个引物池对CYP21A2基因和CYP21A1P基因进行多重PCR扩增。引物序列为:
引物池1:
SEQ ID NO. | 正向引物 | SEQ ID NO. | 对应反向引物 |
8 | TGCTGTGGAACTGGTGGAAGC | 16 | GGTGAGGGCCAGAGCGAGA |
9 | AAAGCCCACAAGAAGCTCACC | 17 | ACAGTGTGAGTTCAGCAGGC |
10 | CCCTGAGCCTCTCCTTGTCC | 18 | CCAGCCTCTCCCCTACAACC |
11 | CTTCCCACAGCTGCATTCTCAT | 19 | AAAGAACCCGCCTCATAGCAA |
12 | ACATGGCTGCAGTGGACCTC | 20 | CTCAGCAACCCAGTGAGCCT |
13 | GCAGGACTCCACCCGATC | 21 | GCAAGGCTAAGGGCACAACG |
14 | GAGGGTTGGGGATGAGTGAGG | 22 | AGCCTCCACCACATTTTCACG |
15 | AGCTCTTCGTGGTGCTGACC | 23 | GAAACTGAGGTACCCGGCTG |
引物池2:
SEQ ID NO. | 正向引物 | SEQ ID NO. | 对应反向引物 |
24 | AGGCCCCAAAACAGTCTACACA | 33 | GCAAGAGGCGGGAGGTGGA |
25 | ATGTGGTGGTGCTGAACTCC | 34 | GGGCAGCATAGCAAGAACCC |
26 | GCCTTCATCAGTTCCCACCC | 35 | CCTCACAGAACTCCTGGGTCA |
27 | GCCTGCTGAACTCACACTGTTTC | 36 | TTTCAGTTCAGGACAAGGAGAGG |
28 | CAGCTGCATTCTCATGCTTCCTG | 37 | AAAGAACCCGCCTCATAGCAA |
29 | CTCCTTTCACCCTCTGCAGGA | 38 | GAGAGGGTGTTTGCTGTGGTC |
30 | GTGCTTGCCTCACCGG | 39 | AGCCGTGCACGGTCCT |
31 | CTCAATGCCACCATCGCC | 40 | GAGGTTCGGAATGATGACTGTG |
32 | CGTGAAAATGTGGTGGAGGCT | 41 | CAGCACCACGAAGAGCTCCA |
引物池3:
SEQ ID NO. | 正向引物 | SEQ ID NO. | 对应反向引物 |
42 | CAGTCTACACAGCAGGAGGGAT | 48 | CAAGCAGATAGATGGGGAGGTC |
43 | CACTTACCTGTAAGGGCCGGG | 49 | ATGGAGTCACGGATGCCCAG |
44 | GCCTGCTGAACTCACACTGTT | 50 | CAGGACAAGGAGAGGCTCAGG |
45 | GCTTCCTGCCGCAGTTCT | 51 | AAAGAACCCGCCTCATAGCAA |
46 | CAGGCCAGCCGCTCAG | 52 | GTGTTTGCTGTGGTCTCAGTGC |
47 | GGCAGGACTCCACCCGATC | 53 | TTCCTCACTCATCCCCAACCC |
引物池4:
SEQ ID NO. | 正向引物 | SEQ ID NO. | 对应反向引物 |
54 | CGGGTCGGTGGGAGGGTAC | 63 | CTTCCACCAGTTCCACAGCAG |
55 | GACCTCCCCATCTATCTGCTTG | 64 | AGTTCAGCACCACCACATCTG |
56 | GTTGGGGAGGCCGAAGAAGG | 65 | ATGGAGTCACGGATGCCCAG |
57 | GCCTCGCCTCTCACAGTAGC | 66 | GGCACCTTGATCTTGTCTCCG |
58 | GGACCTGGAGCCTAGACACC | 67 | GTCCACGTACAGTCCCCACC |
59 | ATGAGGCGGGTTCTTTTGCA | 68 | TTCCAGGAGCTGTCCAGAGC |
60 | CACTGAGACCACAGCAAACACC | 69 | CCTGAGTGCCGGTGAGG |
61 | TCCCGGGTCCCCTACAAG | 70 | CCAACCCTCGGGAGTCAC |
62 | GAACTCCAGAGCTCTGGCCTT | 71 | GCACTTGGAAAGGCTGCATCT |
引物池5:
SEQ ID NO. | 正向引物 | SEQ ID NO. | 对应反向引物 |
