WO2017076300A1 - 多重pcr引物及应用 - Google Patents

多重pcr引物及应用 Download PDF

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WO2017076300A1
WO2017076300A1 PCT/CN2016/104392 CN2016104392W WO2017076300A1 WO 2017076300 A1 WO2017076300 A1 WO 2017076300A1 CN 2016104392 W CN2016104392 W CN 2016104392W WO 2017076300 A1 WO2017076300 A1 WO 2017076300A1
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seq
primer
kras
egfr
multiplex pcr
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French (fr)
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葛良进
邓力蔚
曾立董
刘松
李改玲
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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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, in particular to a multiplex PCR primer and method and application for detecting EGFR, KRAS and/or BRAF gene mutation sites based on next generation sequencing technology.
  • the epidermal growth factor receptor is an expression product of the proto-oncogene C-erbB-1 (HER-1) located on the short arm of chromosome 7, and is the epidermal growth factor receptor family (HER).
  • HER-1 proto-oncogene C-erbB-1
  • HER family plays an important regulatory role in the cellular physiology. Mutations in the EGFR tyrosine kinase region occur mainly in exons 18-21, with exon 19 and 21 mutations accounting for 90% of the total mutation. Abnormalities in EGFR signaling are responsible for a variety of tumors. Studies have shown that EGFR is expressed in a variety of tumors, such as colorectal cancer, breast cancer, pancreatic cancer, prostate cancer and non-small cell lung cancer.
  • the K-ras gene is a proto-oncogene, about 35 kb long, located on the short arm of chromosome 12 and is a member of the ras gene family, encoding the KRAS protein.
  • the common mutation sites of the K-ras gene are located in codon 12 and codon 13 of exon 2 and codon 6l in exon 3, among which there are 7 mutation hotspots: G12C, G12R, G12S, G12V G12D, G12A, G13V/D, accounting for more than 98% of the total mutation of the K-ras gene.
  • somatic K-ras mutations are associated with a variety of human malignancies such as lung cancer, leukemia, mucinous adenocarcinoma, pancreatic cancer, and colorectal cancer, while germ cell K-ras mutations and Noonan syndrome and heart - Associated with cardio-facio-cutaneous (CFC) syndrome.
  • CFC cardio-facio-cutaneous
  • the BRAF gene is a protooncogene that is located on chromosome 7 and encodes a silk/threonine-specific kinase. Approximately 90% of the BRAF gene mutation occurs at nucleotide 1799 of exon 15, and the T mutation is A, resulting in the substitution of proline for glutamate (V600E).
  • the incidence of BRAF gene mutation in melanoma, colorectal cancer, glioma, sarcoma, ovarian cancer, breast cancer and lung cancer cell lines was 59%, 18%, 11%, 9%, 14%, 2%, 3%; in addition, the incidence of BRAF V600E in papillary thyroid carcinoma is as high as 35.8%.
  • BRAF gene mutation is one of the most important prognostic indicators for advanced and recurrent colorectal cancer, and is closely related to the poor overall survival of patients.
  • the currently common methods for detecting these three gene mutations are Sanger sequencing and real-time PCR.
  • Sanger sequencing method a single pair of primers can detect multiple mutations, but multiple mutations, or mutation sites on different exons of the same gene, need to be separately amplified and sequenced, the operation is cumbersome, and the sensitivity is lower. %, the false negative rate is higher. Fluorescence quantification
  • the PCR method has high sensitivity, each pair of primers can only detect one mutation, and each mutation needs to establish a PCR reaction system separately. Simultaneous detection of multiple mutation sites in multiple samples is cumbersome. Both methods require a large amount of sample and are not suitable for detecting multiple gene mutation sites at the same time.
  • the present invention provides a multiplex PCR primer and application.
  • the invention provides a multiplex PCR primer.
