WO2018036176A1 - 一种扩增brca1/2基因的多重pcr引物及一种多重pcr引物的设计方法 - Google Patents

一种扩增brca1/2基因的多重pcr引物及一种多重pcr引物的设计方法 Download PDF

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WO2018036176A1
WO2018036176A1 PCT/CN2017/080814 CN2017080814W WO2018036176A1 WO 2018036176 A1 WO2018036176 A1 WO 2018036176A1 CN 2017080814 W CN2017080814 W CN 2017080814W WO 2018036176 A1 WO2018036176 A1 WO 2018036176A1
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primer
multiplex pcr
primers
pcr primer
brca1
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张纪斌
王辉云
林钊
李伟琴
罗景燕
赖炳权
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广州永诺生物科技有限公司
广州永诺健康科技有限公司
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  • the invention relates to a multiplex PCR primer sequence, in particular to a multiplex PCR primer for amplifying the BRCA1/2 gene and a design method thereof for use in gene detection of hereditary breast cancer.
  • Breast cancer is the number one cancer in women with the highest incidence of malignant tumors in China. According to the World Health Organization (WHO), in 2008, the incidence of breast cancer among Chinese women was 21.6 per 100,000, with 169,000 new cases and 45,000 deaths. According to existing research, about 5% to 10% of breast cancers are caused by mutations in the BRCA1/2 gene.
  • the BRCA1/2 mutation accounts for a large proportion (40% to 45%) of hereditary breast cancer patients, while the BRCA1/2 gene is mutated in 80% of patients with a high incidence of breast cancer. Carriers with BRCA1 mutations have a greater than 80% chance of developing breast cancer in their lifetime, and BRCA2 mutation carriers have a 45% chance of developing breast cancer throughout their lifetime. The detection of these two genetic variants in families with hereditary breast cancer patients and their family members can predict the risk of cancer, prevent early detection and early detection, and improve the cure rate and quality of life.
  • BRCA1/2 is a tumor suppressor gene and is ubiquitous in mammals. BRCA proteins are usually expressed in the breast or other tissues and are primarily responsible for DNA repair. Among them, BRCA1 contains 24 exons, the coding region is about 6K in length; BRCA2 contains 27 exons, and the coding region is about 11K in length. There are thousands of BRCA1/2 mutations reported at present, distributed in each exon of the gene, and there is no hot spot mutation.
  • the traditional BRCA1/2 gene detection method is mainly Sanger sequencing. First, several dozen exons are separately amplified, and then each amplification product is separately sequenced, and finally the sequencing results are analyzed. There is a large amount of manual operation during the detection process, the degree of automation is low, and it is limited by the detection flux.
  • High throughput sequencing also known as next generation sequencing (NGS) or massively parallel sequencing (MPS)
  • NGS next generation sequencing
  • MRS massively parallel sequencing
  • the measurement greatly increases the throughput of sequencing.
  • the analysis of high-throughput sequencing data has been highly automated. Based on this, more and more high-throughput sequencing-based genetic testing methods have emerged on the market, including genetic breast cancer BRCA1/2 detection.
  • the human haploid genome size is about 3 billion base pairs, and the amount of data is huge. Therefore, considering the research objectives and economic benefits, it is generally necessary to perform target sequence enrichment of interest before high-throughput sequencing.
  • the commonly used sequence enrichment methods include chip hybridization capture and multiplex PCR amplification.
  • the core technologies are mainly mastered by foreign biotechnology companies such as Roche, Agilent, and Life Technologies. Among them, hybrid capture is characterized by being able to be even larger in the exome.
  • the target area is captured, but the operation process is complicated, and it needs to rely on specialized equipment.
  • the multiplex PCR operation is simple and flexible, and only needs to be performed by a PCR instrument.
  • the target sequence enrichment and library construction can be completed within a few hours, which is suitable for relatively high Small target sequence enrichment.
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide a multiplex PCR primer for amplifying the BRCA1/2 gene and a design method for a multiplex PCR primer.
  • the technical solution adopted is a multiplex PCR primer for amplifying the BRCA1/2 gene, which includes the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 234.
