WO2017076299A1 - 一种多重pcr引物及应用 - Google Patents

一种多重pcr引物及应用 Download PDF

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WO2017076299A1
WO2017076299A1 PCT/CN2016/104391 CN2016104391W WO2017076299A1 WO 2017076299 A1 WO2017076299 A1 WO 2017076299A1 CN 2016104391 W CN2016104391 W CN 2016104391W WO 2017076299 A1 WO2017076299 A1 WO 2017076299A1
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seq
multiplex pcr
primer
thalassemia
primers
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French (fr)
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葛良进
刘松
李改玲
邓力蔚
林群婷
刘丽春
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深圳市瀚海基因生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention belongs to the field of molecular biology, and particularly relates to a multiplex PCR primer and method and application thereof for detecting ⁇ -thalassemia mutation based on next generation sequencing technology.
  • ⁇ -thalassemia is a monogenic genetic blood disease caused by the imbalance of peptide chain expression caused by ⁇ -globin gene (HBB) mutation, which is caused by point mutation of ⁇ -globin gene.
  • HBB ⁇ -globin gene
  • Beta-thalassemia is one of the most common hereditary in the world, and it is estimated that more than 100 million people worldwide carry the beta-thalassemia gene.
  • ⁇ -thalassemia is also one of the most common and most harmful genetic diseases in the southern provinces of China.
  • the carrying rate of ⁇ -thalassemia in some provinces of China is shown in Table 1.
  • the thalassemia gene is mainly a point mutation, and the ⁇ -thalassemia gene covers 46 point mutations in the southern Chinese population.
  • Table 1 Population carrying rate of ⁇ -thalassaemia in southern China
  • Beta thalassemia carrying rate (%) Guangzhou 2.54 Guangxi 6.78 Sichuan 2.18 Guizhou 4.72 Taiwan 1.10 Hong Kong 3.41
  • the main diagnostic methods for ⁇ -thalassemia are: Sanger sequencing, restriction fragment length polymorphism (RFLP), reverse dot blot (RDB), allele-specific PCR (ARMS-PCR).
  • RFLP restriction fragment length polymorphism
  • RDB reverse dot blot
  • ARMS-PCR allele-specific PCR
  • ARMS-PCR requires the design of corresponding primers for each mutation, and each pair of primers should be optimized for amplification conditions. If multiple mutations are required to be detected simultaneously, the operation is cumbersome, and false positive or false negative results may occur.
  • the present invention provides a multiplex PCR primer and method and application for detecting ⁇ -thalassemia mutation based on next generation sequencing technology.
  • the invention provides a multiplex PCR primer.
  • the multiplex PCR primer comprises at least two pairs of primer pairs: SEQ ID NO: 1 and SEQ ID NO: 2, primer pairs, SEQ ID NO: 3 and SEQ ID NO: Primer pair, primer pairs as shown in SEQ ID NO: 5 and SEQ ID NO: 6, primer pairs shown in SEQ ID NO: 7 and SEQ ID NO: 8, primers shown in SEQ ID NO: 9 and SEQ ID NO: 10. Pairs and primer pairs set forth in SEQ ID NO: 11 and SEQ ID NO: 12.
  • the multiplex PCR primer set provided by the invention can be used for the efficient detection of ⁇ -thalassemia mutation diversity, and can cover 46 point mutation sites of ⁇ -thalassemia mutation in southern China.
  • the multiplex PCR primer described above may further comprise at least one of the following additional technical features:
  • the primer is a primer obtained by extending or truncating the 3' end of one or more primers of SEQ ID NO: 1 to SEQ ID NO: 12 by 0 to 3 bases.
  • a multiplexed PCR primer set (such as a primer set consisting of 6 pairs of primers shown in SEQ ID NO: 1 to SEQ ID NO: 12) is obtained, a better amplification effect is obtained.
  • primers it is also possible to extend or shorten the length of any one of the primers in the multiplex PCR primer set by 0 to 3 bases, and also obtain a good multiplex PCR amplification effect, and should also be incorporated into the protection of the present invention. range.