72 | TCTTGAGCTATAAGTGGCACCT | 81 | CCTGTAGATGGGCCCGAA |
73 | CTTGGGCTGCAAGGTGAGAGG | 82 | CAGGTAAGTGGCTCAGGTCT |
74 | AAGAAGGTCAGGCCCTCAG | 83 | CCTCACAGAACTCCTGGGTCA |
75 | TGAACTCACACTGTTTCTCCACAG | 84 | TTTCAGTTCAGGACAAGGAGAGG |
76 | CGCAGTTCTTCCCCAATCCAG | 85 | ATGCAAAAGAACCCGCCTCA |
77 | GATGGACTACATGCTCCAAGGG | 86 | GTTGCTGGGAAGGAGCCTTTTG |
78 | CTCACTCAGCTCTGAGCACTGT | 87 | CCAGTTCGTGGTCTAGCTCCT |
79 | ACAAGGACCGTGCACG | 88 | CCAACCCTCGGGAGTCAC |
80 | AGATGCAGCCTTTCCAAGTGC | 89 | AGCCTTCTCTGCCAGCGATC |
真基因预扩增:使用预扩增引物和长片段扩增反应体系对受试者基因组DNA进行CYP21A2基因的特异性扩增,扩增产物经过磁珠纯化去除多余的酶、引物、dNTP后,使用Qubit荧光计进行定量,根据预扩增产物长度计算拷贝数。
扩增子测序:以提取的人基因组DNA为模板,与上述预扩增产物按照一定拷贝数比例进行混合,随后使用PCR引物中的至少一种多重引物组合进行一个或多个引物池的多重PCR平行扩增,得到含有CYP21A2基因和CYP21A1P基因序列,以及其他目标基因序列。所述的多重引物上下游5’端均带有通用序列,将一个或多个引物池的扩增产物进行混合后使用磁珠对扩增产物进行纯化,以纯化后的产物为模板,进行第二次PCR扩增,第二次扩增引物为含有上述多重引物5’端的通用序列,测序接头序列,以及区分不同样本的标签序列,第二次扩增后的产物再次经过磁珠纯化后得到测序文库。
数据分析:通过测序数据分析CYP21A2基因和CYP21A1P基因区域内的序列,判断是否存在突变,通过分析突变的比例,判断是否存在CYP21A2拷贝数变异,融合基因以及真假基因微转换,通过分析CYP21A2基因和CYP21A1P基因以外的区域,判断是否有其他基因突变。
目前,二代测序由于读长的限制,通常无法很好的解决假基因的干扰,因此无法直接对CYP21A2基因进行检测。本申请创造性地先扩增出CYP21A2基因全长,并按照一定比例基因组DNA进行混合,对CYP21A2基因进行多重PCR建库后,进行二代测序。同时,多重PCR可以与其他疾病相关的基因检测一同进行,节约时间和流程。
其中,多重引物5’端的通用序列分别为:上游通用序列:ACGGCCAAGGCGA;下游通用序列:TGGGCAGTCGGTGAT。
下面结合具体的实施例进行说明,选取已知的包含正常、CYP21A2基因突变的样本,对样本进行基因组DNA提取,可以采用本领域常用的基因组提取方式进行DNA提取,在此不再详细描述。
预扩增
每个样本取10ng,分别加入预扩增酶、预扩增缓冲液、预扩增引物、dNTP进行预扩增。
组分 | 体积(μL) |
DNA | 6.25 |
KOD FX Neo (1 U/μl) | 0.5 |
2× PCR Buffer for KOD FX Neo | 12.5 |
2 mMdNTPs | 5 |
预扩增引物 | 0.75 |
预扩增引物:
上游引物:SEQ ID NO.7:TTTCCCTTCCTTGCTTCTTGATGGGTGATCA;下游引物:SEQ IDNO.2:CACCTCTCTCGCACCCCAGTATGACT。
预扩增反应条件:
PCR反应结束后对预扩增产物进行1.4倍的磁珠纯化,洗脱后使用Qubit荧光计定量,计算出摩尔浓度,取预扩增产物1×105和对应样本的gDNA 10ng,分别配制引物池1-5的反应体系,CYP21A2相关的引物序列分别为上述引物池1-5中的序列。
每个样本每个引物池取10ng人类基因组DNA,分别配制各个样本的反应体系,见下表:
组分 | 体积(μL) |
DNA | 6 |
引物1混合物Primer 3/ Primer 4 | 4 |
Taq DNA聚合酶混合物 | 10 |
反应体系配置完成后,将样本放入Bio-Rad T100 Thermal Cycler PCR仪中,进行PCR1反应。程序为:94°C,5min;94°C 15s , 60°C 4min进行15个循环,72°C 10min,4°C保持,见下表:
PCR1反应结束后将取同样本的5个引物池扩增产物各4μL进行混合,对混合产物进行两步磁珠纯化,第一步加入1.4倍体积的磁珠进行纯化,第二步加入1.8倍体积的磁珠进行纯化,洗脱后的产物取15μL进行PCR2,见下表:
组分 | 体积(μL) |
PCR 1纯化产物 | 15 |
引物2 | 5 |
Taq DNA聚合酶混合物 | 20 |
引物2的上游引物(F)为:
SEQ ID NO.90:CCATCTCATCCCTGCGTGTCTCCGACTCAGCTAAGGTAACACGGCCAAGGCGA |
SEQ ID NO.91:CCATCTCATCCCTGCGTGTCTCCGACTCAGTAAGGAGAACACGGCCAAGGCGA |
SEQ ID NO.92:CCATCTCATCCCTGCGTGTCTCCGACTCAGAAGAGGATTCACGGCCAAGGCGA |
SEQ ID NO.93:CCATCTCATCCCTGCGTGTCTCCGACTCAGTACCAAGATCACGGCCAAGGCGA |
SEQ ID NO.94:CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCGTGATTCACGGCCAAGGCGA |
SEQ ID NO.95:CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCCGATAACACGGCCAAGGCGA |
SEQ ID NO.96:CCATCTCATCCCTGCGTGTCTCCGACTCAGTGAGCGGAACACGGCCAAGGCGA |
引物2的下游引物(R)SEQ ID NO.97:CCTCTCTATGGGCAGTCGGTGAT。
其中,上游引物中的CCATCTCATCCCTGCGTGTCTCCGACTCAG序列为测序接头,ACGGCCAAGGCGA序列为通用序列。下游引物中的CCTCTCTA序列为测序接头,TGGGCAGTCGGTGAT序列为通用序列。
反应体系配置完成后,将样本放入Bio-Rad T100 Thermal Cycler PCR仪中,进行PCR2反应。程序为:94°C,5min;94°C 15s , 58°C 2min进行10个循环,72°C 10min,4°C保持。见下表:
PCR2反应结束后对产物进行一步1.4倍磁珠纯化,洗脱后的产物使用Qubit荧光计定量,计算投入量后使用Thermo Fisher的Ion Proton进行半导体测序。
CYP21A2基因可以与其他基因一同进行二代测序的检测,其他二代测序基因包括且不限于:AGL、DBT、FCGR2C、GBA、GJB3、MMACHC、NTNG1、HBB、PTS、SLC37A4、SMPD1、PAH、ATP7B、GJB2、PCCA、FOXG1、NPC2、ETFA、FAH、IVD、ACADVL、COL1A1、G6PC、GAA、NPC1、BCKDHA、ETFB、GCDH、HLCS、BTD、GLB1、PCCB、ETFDH、FGFR3、IDUA。
测序数据进行多克隆、低质量序列过滤,接头序列拆分后,将序列文件比对到参考基因组(GRCh37/hg19)上的目标区域,计算CYP21A2和CYP21A1P扩增子覆盖度,根据两者覆盖度比值差异判断预扩增比例是否达到预期水平。将比对到CYP21A1P的读出结果(reads)进行重比对到CYP21A2基因上,使用TVC软件找单核苷酸位点变异(SNV)和小的插入与缺失,采用ACMG分类原则判断变异的致病性,由于真基因进行了预扩增,真基因被显著富集,可准确地检测真基因的致病变异。将CYP21A2扩增子覆盖度与参考样本的参考扩增子进行比较,计算出CYP21A2基因的拷贝数。当CYP21A2基因存在缺失时,预扩增仅能扩增未缺失的单倍型,基因拷贝数从2变为1,通过与参考样本进行对比,可进行准确检测。本申请也可检测发生在真基因上的微转换和基因融合现象,根据真假基因差异位点等位基因频率,当致病变异上下游多个位点变异碱基的频率明显升高且接近0.5时,表明该位点附近的真假基因之间发生了微转换或基因融合。
图1示出了根据本申请的实施例的CYP21A2拷贝数的检测。从图1可知,样本S1-S5为CYP21A2基因1-7号外显子缺失,S6为正常样本。
图2至图9示出了根据本申请的实施例的真假基因微转换及融合基因的检测。从图2至图9可知,样本S7-S14为存在微转换或者融合基因的样本,融合位点所在区间为32007593(A)- 32007624(GT)- 32007790(G)-32007887(T)- 32007966(TT)-32007993(C)-32008198(T)- 32008312(T)- 32008574(A)- 32008896(A)- 32008904(A)。经建库检测及数据分析,不同样本差异位点频率变化均与相应融合区域一致,均能正确检出。