  • the invention provides a design method of multiplex PCR primers, and the design method comprises the following steps:
  • the target region is divided into a plurality of small target regions, the length of the small target region is controlled within 200 bp, and then multiple pairs of primers are designed for each small target region. Forming the primer library;
  • the number of base complements at the 3' end between the primer and the primer is not higher than 10, and the overall complementarity between the primer and the primer is not higher than 75%.
  • the target region is a hereditary breast cancer gene.
  • the target region is the BRCA1/2 gene.
  • the design software is Primer3.
  • the present invention provides the use of the multiplex PCR primers described above for the preparation of a reagent for detecting a genetic breast cancer gene.
  • the present invention provides the use of the multiplex PCR primer described above for the preparation of a kit for detecting a genetic breast cancer gene.
  • the present invention provides an agent for detecting a hereditary breast cancer gene, which comprises the multiplex PCR primer described above.
  • the invention provides a kit for detecting a genetic breast cancer gene, the kit comprising the multiplex PCR primer described above.
  • the present invention provides a multiplex PCR primer, which is capable of achieving highly automated, high-throughput BRCA1/2 gene detection in a highly automated, high-throughput sequence based on multiplex PCR sequence enrichment + NGS sequencing.
  • the present invention has made an important breakthrough in the sequence enrichment technology and has reached the international leading level.
  • the genetic detection of hereditary breast cancer based on the invention has the characteristics of short cycle, simple operation, high degree of automation and high throughput, and is convenient for clinical application.
  • the enrichment of the BRCA1/2 target sequence can be completed quickly, and then the NGS library can be constructed and sequenced to achieve highly automated, high-throughput BRCA1/2 gene detection in hereditary breast cancer.
  • Example 1 is a qPCR amplification melting curve in Example 1 of the present invention
  • Figure 3 is a 2100 result of the 293T-2 library in Example 1 of the present invention.
  • Figure 4 is a result of 2100 of 293T-3 library in Example 1 of the present invention.
  • Figure 5 is a result of 2100 293T-4 library in Example 1 of the present invention.
  • the input of the multiplex PCR primer design software is the genomic region (bed file format, each line of contents is "chromosome-tab-start coordinate-tab-termination coordinate"), and the output is designed to be Multiple pairs of primers for amplification in one reaction
  • the specific implementation ideas are as follows: 1) using Primer3 software, designing as many primers as possible for each target region as an alternative; 2) according to the specificity of primer amplification, Whether the primer contains a simple repeat sequence, on the primer Whether there are high-frequency SNP sites (defined as sites with smaller allele frequencies in dbSNP above 5/1000) filter the primers designed in step 1; 3) for the previous filter Primers, the pair of primers with the highest value of Primer3 were selected for each target region, and the primers selected from different target regions should meet the principle of “minimum interaction between primers”, that is, minimize the 3′ end of primers and the overall complementarity of primers. .
  • the specific requirement is generally that the number of base complements at the 3' end between the
  • the target region is the coding exon and the exon-intron boundary of the BRCA1 and BRCA2 genes are 10 bases. Because the longest coding exon of BRCA1 and BRCA2 genes is up to several K, a single pair of primers cannot amplify a region of this length, and the sequencing read length does not reach this length, so it is necessary to divide the exon having an excessive length. , divided into two PCR reaction systems.
  • the final BRCA1/2 multiplex PCR primer design is summarized in Table 1.
  • the 293T cell line was used as a standard, and each pair of primers was subjected to qPCR amplification verification.
  • the melting curve is used to determine whether a single pair of primers can amplify a specific band (Fig. 1) and whether the Ct value is appropriate to ensure separate amplification of each pair of primers.
  • experiment was performed in parallel with the standard 293T cell line using the BRCA1/2 multiplex PCR sequence enrichment kit of one of the most popular and foreign companies on the market (two replicates, 293T-3 and 293T-4).
  • the above library was sequenced on Illumina hiseq3000 with a sequencing mode of PE150.
  • sequencing data after bioinformatics analysis, on one hand, statistical data indicators are used to evaluate the enrichment effect of the sequence, and on the other hand, single nucleotide polymorphism (SNP) and short insertion deletion (InDel) mutation detection are performed to evaluate the hereditary mammary gland. Feasibility of detection of cancer BRCA1/2 gene.