  • the invention proposes the use of the multiplex PCR primers described above for obtaining and/or detecting a HBB gene sequence.
  • the invention proposes a kit.
  • the kit comprises the multiplex PCR primers described above.
  • the invention provides the use of the kit described above in at least one of the following: obtaining and/or detecting a HBB gene sequence; detecting beta-thalassemia; detecting a beta-thalassemia mutant polymorphism.
  • the invention proposes a method of multiplex PCR.
  • the method comprises performing the multiplex PCR using the multiplex PCR primers described above.
  • the invention proposes a method for detecting HBB genes.
  • the method comprises: (1) amplifying at least a portion of the nucleic acid in the sample to be tested using the method described above to obtain an amplification product; and (2) analyzing the amplification product to obtain HBB gene test results.
  • the above method for detecting HBB gene may further include the following additional technical features. At least one:
  • the nucleic acid is DNA and/or RNA.
  • the system for performing multiplex PCR is configured with reference to a general PCR system.
  • the nucleic acid is RNA
  • step (1) comprises: (1-1) reverse transcription of the RNA into cDNA using upstream or downstream primers in the multiplex PCR primer; and (1) 2) Amplifying the cDNA using the corresponding downstream or upstream primers in the multiplex PCR primer to obtain an amplification product.
  • the reverse transcription synthesis cDNA is first synthesized, and then the second strand DNA is synthesized.
  • the step of reverse transcription synthesis of cDNA is equivalent to one cycle of multiplex PCR (only Using the upstream primer set or the downstream primer set), the step of synthesizing the second strand DNA is also equivalent to one cycle of multiplex PCR (using only the downstream primer set or the upstream primer set).
  • each upstream primer in the upstream primer set is equimolar mixed; each downstream primer in the downstream primer set is equimolar mixed.
  • the multiplex PCR reaction system has a template amount of 50 ng to 1 ⁇ g/50 ⁇ l of the system.
  • the procedure of the multiplex PCR reaction is:
  • the above procedure is specifically: pre-denaturation at 95 ° C for 15 min, denaturation at 95 ° C for 15 s, annealing at 60 ° C for 2 min, extension at 72 ° C for 3 min, cycling 30 to 40 times (preferably 35 times), and finally extending at 72 ° C for 10 min.
  • the DNA fragment of the fragment length of 240-270 bp is recovered by electrophoresis and tapping.
  • the "upstream primer set” or the “downstream primer set” is an upstream primer set or a downstream primer set consisting of an upstream primer or a downstream primer of each primer pair in the multiplex PCR primer according to the first aspect;
  • the present invention uses "F” for the upstream primer and "R” for the downstream primer, as shown in Table 2.
  • step (2) comprises: (2-1) sequencing the amplification product; and (2-2) aligning the sequencing result with a wild type sequence of the HBB gene to determine the HBB gene Mutation site.
  • the invention provides a method of detecting beta-thalassemia.
  • the method comprises: performing HBB gene detection using the HBB gene detection method described above, obtaining a detection result, the sample to be tested is from a subject; and evaluating the subject according to the detection result Suffering from the risk of beta-thalassemia.
  • the above method for detecting ⁇ -thalassemia may further include the following additional techniques At least one of the features:
  • the HBB gene detection method is carried out by using the HBB gene detection method described above, and the detection result is obtained by constructing the obtained multiplex PCR product, performing high-throughput sequencing, and passing biological information.
  • the analysis obtained high-throughput sequencing results.
  • the ⁇ -thalassemia mutation was analyzed to assess the risk of ⁇ -thalassemia in the subject.
  • the test result has at least one of the mutation sites shown in Table 3, which is an indication that the patient has beta thalassemia.
  • the method for expressing the mutation site shown in Table 3 is indicated by the position of the site on the reference cDNA according to the nomenclature such as HGVS, and one mutation site may have other representations, such as GenBank SNP.
  • the naming method of the database is a SNP site representation beginning with "rs". For example, rs671 and the ALDH2 gene c.1510G>A (G1510A) represent the same site.