  • the primer comprises at least two pairs of the following a)-f) primer pairs:
  • Primer pairs for amplifying the region of exon 18 of the EGFR gene primer pairs as shown in SEQ ID NO: 1 and SEQ ID NO: 2 and/or SEQ ID NO: 3 and SEQ ID NO: 4 Primer pair
  • Primer pairs for amplifying the region of exon 19 of the EGFR gene primer pairs as shown in SEQ ID NO: 5 and SEQ ID NO: 6 and/or SEQ ID NO: 7 and SEQ ID NO: 8 Primer pair
  • Primer pair for amplifying the region of exon 20 of the EGFR gene Primer pair shown in SEQ ID NO: 9 and SEQ ID NO: 10, primer pair shown in SEQ ID NO: 11 and SEQ ID NO: And one or more of the primer pairs shown in SEQ ID NO: 13 and SEQ ID NO:
  • Primer pair for amplifying the region of exon 21 of the EGFR gene Primer pair shown in SEQ ID NO: 15 and SEQ ID NO: 16, primer pair shown in SEQ ID NO: 17 and SEQ ID NO: 18. And one or more of the primer pairs shown in SEQ ID NO: 19 and SEQ ID NO: 20;
  • Primer pairs for amplifying the region of exon 2 of the KRAS gene primer pairs as shown in SEQ ID NO: 21 and SEQ ID NO: 22 and/or SEQ ID NO: 23 and SEQ ID NO: 24 Primer pair
  • Primer pair for amplifying the region of exon 15 of the BRAF gene primer pairs as shown in SEQ ID NO: 25 and SEQ ID NO: 26 and/or SEQ ID NO: 27 and SEQ ID NO: 28 Primer pair.
  • the multiplex PCR primer according to the embodiment of the present invention can be used for the efficient detection of EGFR/KRAS/BRAF gene mutation site diversity, and can cover EGFR 18-21 exon, KRAS exon 2 and BRAF exon 15 .
  • the multiplex PCR primer described above may further comprise at least one of the following additional technical features:
  • the multiplex PCR primer comprises at least a primer pair that amplifies any two of the EGFR, KRAS and BRAF genes, and the primer pair can be used to amplify at least one of the genes Sub-area.
  • the multiplex PCR primer is two or more pairs of primer pairs: SEQ ID NO: 1 and SEQ ID NO: 2, primer pairs, SEQ ID NO: 5 and SEQ ID NO: Primer pair shown in Figure 6, primer pair shown in SEQ ID NO: 9 and SEQ ID NO: 10, primer pair shown in SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: Primer pair, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 25 and SEQ ID NO: The primer pair shown in 26.
  • the multiplex PCR primer is two or more pairs of primer pairs: SEQ ID NO: 3 and SEQ ID NO: 4, primer pairs, SEQ ID NO: 7 and SEQ ID NO: Primer pair shown in Figure 8, primer pairs shown in SEQ ID NO: 11 and SEQ ID NO: 12, primer pairs shown in SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 Primer pairs, primer pairs as shown in SEQ ID NO: 23 and SEQ ID NO: 24, primer pairs as shown in SEQ ID NO: 27 and SEQ ID NO: 28 are shown.
  • the multiplex PCR primer is a primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 28.
  • a multiplexed PCR primer set for example, a primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 28
  • a multiplexed PCR primer set obtains a better amplification effect
  • the scope of protection of the invention is not limited to the primers, a primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 28.
  • the invention provides the use of the multiplex PCR primers described above for obtaining and/or detecting EGFR, KRAS and/or BRAF gene sequences.
  • the invention provides a kit.
  • the kit comprises the multiplex PCR primers described above.
  • the invention provides the use of a kit as described above for obtaining and/or detecting EGFR, KRAS and/or BRAF gene sequences.
  • the invention provides a multiplex PCR method.
  • the method comprises performing the multiplex PCR using the multiplex PCR primers described above.
  • the multiplex PCR method may further include at least one of the following additional technical features:
  • each primer is equimolarly mixed.
  • the amount of the template is 50 ng to 1 ⁇ g/50 ⁇ l.
  • the multiplex PCR reaction procedure 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, It is circulated 30 to 40 times (preferably 35 times), and is extended for 10 minutes after the last 72 °C.
  • the invention provides a method of detecting EGFR, KRAS and/or BRAF genes.
  • the method comprises: (1) amplifying at least a portion of the nucleic acid in the sample using the multiplex PCR method described above to obtain an amplification product; (2) analyzing the amplification product to The EGFR, KRAS and/or BRAF gene test results were obtained.
  • the above method for detecting EGFR, KRAS and/or BRAF genes may further comprise at least one of the following additional technical features:
  • the nucleic acid is DNA and/or RNA.
  • the RNA is a total RNA obtained by extracting human peripheral blood mononuclear cells, preferably using an RNA kit.
  • 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 step (1) comprises: first synthesizing cDNA by using a downstream primer set as a reverse transcription primer; then, using the synthesized cDNA as a template, adding an upstream primer set for multiplex PCR The cDNA was amplified to obtain multiplex PCR products.
  • the step (1) comprises: first synthesizing cDNA by using an upstream primer set as a reverse transcription primer; then, using the synthesized cDNA as a template, adding a downstream primer set to perform Multiplex PCR, amplification of cDNA, and multiplex PCR products were obtained.