  • the BRCA1/2 target region was approximately 17 Kb in size, and the two libraries 293T-1 and 293T-2 of the present invention were sequenced to produce more than 3.6 million read sequences with a sequencing data volume of approximately 500 Mb. More than 95% of the short sequences in the sequencing data were able to align the upper genome, and more than 99% of the data in the aligned genome were aligned to the target region, demonstrating the high specificity of multiplex PCR enrichment. In addition, the enrichment effect of the BRCA1/2 target sequence of the present invention reaches 100% coverage, and the average sequencing depth of the target region is more than 20,000X.
  • the multiplex PCR sequence enrichment technique of the present invention is an efficient and reliable high-throughput sequencing sequence enrichment method.
  • the company's sequence enrichment requires three PCR reactions, each requiring 10 ng of DNA starting, for a total of 30 ng of DNA.
  • the present invention only needs to be carried out in two PCR reaction systems, on the one hand, the experimental procedure is reduced, and the initial amount of the sample is also reduced to 20 ng (Table 1).
  • the two methods have relatively consistent performance indicators, and the target area has reached the enrichment rate of 100%.
  • the present invention has better amplification uniformity than the enrichment method of foreign companies.
  • Sexuality able to produce higher quality, more suitable for subsequent bioinformatics analysis.
  • the present invention will have higher accuracy and better application.
  • the purpose of enrichment of the BRCA1/2 sequence in hereditary breast cancer is to perform genetic testing for the analysis of the presence of functional mutations in the BRCA1/2 gene, including single nucleotide polymorphisms (SNPs) and short insertions. Missing (InDel). Sequencing data mutation analysis detected 12 SNPs in 293T-1, 293T-2, 293T-3, and 293T-4, and InDel was not detected, and the consistency of the four results was 100%. After further analysis of these 12 SNPs, their functional classifications belong to normal polymorphism.
  • SNPs single nucleotide polymorphisms