  • rs671 and the ALDH2 gene c.1510G>A represent the same site.
  • One of ordinary skill in the art will recognize that it is within the scope of the invention to employ other nomenclatures such as labeling the same mutations on the reference gDNA as referring to the same mutations as the present invention.
  • the beneficial effects of the multiplex PCR primers and methods for detecting ⁇ -thalassemia mutation based on next-generation sequencing technology provided by the present invention are as follows: 1) covering 46 point mutation sites in the southern population; 2) the average amplification product length is about 250 bp. The amplified product is available for all next generation sequencing platforms.
  • FIG. 1 is an agarose gel electrophoresis diagram of PCR of a single primer according to an embodiment of the present invention
  • FIG. 2 is an agarose gel electrophoresis diagram of multiplex PCR according to an embodiment of the present invention
  • FIG. 3 is a result of bioinformatics analysis of sequencing depth according to an embodiment of the present invention.
  • the reagents used in the embodiments of the present invention are all commercially available products, and the databases used in the embodiments of the present invention are all public online databases.
  • primers of the present invention are shown in Table 2:
  • Design primers Oligo 7.0 and MFEprimer-2.0 were used to analyze primer dimers and stem-loop mismatches, and primers were designed at both ends of the exon containing the mutation site.
  • the average length of the amplified sequences was about 250 bp, and 6 pairs.
  • the annealing temperatures of the primers are basically the same.
  • the primer set provided in this example covers 46 point mutation sites in the southern population. Due to the small sequence variation, the amplification effect of the primers was significantly reduced.
  • the inventors designed multiple sets of multiplex PCR primer sets for different segments of different target regions. After pre-experiment screening, the length of the integrated product fragments and point mutation coverage Scope, the present invention selects the primer set with the best amplification effect, as shown in Table 2.
  • Table 3 shows 46 kinds of beta thalassemia point mutations which resulted in decreased expression in the southern Chinese population and corresponding primers of the present invention.
  • Example 1 provides a method for preparing a DNA sample to be tested for ⁇ -thalassemia mutation, comprising the following steps:
  • the collected fresh peripheral blood samples were each 2 ml (ml), and the genomic DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Cat. No: 51304) kit, and the concentration and purity of the DNA were measured with Nanodrop 2000 (Thermo), and then the genomic DNA was preserved.
  • QIAamp DNA Mini Kit Qiagen, Cat. No: 51304
  • Example 2 provides a method for constructing a ⁇ -thalassemia mutation sequencing library using multiplex PCR primers for detecting ⁇ -thalassemia mutations, comprising the following steps:
  • Example 2 Using the genomic DNA obtained in Example 1 as an amplification template, a total of 6 pairs of primer pairs shown in SEQ ID NO: 1 to SEQ ID NO: 12 were used, and then QIAGEN Multiplex PCR Kit (Cat. No. 206143) was used. The kit instructions configure a multiplex PCR system.
  • Multiplex Buffer 2 ⁇
  • Q solvent Q solution, 5 ⁇
  • DNA 10 ⁇ l Total 50 ⁇ l
  • Each primer was mixed in an equimolar amount, and the total concentration of the primer was 10 micromoles, and the amount of the template was adjusted, and 200 ng was used in this example.
  • the PCR instrument program is set up according to the following multiplex PCR conditions to perform multiplex PCR:
  • the PCR product was stored at 4 ° C and detected by electrophoresis, and the target fragment of about 240-270 bp was cut under ultraviolet light.
  • the remaining PCR product was directly recovered to obtain 27 uL of the purified product, and the recovery step was carried out by QIAGEN QIAquick Gel Purification Kit according to routine laboratory procedures).
  • the present invention separately performs PCR pre-experiment on the 6 pairs of primers in Table 2, and the PCR system and the cycle parameters are the same as the above multiplex PCR, except that the primers are respectively replaced with P1 of Table 2 ( HBB-1F and HBB-1R), P2 (HBB-2F and HBB-2R), P3 (HBB-3F and HBB-3R), P4 (HBB-4F and HBB-4R), P5 (HBB-5F and HBB- 5R) and P6 (HBB-6F and HBB-6R) have 6 pairs of primers.