本申请采用了特异性的预扩增CYP21A2基因与二代测序的方法,通过预扩增产物与基因组DNA按照一定比例进行混合,从而基于二代测序结果即可判断突变来源于真基因还是假基因上。另外,除检测点突变外,可同时检测CYP21A2拷贝数,融合基因以及真假基因微转换。在本申请中,对预扩增产物按照一定拷贝数投入到样本gDNA中,投入过高则无法判断假基因是否有突变,过低则无法区分突变来源。另外,通过CYP21A2基因与其他基因相结合的方式,可以同时检测多种疾病类型。
因此,本申请提供的方法能够满足同时对CYP21A2基因多种类型的致病变异检测的需求。通过采用巧妙的特异性预扩增,结合二代测序的方法,解决了CYP21A2基因容易被假基因干扰的问题,对于多种疾病类型的基因检测,无需采用多种不同的检测手段,显著提高了处理样本的效率,节约了运行成本。本申请的方法成本低,操作简单,检测周期短,并且分析简单。
本领域技术人员应理解,以上实施例仅是示例性实施例,在不背离本申请的精神和范围的情况下,可以进行多种变化、替换以及改变。
Claims (3)
1.一种用于检测CYP21A2基因的试剂盒,其特征在于,包括:
第一引物组,所述第一引物组包括SEQ ID NO.2和SEQ ID NO.7;
第二引物组,所述第二引物组包括:
第一引物池,所述第一引物池包括引物对SEQ ID NO.8和SEQ ID NO.16、引物对SEQ IDNO.9和SEQ ID NO.17、引物对SEQ ID NO.10和SEQ ID NO.18、引物对SEQ ID NO.11和SEQID NO.19、引物对SEQ ID NO.12和SEQ ID NO.20、引物对SEQ ID NO.13和SEQ ID NO.21、引物对SEQ ID NO.14和SEQ ID NO.22、引物对SEQ ID NO.15和SEQ ID NO.23;
第二引物池,所述第二引物池包括引物对SEQ ID NO.24和SEQ ID NO.33、引物对SEQID NO.25和SEQ ID NO.34、引物对SEQ ID NO.26和SEQ ID NO.35、引物对SEQ ID NO.27和SEQ ID NO.36、引物对SEQ ID NO.28和SEQ ID NO.37、引物对SEQ ID NO.29和SEQ IDNO.38、引物对SEQ ID NO.30和SEQ ID NO.39、引物对SEQ ID NO.31和SEQ ID NO.40、引物对SEQ ID NO.32和SEQ ID NO.41;
第三引物池,所述第三引物池包括引物对SEQ ID NO.42和SEQ ID NO.48、引物对SEQID NO.43和SEQ ID NO.49、引物对SEQ ID NO.44和SEQ ID NO.50、引物对SEQ ID NO.45和SEQ ID NO.51、引物对SEQ ID NO.46和SEQ ID NO.52、引物对SEQ ID NO.47和SEQ IDNO.53;
第四引物池,所述第四引物池包括引物对SEQ ID NO.54和SEQ ID NO.63、引物对SEQID NO.55和SEQ ID NO.64、引物对SEQ ID NO.56和SEQ ID NO.65、引物对SEQ ID NO.57和SEQ ID NO.66、引物对SEQ ID NO.58和SEQ ID NO.67、引物对SEQ ID NO.59和SEQ IDNO.68、引物对SEQ ID NO.60和SEQ ID NO.69、引物对SEQ ID NO.61和SEQ ID NO.70、引物对SEQ ID NO.62和SEQ ID NO.71;
第五引物池,所述第五引物池包括引物对SEQ ID NO.72和SEQ ID NO.81、引物对SEQID NO.73和SEQ ID NO.82、引物对SEQ ID NO.74和SEQ ID NO.83、引物对SEQ ID NO.75和SEQ ID NO.84、引物对SEQ ID NO.76和SEQ ID NO.85、引物对SEQ ID NO.77和SEQ IDNO.86、引物对SEQ ID NO.78和SEQ ID NO.87、引物对SEQ ID NO.79和SEQ ID NO.88、引物对SEQ ID NO.80和SEQ ID NO.89;
第三引物组,所述第三引物组包括SEQ ID NO. 90-97;
其中,所述第二引物组的所有引物对的上下游引物5’端均带有通用序列,上游引物5'端带有的通用序列为:ACGGCCAAGGCGA;下游引物5'端带有的通用序列为:TGGGCAGTCGGTGAT。
2.根据权利要求1所述的用于检测CYP21A2基因的试剂盒,其特征在于,所述第一引物组还包括SEQ ID NO.3-6。
3.根据权利要求1所述的用于检测CYP21A2基因的试剂盒,其特征在于,所述第一引物池、所述第二引物池、所述第三引物池、所述第四引物池和所述第五引物池分别放置在不同的容器中。
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