  • the present invention proposes a design method of multiplex PCR primers and applies it to genetic breast cancer gene detection, and designs a technical system from the beginning to evaluate the multiplex PCR sequence enrichment effect.
  • the multiplex PCR sequence enrichment technology has made important breakthroughs and reached the international leading level.
  • the genetic detection of hereditary breast cancer based on the invention has the characteristics of short cycle, simple operation, high degree of automation and high throughput, and is convenient for clinical application.

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Abstract

本发明提供了一种扩增BRCA1/2基因的多重PCR引物及一种多重PCR引物的设计方法，所述多重PCR引物包括SEQ ID NO：1～SEQ ID NO：234所示的核苷酸序列。本发明提供的多重PCR引物可以快速的完成BRCA1/2目标序列的富集，进而进行NGS文库构建和测序，实现高度自动化、高通量的遗传性乳腺癌BRCA1/2基因检测。

Description

一种扩增BRCA1/2基因的多重PCR引物及一种多重PCR引物的设计方法 技术领域
本发明涉及一种多重PCR引物序列，具体涉及一种扩增BRCA1/2基因的多重PCR引物及其设计方法在遗传性乳腺癌基因检测中的应用。
背景技术
乳腺癌是我国妇女恶性肿瘤发病率排第一的癌症。根据世界卫生组织(WHO)的数据显示，2008年，中国妇女乳腺癌的发病率为21.6/10万人，新增病例达16.9万例，死亡人数为4.5万例。根据现有研究报道，约有5％～10％的乳腺癌是由BRCA1/2基因突变引起的。BRCA1/2突变在遗传性乳腺癌患者中占有很大的比例(40％～45％)，而在乳腺癌高发家族中80％的患者的BRCA1/2基因存在突变。BRCA1突变携带者一生患乳腺癌的几率大于80％，BRCA2突变携带者一生发生乳腺癌的机率为45％。对于家族遗传性乳腺癌患者及其家庭成员进行这两个基因变异的检测，能够预测癌症患病风险，做到早预防和早发现，提高治愈率和生存质量。
BRCA1/2是抑癌基因，在哺乳动物中普遍存在。BRCA蛋白通常在乳腺或其它组织中表达，主要负责DNA修复。其中，BRCA1包含24个外显子，编码区长度约6K；BRCA2包含27个外显子，编码区长度约11K。目前已报道的BRCA1/2突变种类达数千种，分布在基因的各个外显子上，不存在热点突变。
传统的BRCA1/2基因检测方法主要是Sanger测序，首先对几十个外显子分别进行扩增，然后每个扩增产物单独进行测序，最后再对测序结果进行分析。检测过程中存在大量的人工操作，自动化程度低，并且受限于检测通量。
高通量测序技术(high throughput sequencing)又称下一代测序(next generation sequencing，NGS)或大规模并行测序(massivelyparallel sequencing，MPS)，能一次并行对几十万到几百万条DNA分子进行序列测定，大大提高了测序的通量。同时，随着生物信息学的快速发展，高通量测序数据的分析已经实现了高度自动化。基于此，市场上涌现出越来越多的基于高通量测序的基因检测方法，包括遗传性乳腺癌BRCA1/2检测。
人单倍体基因组大小为30亿碱基对左右，数据量庞大。因此，综合考虑研究目的和经济效益，一般需要在高通量测序之前先进行感兴趣的目标序列富集。目前常用的序列富集方法包括芯片杂交捕获和多重PCR扩增，核心技术主要掌握在国外大生物科技公司手上，如Roche、Agilent、Life Technologies等。其中，杂交捕获的特点是能够对外显子组甚至更大的 目标区域进行捕获，但操作流程较复杂，需要依赖专门仪器设备；多重PCR操作简单灵活，只需要PCR仪即可进行，能在几个小时内完成目标序列富集和文库构建，适用于相对较小的目标序列富集。
发明内容
本发明的目的在于克服现有技术存在的不足之处而提供了一种扩增BRCA1/2基因的多重PCR引物以及一种多重PCR引物的设计方法。
为实现上述目的，所采取的技术方案：一种扩增BRCA1/2基因的多重PCR引物，所述多重PCR引物包括SEQ ID NO：1～SEQ ID NO：234所示的核苷酸序列。
本发明提供了一种多重PCR引物的设计方法，所述设计方法包括以下步骤：
(1)使用引物设计软件设计目标区域的引物库，将目标区域被分割成多个小目标区域，所述小目标区域的长度控制在200bp以内，然后针对每个小目标区域设计多对引物，构成所述引物库；
(2)对步骤(1)设计的引物库中的引物进行筛选，保留特异性强、不包含简单重复序列和不存在高频SNP位点的引物；