  • P1 of Table 2 HBB-1F and HBB-1R
  • P2 HBB-2F and HBB-2R
  • P3 HBB-3F and HBB-3R
  • P4 HBB-4F and HBB-4R
  • P5 HBB-5F and HBB- 5R
  • P6 HBB-6F and HBB-6R
  • the electrophoresis patterns of the amplification products of 6 pairs of primers alone PCR are shown in Fig. 1.
  • the annealing temperatures of the 6 pairs of primers were basically the same, the amplification efficiency was consistent, the length of the amplified products was basically close, and the average length was about 250 bp.
  • the amplification products of 6 pairs of primers covered 46 point mutation sites of ⁇ -thalassemia mutation.
  • the system was placed at 37 ° C for 30 min.
  • the A-tailed PCR product was purified using QIAGEN QIAquick Glue Purification Kit.
  • sequence of the Adaptor is as shown in SEQ ID NO: 13:
  • the configured system was subjected to PCR reaction as follows:
  • the size of the amplified fragment of the PCR product was verified by 1.5% agarose gel electrophoresis, and the 370 bp target fragment was selected for gel extraction and purified to 30 ⁇ L.
  • step 4 The purified product obtained in step 4 was directly sequenced by sequencing with the Miseq platform.
  • the sequencing result is data in the fastq format, and the ⁇ -thalassemia point mutation is obtained by bioinformatics analysis.
  • the sequencing depth is shown in Figure 3.
  • the PE150 kit was used for sequencing, that is, 150 bp was sequenced at both ends of the fragment.
  • Bioinformatics analysis The sequencing depth is shown in Figure 3.
  • the abscissa is the amplification product obtained with each pair of primers, and the ordinate is the sequencing depth.
  • the sequencing results of this example covered the amplification products of all primer pairs, and the distribution was relatively average.

Abstract

提供了一种检测β-地中海贫血突变的多重PCR引物，所述多重PCR引物为如下引物对中的两对或多对：SEQ ID NO:1和SEQ ID NO:2所示引物对、SEQ ID NO:3和SEQ ID NO:4所示引物对、SEQ ID NO:5和SEQ ID NO:6所示引物对、SEQ ID NO:7和SEQ ID NO:8所示引物对、SEQ ID NO:9和SEQ ID NO:10所示引物对、SEQ ID NO:11和SEQ ID NO:12所示引物对。