  • step (2) comprises: (2-1) sequencing the amplification product; and (2-2) comparing the sequencing result to the EFGR, KRAS and/or BRAF gene wild type sequence, respectively To determine the EFGR, KRAS and/or BRAF gene mutation sites.
  • the "EGFR, KRAS and/or BRAF gene mutation site” or “EGFR/KRAS/BRAF gene mutation site” means one or more of the three genes EGFR, KRAS, BRAF.
  • the invention provides a method of detecting cancer, according to an embodiment of the invention, the method comprising: performing EFGR, KRAS and/or using the methods of EGFR, KRAS and/or BRAF gene detection as described above.
  • the BRAF gene is detected, the test result is obtained, the sample to be tested is from the subject; and the risk of cancer of the subject is evaluated according to the test result.
  • the above method for detecting cancer may further include at least one of the following additional technical features:
  • the detection result comprises at least one of the following mutations: EGFR c. 2156G>A, EGFR c.2235_2249del15, EGFR c.2240_2257del18, EGFR c.2369C>T, EGFR c.2573T>G, EGFR c.2582T>A, EGFR c.34G>T, KRAS c.34G>T, KRAS c.34G> A, KRAS c.34G>C, KRAS c.35G>T, KRAS c.35G>A, KRAS c.35G>C, KRAS c.38G>A and BRAF c.1799T>A, indicating the subject suffers There is cancer.
  • a mutation site may have other representations, such as the naming method of the GenBank SNP database, which is "rs".
  • the initial SNP site representation for example, rs671 and the ALDH2 gene c.1510G>A (G1510A) represent the same site.
  • nomenclatures such as labeling the same mutations on the reference gDNA as referring to the same mutations as the present invention.
  • the cancer comprises a colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, non-small cell lung cancer, leukemia, mucinous adenocarcinoma, melanoma, glioma, sarcoma, ovarian cancer. And at least one of breast cancer.
  • the multiplex PCR primers and methods for detecting EGFR/KRAS/BRAF gene mutation sites based on next-generation sequencing technology have the beneficial effects that the present invention provides multiplex PCR primers for detecting EGFR, KRAS and BRAF gene mutation sites, including Primer pairs that amplify one or more exon regions of these three genes, respectively, preferably cover EGFR 18-21 exons, KRAS exon 2 and BRAF exon 15, including EGFR, KRAS and The main mutation site of the BRAF gene.
  • the designed target sequence was efficiently amplified in each DNA sample, and mutations were detected in each exon of the designed KRAS, EGFR and BRAF genes, reflecting that the provided detection scheme is better. Specificity and applicability.
  • 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.
  • the primers of the present invention are shown in Table 1.
  • Design primers Oligo 7.0 and MFEprimer-2.0 were used to analyze the primer dimer and stem-loop mismatch, and primers were designed at both ends of the exon containing the mutation site.
  • the average length of the amplified sequence was about 150 bp, and 14 pairs.
  • the annealing temperatures of the primers are basically the same.
  • the primer set provided in this example covers exon 18-21 of exon EGFR, exon KRAS 2 and exon gene of BRAF 15 exon. 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 1 above.
  • the reaction system is shown in Table 2: It is configured according to QIAGEN Multiplex PCR Kit (Cat. No. 206143).
  • Multiplex Buffer 2 ⁇
  • Q solvent Q solution, 5 ⁇
  • DNA 10 ⁇ l Total 50 ⁇ l
  • Embodiment 1 of the present invention provides a method for preparing a DNA sample to be tested, comprising the following steps:
  • Tissues containing cancer cells were obtained from the hospital, genomic DNA was extracted using the QIAamp DNA Mini Kit (51304) kit, and the concentration and purity of the DNA were measured using Nanodrop2000 (Thermo), and then the genomic DNA was preserved.
  • Embodiment 2 of the present invention provides a method for constructing an EGFR/KRAS/BRAF gene mutation sequencing library by using multiplex PCR primers for detecting EGFR/KRAS/BRAF gene mutation sites, comprising the following steps:
  • Example 3 Using the genomic DNA obtained in Example 1 as an amplification template, 14 pairs of primer pairs shown in SEQ ID NO: 1 to SEQ ID NO: 28 were used, and then QIAGEN Multiplex PCR Kit (Cat. No. 206143) was used, and two kits were configured according to the kit instructions.
  • the multiplex PCR system and the multiplex PCR system are shown in Table 3.