(3)对于步骤(2)中保留的引物中扩增相同小目标区域的引物，挑选引物设计软件中分值最高的一对引物；
(4)在步骤(3)中获得的引物中挑选满足“引物间相互作用最小化”的原则的引物，即得所述多重PCR引物；
其中所述“引物间相互作用最小化”的原则为：
引物和引物之间3’末端碱基互补数不高于10，引物和引物之间整体互补率不高于75％。
优选地，所述目标区域为遗传性乳腺癌基因。
优选地，所述目标区域为BRCA1/2基因。
优选地，所述设计软件为Primer3。
本发明提供了上述所述的多重PCR引物在制备检测遗传性乳腺癌基因的试剂中用途。
本发明提供了上述所述的多重PCR引物在制备检测遗传性乳腺癌基因的试剂盒中用途。
本发明提供了一种检测遗传性乳腺癌基因的试剂，所述试剂包括上述所述的多重PCR引物。
本发明提供了一种检测遗传性乳腺癌基因的试剂盒，所述试剂盒包括上述所述的多重PCR引物。
通过熔解曲线判断单对引物能否扩增出特异的条带，如果能扩增出特异的条带，则为特 异性强的引物；如果不能扩增出条带或者扩增出多条条带，则为特异性不强的引物。本发明的有益效果在于：
1、本发明提供了一种多重PCR引物，与Sanger测序方法相比，基于多重PCR序列富集+NGS测序的方法能实现高度自动化、高通量的遗传性乳腺癌BRCA1/2基因检测。
2、与市场上最流行、国外某大公司的BRCA1/2多重PCR序列富集方案相比：1)国外某大公司的序列富集需要分3个PCR反应体系进行，每个反应10ng DNA起始量，共需30ngDNA。本发明仅需在2个PCR反应体系进行，一方面减少了实验操作步骤，同时也将样本起始量降低到了20ng；2)测序数据分析比较发现，本发明的序列富集方法对目标区域达到了100％的富集率，同时相比国外公司的富集方法有更好的扩增均一性，能够产出更高质量、更适合后续生物信息分析的数据。
因此，本发明在序列富集技术方面取得了重要突破，达到了国际领先水平。基于本发明开发出的遗传性乳腺癌基因检测具有周期短、操作简单、自动化程度高、通量高等特点，便于临床应用。可以快速的完成BRCA1/2目标序列的富集，进而进行NGS文库构建和测序，实现高度自动化、高通量的遗传性乳腺癌BRCA1/2基因检测。
附图说明
图1为本发明实施例1中qPCR扩增熔解曲线；
图2为本发明实施例1中293T-1文库2100结果；
图3为本发明实施例1中293T-2文库2100结果；
图4为本发明实施例1中293T-3文库2100结果；
图5为本发明实施例1中293T-4文库2100结果。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。
实施例1
本发明的具体实施过程如下：
一、多重PCR引物设计软件开发及遗传性乳腺癌BRCA1/2引物设计。
多重PCR引物设计软件的输入为需要进行引物设计的基因组区域(bed文件格式，每行内容为“染色体-制表符-起始坐标-制表符-终止坐标”)，输出为设计好可在一个反应中进行扩增的多对引物，其具体实现思路如下：1)使用Primer3软件，针对每个目标区域设计出尽可能多的可用引物作为备选；2)根据引物扩增的特异性、引物是否包含简单重复序列、引物上 是否存在高频的SNP位点(定义为dbSNP中较小等位基因频率在千分之五以上的位点)等条件对步骤1中设计好的引物进行过滤；3)对于满足上一步过滤条件的引物，每个目标区域挑选Primer3分值最高的一对引物，不同目标区域挑选的引物之间要满足“引物间相互作用最小化”的原则，即最小化引物3’末端及引物整体互补率。具体要求一般为引物-引物之间3’末端碱基互补数不高于10，引物-引物之间整体互补率不高于75％，该原则可有效避免引物二聚体的形成。
以遗传性乳腺癌BRCA1/2引物设计为例，目标区域为BRCA1和BRCA2基因的编码外显子和外显子-内含子边界10碱基。因为BRCA1和BRCA2基因的最长编码外显子长达几K，单对引物无法扩增该长度的区域，且测序读长达不到该长度，所以需要将长度过大的外显子进行划分，分到两个PCR反应体系中进行。最终的BRCA1/2多重PCR引物设计概况如表1所示。
表1 BRCA1/2多重PCR引物设计概况
Figure PCTCN2017080814-appb-000001
二、单对引物qPCR验证。
对步骤一设计好的117对引物，采用293T细胞系作为标准品，每对引物都进行qPCR扩增验证。通过熔解曲线判断单对引物能否扩增出特异的条带(图1)，以及Ct值是否合适，以确保每对引物单独的扩增效果。
三、多重PCR扩增，NGS测序及数据分析。
将117对引物按设计方案混合成2个mix，以293T细胞系为标准品进行BRCA1/2目标序列富 集和文库构建(设2个重复，分别为293T-1和293T-2)。
为进行客观比较，该实验同时采用市场上最流行、国外某大公司的BRCA1/2多重PCR序列富集试剂盒对标准品293T细胞系进行平行测试(设2个重复，分别为293T-3和293T-4)。
上述两个平行测试仅多重PCR扩增时使用的引物不同，其它所有实验条件保持相同。
293T-1和293T-2高通量测序文库2100分析的结果如图2、3，293T-3和293T-4高通量测序文库2100分析的结果如图4、5，两种方案都产生了高质量的测序文库，且文库的长度分布与各自的设计预期相符。