Description

一种多重PCR引物及应用
优先权信息
本申请请求2015年11月4日向中国国家知识产权局提交的、专利申请号为201510740909.5的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明属于分子生物学领域，特别涉及一种基于下一代测序技术检测β-地中海贫血突变的多重PCR引物和方法及应用。
背景技术
β-地中海贫血是一种由于β-珠蛋白基因(HBB)突变导致肽链表达失衡而产生的单基因遗传血液病，多由β-珠蛋白基因点突变所致。β-地中海贫血是世界上最常见的遗传性之一，据统计全世界约有1亿多人携带β-地中海贫血基因。β-地中海贫血也是我国南方各省最常见、危害最大的遗传病之一，我国部分省份的β-地中海贫血携带率见表1。地中海贫血基因主要为点突变，在中国南方人群中β-地中海贫血基因覆盖了46种点突变。
表1:中国南方β地贫的人群携带率
地区 β地贫携带率（％）
广州 2.54
广西 6.78
四川 2.18
贵州 4.72
台湾 1.10
香港 3.41
β-地中海贫血的基因诊断方法主要有：Sanger测序法、限制性片段长度多态性(RFLP)、反向点杂交(RDB)、等位基因特异性PCR(ARMS-PCR)。Sanger测序方法操作较繁琐，极大地限制了其在临床诊断中的应用，测序的检测通量也有限，并且对检测结果需要人工分析，分析耗时耗力。RFLP是通过识别特异性序列位点进行酶切反应，方法简便成本低廉，但其局限性是只能对有限的可产生酶切位点的突变进行检测，由于不完全或部分酶切反应还会导致假阳性或假阴性结果。反向点杂交技术的结果是用肉眼判读，失误率高，往往造成一份样本反复检测。ARMS-PCR需针对每个突变设计相应引物，每一对引物扩增条件都需优化，若需同时检测多种突变时则操作繁琐，而且也会出现假阳性或假阴性结果。
基于地中海贫血缺陷基因的市场和社会检测需求，以及目前地中海贫血缺陷基因检测 现状的落后，利用高通量测序技术开发地中海贫血基因突变位点检测试剂盒势在必行。
发明内容
鉴于此，本发明提供了一种基于下一代测序技术检测β-地中海贫血突变的多重PCR引物和方法及应用。
第一方面，本发明提供了一种多重PCR引物。根据本发明的实施例，所述多重PCR引物包括如下引物对中的至少两对：SEQ ID NO:1和SEQ ID NO:2所示引物对、SEQ ID NO:3和SEQ ID NO:4所示引物对、SEQ ID NO:5和SEQ ID NO:6所示引物对、SEQ ID NO:7和SEQ ID NO:8所示引物对、SEQ ID NO:9和SEQ ID NO:10所示引物对以及SEQ ID NO:11和SEQ ID NO:12所示引物对。
本发明提供的多重PCR引物组能用于β-地中海贫血突变多样性的高效检测，可覆盖中国南方人群β-地中海贫血突变的46个点突变位点。
根据本发明的实施例，上述多重PCR引物还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述引物为SEQ ID NO:1～SEQ ID NO:12中的一条或多条引物3’端延长或截短0～3个碱基后所得的引物。
本领域技术人员可以理解的，若摸索的多重PCR引物组(比如SEQ ID NO:1～SEQ ID NO:12所示的6对引物组成的引物组)获得了较佳的扩增效果，一般情况下，设计引物的时候，适当的将多重PCR引物组中任一一条引物的长度延长或截短0～3个碱基，也可以获得不错的多重PCR扩增效果，也应纳入本发明保护范围。
第二方面，本发明提出了前面所述的多重PCR引物在获得和/或检测HBB基因序列中的用途。
第三方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括前面所述的多重PCR引物。
第四方面，本发明提出了前面所述的试剂盒在下列至少之一中的用途：获得和/或检测HBB基因序列；检测β-地中海贫血症；检测β-地中海贫血突变多态性。
第五方面，本发明提出了一种多重PCR的方法。根据本发明的实施例，所述方法包括利用前面所述的多重PCR引物进行所述多重PCR。
第六方面，本发明提出了一种HBB基因检测的方法。根据本发明的实施例，所述方法包括：(1)利用前面所述的方法对待测样品中的至少一部分核酸进行扩增，获得扩增产物；(2)分析所述扩增产物，以获得HBB基因检测结果。