  • Multiplex Buffer 2 ⁇
  • Q solvent Q solution, 5 ⁇
  • DNA 10 ⁇ l Total 50 ⁇ l
  • the first set of primers includes: primer numbers 1, 2, 5, 6, 9, 10, 13, 14, 15, 16, 21, 22, 25, and 26; the second set of primers includes: primer numbers 3, 4, 7, 8, 11, 12, 17, 18, 19, 20, 23, 24, 27 and 28.
  • the amount of the template can be adjusted, and 200 ng is used in this embodiment.
  • 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 150 bp was cut under ultraviolet light.
  • the PCR products of the first set of primers and the second set of primers were combined and recovered to obtain 32 uL of the purified product.
  • the gel recovery step was carried out by QIAGEN QIAquick gel purification kit according to a routine laboratory operation.
  • the system was placed at 37 ° C for 30 min.
  • the A-tailed PCR product was purified using QIAGEN QIAquick Glue Purification Kit.
  • a linker for sequencing was added to both ends of the DNA, and the configuration system is shown in Table 5 (where Quick ligase was purchased from NEB, M2200L).
  • 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 270 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 fastq format, and the multiple mutation sites of EGFR, BRAS and KRAF are 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 from each pair of primers of EGFR, BRAS and KRAF, 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.
  • Example 7 100 cases of lung cancer samples (samples containing cancer cells) were subjected to multiplex PCR, addition of A tail, addition of a linker, tagging sequence, high-throughput sequencing and bioinformatics analysis, and the following results were obtained. As shown in Table 7:

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Abstract

提供了一种基于下一代测序技术检测EGFR、KRAS和/或BRAF基因突变位点的多重PCR引物，包括SEQ ID NO:1-SEQ ID NO:28所示14对引物对中的至少两对；优选包括至少包括扩增EGFR、KRAS和BRAF基因中任意两个或三个基因，其中，每个基因扩增一个或多个外显子的区域，每个外显子区域采用一对或多对引物对。

Description

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201510740906.1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，特别是涉及一种基于下一代测序技术检测EGFR、KRAS和/或BRAF基因突变位点的多重PCR引物和方法及应用。
背景技术
表皮生长因子受体(epidermal growth factor receptor,EGFR)是位于7号染色体短臂上的原癌基因C-erbB-1(HER-1)的表达产物，是表皮生长因子受体家族(HER)中的4个成员之一，HER家族在细胞生理过程中发挥重要的调节作用。EGFR酪氨酸激酶区域的突变主要发生在18～21号外显子，其中19和21号外显子突变占总突变的90％。EGFR信号传导的异常是导致多种肿瘤发生的原因。研究表明，EGFR在多种肿瘤中都有表达，如结直肠癌、乳腺癌、胰腺癌、前列腺癌和非小细胞肺癌等。
K-ras基因是一种原癌基因，长约35kb，位于12号染色体短臂上，是ras基因家族成员之一，编码KRAS蛋白。K-ras基因常见的突变位点位于2号外显子的12号密码子和13号密码子上、3号外显子的6l号密码子，其中有7个突变热点：G12C、G12R、G12S、G12V、G12D、G12A、G13V/D，占K-ras基因总突变的98％以上。研究表明体细胞K-ras基因突变与多种人类恶性肿瘤，如肺癌、白血病、黏蛋白腺癌、胰腺癌、结直肠癌有关，而生殖细胞K-ras基因突变与努南综合征和心脏-面部-皮肤(cardio-facio-cutaneous，CFC)综合征相关。
BRAF基因是一种原癌基因，定位于7号染色体上，编码丝/苏氨酸特异性激酶。约90％的BRAF基因突变发生在外显子15的1799位核苷酸上，T突变为A，导致缬氨酸被谷氨酸取代(V600E)。BRAF基因突变在黑色素瘤、结直肠癌、神经胶质瘤、肉瘤、卵巢癌、乳腺癌以及肺癌等肿瘤细胞系中的发生率依次为59％、18％、11％、9％、14％、2％、3％；此外，BRAF V600E在甲状腺乳头状癌中的发生率高达35.8％。BRAF基因突变是晚期及复发性结直肠癌最重要的预后指标之一，与患者较差的总体生存期密切相关。