上述文库在Illumina hiseq3000进行上机测序，测序模式为PE150。对于测序数据，经过生物信息分析，一方面统计数据指标用于评价序列富集效果，另一方面进行单核苷酸多态性(SNP)和短插入缺失(InDel)突变检测，评估遗传性乳腺癌BRCA1/2基因检测的可行性。
表2 测序数据统计
Figure PCTCN2017080814-appb-000002
如表2所示，BRCA1/2目标区域大小约为17Kb，本发明两个文库293T-1和293T-2测序产生了超过360万条read序列，测序数据量约500Mb。测序数据中超过95％的短序列能够比对上基因组，比对上基因组的数据中99％以上比对到了目标区域，证明了多重PCR序列富集的高度特异性。此外，本发明对BRCA1/2目标序列的富集效果达到了100％覆盖度，并且目标区域平均测序深度达2万X以上。
综上，本发明中的多重PCR序列富集技术是一种高效、可靠的高通量测序序列富集方法。
四、与市场上最流行的富集方案进行平行比较。
为进行客观比较，同时采用市场上最流行、国外某大公司的BRCA1/2多重PCR序列富 集试剂盒进行了平行实验。该多重PCR试剂盒的设计概况如表3所示：
表3 国外某大公司BRCA1/2多重PCR引物设计概况
Figure PCTCN2017080814-appb-000003
该公司的序列富集需要分3个PCR反应体系进行，每个反应10ng DNA起始量，共需30ngDNA。而本发明仅需在2个PCR反应体系进行，一方面减少了实验操作步骤，同时也将样本起始量降低到了20ng(表1)。
从表2的测序数据分析比较发现，两种方法有较一致的性能指标，目标区域都达到了100％的富集率；同时本发明相比国外公司的富集方法有更好的扩增均一性，能够产出更高质量、更适合后续生物信息分析的数据。例如，对于基因拷贝数分析这种需要对模板进行精确定量的分析，本发明将有更高的准确性和更好的应用。
最后，遗传性乳腺癌BRCA1/2序列富集的目的是为了对其进行基因检测，即分析BRCA1/2基因上是否存在功能性的突变，包括单核苷酸多态性(SNP)和短插入缺失(InDel)。测序数据突变分析在293T-1、293T-2、293T-3、293T-4中均检测到12个SNP，未检测到InDel，四个结果的一致性100％。经过对这12个SNP的进一步分析，其功能分类均属于正常多态性。
综合以上四点，本发明提出了出了一种多重PCR引物的设计方法并将其应用于遗传性乳腺癌基因检测，并从头设计了一套技术体系用于评估多重PCR序列富集效果，在多重PCR序列富集技术方面取得了重要突破，达到了国际领先水平。基于本发明开发出的遗传性乳腺癌基因检测具有周期短、操作简单、自动化程度高、通量高等特点，便于临床应用。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (9)

  1. 一种扩增BRCA1/2基因的多重PCR引物，其特征在于，所述多重PCR引物包括SEQ ID NO：1～SEQ ID NO：234所示的核苷酸序列。
  2. 一种多重PCR引物的设计方法，其特征在于，所述设计方法包括以下具体步骤：
    (1)使用引物设计软件设计目标区域的引物库，将目标区域分割成多个小目标区域，所述小目标区域的长度控制在200bp以内，然后针对每个小目标区域设计多对引物，构成所述引物库；
    (2)对步骤(1)设计的引物库中的引物进行筛选，保留特异性强、不包含简单重复序列和不存在高频SNP位点的引物；
    (3)对于步骤(2)中保留的引物中扩增相同小目标区域的引物，挑选引物设计软件中分值最高的一对引物；
    (4)在步骤(3)中获得的引物中挑选满足“引物间相互作用最小化”的原则的引物，即得所述多重PCR引物；
    其中所述“引物间相互作用最小化”的原则为：
    引物和引物之间3’末端碱基互补数不高于10，引物和引物之间整体互补率不高于75％。
  3. 根据权利要求2所述的设计方法，其特征在于，所述目标区域为遗传性乳腺癌基因。
  4. 根据权利要求2所述的设计方法，其特征在于，所述目标区域为BRCA1/2基因。
  5. 根据权利要求2所述的设计方法，其特征在于，所述引物设计软件为Primer3。
  6. 如权利要求1所述的多重PCR引物在制备检测遗传性乳腺癌基因的试剂中用途。
  7. 如权利要求1所述的多重PCR引物在制备检测遗传性乳腺癌基因的试剂盒中用途。
  8. 一种检测遗传性乳腺癌基因的试剂，其特征在于，所述试剂包括如权利要求1所述的多重PCR引物。
  9. 一种检测遗传性乳腺癌基因的试剂盒，其特征在于，所述试剂盒包括如权利要求1所述的多重PCR引物。
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CN113088571A (zh) * 2021-05-11 2021-07-09 郑州普利莱医学检验所股份有限公司 一种scn5a基因检测试剂盒及检测方法
CN113151448A (zh) * 2021-05-11 2021-07-23 郑州普利莱医学检验所股份有限公司 一种kcnh2基因检测试剂盒及检测方法
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