根据本发明的实施例，上述HBB基因检测的方法还可以进一步包括如下附加技术特征 至少之一：
根据本发明的实施例，所述核酸为DNA和/或RNA。
根据本发明的具体实施例，当所述核酸为DNA时，进行多重PCR的体系参照普通PCR体系进行配置。
根据本发明的实施例，所述核酸为RNA，步骤(1)包括：(1-1)利用所述多重PCR引物中的上游或下游引物将所述RNA反转录为cDNA；以及(1-2)利用所述多重PCR引物中相应的下游或上游引物对所述cDNA进行扩增，获得扩增产物。根据本发明的具体实施例，当所述取核酸为RNA时，要先进行逆转录合成cDNA，再合成第二链DNA，此时，逆转录合成cDNA的步骤相当于一个循环的多重PCR(只采用上游引物组或下游引物组)，合成第二链DNA的步骤也相当于一个循环的多重PCR(只采用下游引物组或上游引物组)。
根据本发明的实施例，所述多重PCR反应的体系中，上游引物组中的各上游引物等摩尔混合；下游引物组中的各下游引物等摩尔混合。
根据本发明的具体实施例，所述多重PCR反应的体系中，模板量50ng～1μg/50μl体系。
根据本发明的具体实施例，所述多重PCR反应的程序为：
Figure PCTCN2016104391-appb-000001
上述程序具体为：95℃预变性15min，95℃变性15s，60℃退火2min，72℃延伸3min，循环30～40次(优选为35次)，最后72℃后延伸10min。
根据本发明的再一具体实施例，所述多重PCR反应结束后，电泳，割胶回收片段长度为240-270bp的DNA片段。
如本发明所述的，“上游引物组”或“下游引物组”为如第一方面所述的多重PCR引物中各引物对的上游引物或下游引物组成的上游引物组或下游引物组；具体地，本发明用“F”表示上游引物，用“R”表示下游引物，如表2所示。
根据本发明的实施例，步骤(2)包括：(2-1)对所述扩增产物进行测序；以及(2-2)将测序结果与HBB基因野生型序列比对，以确定HBB基因的突变位点。
第七方面，本发明提出了一种检测β-地中海贫血症的方法。根据本发明的实施例，所述方法包括：利用前面所述的HBB基因检测方法进行HBB基因检测，获得检测结果，所述待测样品来自受检者；以及依据检测结果，评估受检者的患β-地中海贫血症风险。
根据本发明的实施例，上述检测β-地中海贫血症的方法还可以进一步包括如下附加技 术特征至少之一：
根据本发明的实施例，利用前面所述的HBB基因检测方法进行HBB基因检测，获得检测结果是通过如下方式实现的：将所得多重PCR产物进行建库，进行高通量测序，并通过生物信息学分析获得高通量测序结果。进而依据测序结果，分析β-地中海贫血突变，评估受检者的患β-地中海贫血症风险。
根据本发明的具体实施例，所述检测结果具有表3所示的突变位点的至少之一，是患者患有β地中海贫血症的指示。需要说明的是，表3所示的突变位点的表示方法是根据HGVS等命名法标注该位点在参考cDNA上的位置来表示的，一个突变位点还可以有其它表示方式，如GenBank SNP数据库的命名方法，是以“rs”开头的SNP位点表示方式，例如，rs671与ALDH2基因c.1510G>A(G1510A)表示同一个位点。本领域普通技术人员知道，采用其它命名方式比如以该突变在参考gDNA上的位置来标注指代与本发明一样的突变也属于本发明范围。
本发明提供的基于下一代测序技术检测β-地中海贫血突变的多重PCR引物及方法的有益效果为：1)覆盖了南方人群46种点突变位点；2)平均扩增产物的长度在250bp左右，扩增的产物可用于所有的下一代测序平台。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为本发明实施例提供的单独引物PCR的琼脂糖凝胶电泳图；
图2为本发明实施例提供的多重PCR的琼脂糖凝胶电泳图；以及
图3为本发明实施例提供的测序深度的生物信息学分析结果。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
材料及试剂说明：
β地中海贫血患者：来源于深圳市人民医院，患者知情同意。非特殊说明，本发明实施例采用的试剂均为市售商品，本发明实施例采用的数据库均为公开的在线数据库。
具体地，本发明引物如表2所示：
表2：多重PCR引物序列
Figure PCTCN2016104391-appb-000002
设计引物：采用Oligo 7.0和MFEprimer-2.0对引物二聚体以及茎环错配进行分析，在包含突变位点的外显子两端设计引物，扩增的序列平均长度在250bp左右，且6对引物的退火温度基本一致。
本实施例提供的引物组覆盖了南方人群46个点突变位点。由于很小的序列变化将导致引物扩增效果显著降低，发明人分别针对不同目的区域的不同区段设计了多组多重PCR引物组，在经过预实验筛选后，综合产物片段长度和点突变覆盖范围，本发明选取了扩增效果最佳的引物组，如表2所示。
表3为46种见于中国南方人群导致表达降低的β地中海贫血点突变及对应的本发明的引物。
表3：
Figure PCTCN2016104391-appb-000003
Figure PCTCN2016104391-appb-000004
实施例1
实施例1提供了一种β-地中海贫血突变待测DNA样品的制备方法，包括如下步骤：
收集的新鲜外周血样本各2毫升(ml)，采用QIAamp DNA Mini Kit(Qiagen,Cat.No:51304)试剂盒提取基因组DNA，并用Nanodrop2000(Thermo)测定DNA的浓度及纯度，然后保存基因组DNA。
实施例2
实施例2提供了一种采用检测β-地中海贫血突变的多重PCR引物构建β-地中海贫血突变测序文库的方法，包括如下步骤：
1、多重PCR：
以实施例1所得基因组DNA为扩增模板，采用SEQ ID NO:1～SEQ ID NO:12所示共6对引物对，再采用QIAGEN公司Multiplex PCR试剂盒(货号：206143)，按试 剂盒说明书配置多重PCR体系。
反应体系如表4所示。
表4：
多重PCR缓冲液(Multiplex Buffer,2×) 25μl
Q溶剂(Q solution,5×) 10μl
引物 5μl
DNA 10μl
Total 50μl
各引物等摩尔混合，引物总浓度是10微摩尔，模板量可以调整，本实施例中采用200ng。
再按下述多重PCR的条件设置PCR仪器程序，进行多重PCR：
Figure PCTCN2016104391-appb-000005
PCR结束后，4℃保存PCR产物并电泳检测，在紫外下切下约240-270bp左右的目的片段。直接回收剩余的PCR产物，得到27uL纯化后的产物，回收步骤采用QIAGEN公司QIAquick胶纯化试剂盒，按常规实验室操作进行)。
为充分说明书本发明实施例的有益效果，本发明单独对表2中的6对引物进行PCR预实验，PCR体系和循环参数等均与上述多重PCR一样，除引物分别替换为表2的P1(HBB-1F和HBB-1R)、P2(HBB-2F和HBB-2R)、P3(HBB-3F和HBB-3R)、P4(HBB-4F和HBB-4R)、P5(HBB-5F和HBB-5R)和P6(HBB-6F和HBB-6R)共6对引物。
6对引物单独PCR的扩增产物电泳图谱如图1所示。6对引物的退火温度基本一致，扩增效率一致，扩增产物长度基本接近，平均长度在250bp左右。6对引物的扩增产物覆盖了β-地中海贫血突变的46种点突变位点。
2、加尾：
取纯化后的PCR产物，在产物3’末端加A尾，配置体系如表5所示(其中，Klenow exo-购自NEB，货号：M0212)：
表5：
10×NEB2 BUFFER 5μl
dATP(1mM) 10μl
Klenow exo- 3μl
DNA 32μl
Total 50μl
将该体系置于37℃下30min。利用QIAGEN公司QIAquick胶纯化试剂盒纯化加A尾的PCR产物。
3、加接头：
在DNA两端加上测序用的接头，配置体系如表6所示(其中，Quick ligase购自NEB，M2200L)：
表6：
5×quick ligase buffer 10μl
Adaptor 1μl
Quick ligase 5μl
加A尾的PCR产物 25μl
H2O 9μl
Total 50μl
其中，Adaptor的序列如SEQ ID NO：13所示：
Figure PCTCN2016104391-appb-000006
随后将该体系置于20℃下15min。然后加入3μL的USER，37℃放置15min。最后通过QIAGEN公司QIAquick胶回收试剂盒纯化连接产物。
4、产物上加入测序用的标签序列，配置体系如表7所示(具体步骤参照illumina高通量测序文库构建说明书)
表7：
5×Q5 Solution buffer 10μl
dNTP(10mM) 1μl
P1(P5序列+common序列+接头序列) 1μl
Index(P7序列+标签序列Index+接头序列) 1μl
Q5酶 0.5μl
纯化后的连接产物 36.5μl
Total 50μl
将配置的体系按下述程序进行PCR反应：
Figure PCTCN2016104391-appb-000007
PCR结束后，用1.5％琼脂糖凝胶电泳验证PCR产物富集片段的大小，选取370bp的目的片段进行切胶回收，并纯化至30μL。
琼脂糖凝胶电泳结果图2所示。在图2中，T1泳道可以明显看到在300bp-400bp之间有一条带，为加上接头的片段(370bp，箭头所指处)，与理论相符。
5、将步骤4获得的纯化产物直接用Miseq平台测序进行测序。
测序结果是fastq格式的数据，通过生物信息学分析获得β地中海贫血点突变情况。测序深度见图3。本实施例使用PE150试剂盒进行测序，即片段两端各测序150bp。生物信息学分析测序深度如图3所示，横坐标是利用每对引物得到的扩增产物，纵坐标是测序深度。由图3可知，本实施例测序结果中覆盖了所有引物对的扩增产物，并且分布比较平均。
效果实施例1
采用实施例2的方法，对14例样品分别进行多重PCR、加A尾、加接头、加标签序列、高通量测序及生物信息学分析，获得如下结果，部分结果如表8所示：
表8：β-地中海贫血突变检测结果
Figure PCTCN2016104391-appb-000008
Figure PCTCN2016104391-appb-000009
如表8所示，在14例样品中，共检测到11处突变。特别值得注意的是，经过对样品测序的数据分析，证实所设计的目标序列在每个DNA样品中均得到有效的扩增，从而反映出所提供的检测方案具有较好的特异性和适用性。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨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，本发明的范围由权利要求及其等同物限定。

Claims (12)

  1. 一种多重PCR引物，其特征在于，包括如下引物对中的至少两对：
    SEQ ID NO:1和SEQ ID NO:2所示引物对、
    SEQ ID NO:3和SEQ ID NO:4所示引物对、
    SEQ ID NO:5和SEQ ID NO:6所示引物对、
    SEQ ID NO:7和SEQ ID NO:8所示引物对、
    SEQ ID NO:9和SEQ ID NO:10所示引物对以及
    SEQ ID NO:11和SEQ ID NO:12所示引物对。
  2. 如权利要求1所述的多重PCR引物，其特征在于，所述引物为SEQ ID NO:1～SEQ ID NO:12中的一条或多条引物的3’端延长或截短0～3个碱基后所得的引物。
  3. 权利要求1～2任一项所述的多重PCR引物在获得和/或检测HBB基因序列中的用途。
  4. 一种试剂盒，其包括权利要求1～2任一所述的多重PCR引物。
  5. 权利要求4的所述的试剂盒在下列至少之一中的用途：
    获得和/或检测HBB基因序列；
    检测β-地中海贫血症；
    检测β-地中海贫血突变多态性。
  6. 一种多重PCR的方法，其特征在于，利用权利要求1～2任一所述的多重PCR引物进行所述多重PCR。
  7. 一种HBB基因检测的方法，其特征在于，包括：
    (1)利用权利要求6的所述的方法对待测样品中的至少一部分核酸进行扩增，获得扩增产物；
    (2)分析所述扩增产物，以获得HBB基因检测结果。
  8. 如权利要求7所述的方法，其特征在于，所述核酸为DNA和/或RNA。
  9. 如权利要求8所述的方法，其特征在于，所述核酸为RNA，步骤(1)包括：
    (1-1)利用所述多重PCR引物中的上游或下游引物将所述RNA反转录为cDNA；以及
    (1-2)利用所述多重PCR引物中相应的下游或上游引物对所述cDNA进行扩增，获得扩增产物。
  10. 如权利要求8所述的方法，其特征在于，步骤(2)包括：
    (2-1)对所述扩增产物进行测序；以及
    (2-2)将测序结果与HBB基因野生型序列比对，以确定HBB基因的突变位点。
  11. 一种检测β-地中海贫血症的方法，其特征在于，包括：
    利用权利要求7～10任一项所述的方法进行HBB基因检测，获得检测结果，所述待测样品来自受检者；以及
    依据检测结果，评估受检者的患β-地中海贫血症风险。
  12. 根据权利要求11所述的方法，其特征在于，所述检测结果具有表3所示的突变位点的至少之一，是患者患有β地中海贫血症的指示。
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