目前常见的检测这三个基因突变的方法有Sanger测序法和荧光定量PCR法。Sanger测序法中，单对引物可检测多个突变，但对多个基因、或同一基因的不同外显子上的突变位点就需分别进行扩增测序，操作繁琐，并且灵敏度较低约20％，假阴性率较高。荧光定量 PCR法虽然灵敏度高，但每对引物只能检测一种突变，且每种突变需单独建立一个PCR反应体系，同时检测多个样本多个突变位点操作繁琐。这两种方法对样本的需要量都较大，不适合同时检测多个基因突变位点。
发明内容
鉴于此，本发明提供了一种多重PCR引物及应用。
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，所述引物包括如下a)-f)引物对中的至少两对：
a)用于扩增EGFR基因的外显子18的区域的引物对：SEQ ID NO:1和SEQ ID NO:2所示引物对和/或SEQ ID NO:3和SEQ ID NO:4所示引物对；
b)用于扩增EGFR基因的外显子19的区域的引物对：SEQ ID NO:5和SEQ ID NO:6所示引物对和/或SEQ ID NO:7和SEQ ID NO:8所示引物对；
c)用于扩增EGFR基因的外显子20的区域的引物对：SEQ ID NO:9和SEQ ID NO:10所示引物对、SEQ ID NO:11和SEQ ID NO:12所示引物对和SEQ ID NO:13和SEQ ID NO:14所示引物对中的一种或多种；
d)用于扩增EGFR基因的外显子21的区域的引物对：SEQ ID NO:15和SEQ ID NO:16所示引物对、SEQ ID NO:17和SEQ ID NO:18所示引物对和SEQ ID NO:19和SEQ ID NO:20所示引物对中的一种或多种；
e)用于扩增KRAS基因的外显子2的区域的引物对：SEQ ID NO:21和SEQ ID NO:22所示引物对和/或SEQ ID NO:23和SEQ ID NO:24所示引物对；
f)用于扩增BRAF基因的外显子15的区域的引物对：SEQ ID NO:25和SEQ ID NO:26所示引物对和/或SEQ ID NO:27和SEQ ID NO:28所示引物对。
利用根据本发明实施例的多重PCR引物，能用于EGFR/KRAS/BRAF基因突变位点多样性的高效检测，可覆盖EGFR 18～21号外显子、KRAS 2号外显子和BRAF 15号外显子。
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，所述多重PCR引物中，至少包括扩增EGFR、KRAS和BRAF基因中任意两个基因的引物对，所述引物对能够用于扩增所述基因的至少一个外显子区域。
根据本发明的实施例，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:5和SEQ ID NO:6所示引物对、SEQ ID NO:9和SEQ ID NO:10所示引物对、SEQ ID NO:13和SEQ ID NO:14所示引物对、SEQ ID NO:15和SEQ ID NO:16所示引物对、SEQ ID NO:21和SEQ ID NO:22、SEQ ID NO:25和SEQ ID  NO:26所示引物对。
根据本发明的实施例，所述多重PCR引物为如下引物对中的两对或多对：SEQ ID NO:3和SEQ ID NO:4所示引物对、SEQ ID NO:7和SEQ ID NO:8所示引物对、SEQ ID NO:11和SEQ ID NO:12所示引物对、SEQ ID NO:17和SEQ ID NO:18所示引物对、SEQ ID NO:19和SEQ ID NO:20所示引物对、SEQ ID NO:23和SEQ ID NO:24所示引物对、SEQ ID NO:27和SEQ ID NO:28所示引物对。
根据本发明的实施例，所述多重PCR引物为SEQ ID NO:1～SEQ ID NO:28所示的核苷酸序列组成的引物组。
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:28所示的核苷酸序列组成的引物组)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，本发明提供了前面所述的多重PCR引物在获得和/或检测EGFR、KRAS和/或BRAF基因序列中的用途。
第三方面，本发明提供了一种试剂盒。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，本发明提供了前面所述的试剂盒在获得和/或检测EGFR、KRAS和/或BRAF基因序列中的用途。
第五方面，本发明提供了一种多重PCR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。
根据本发明的实施例，上述多重PCR方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的具体实施例，所述多重PCR反应的体系中，各引物等摩尔混合。
根据本发明的再一具体实施例，所述多重PCR反应的体系中，模板量为50ng～1μg/50μl体系。
根据本发明的再一具体实施例，所述多重PCR反应的程序为：
Figure PCTCN2016104392-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。
根据本发明的具体示例，所述多重PCR反应结束后，电泳，割胶回收片段长度为150bp的DNA片段。
第六方面，本发明提供了一种EGFR、KRAS和/或BRAF基因检测的方法。根据本发明的实施例，所述方法包括：(1)利用前面所述的多重PCR方法对待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EGFR、KRAS和/或BRAF基因检测结果。
根据本发明的实施例，上述EGFR、KRAS和/或BRAF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。
根据本发明的实施例，所述RNA为(优选采用RNA试剂盒)提取人外周血单个核细胞获得的总RNA。
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，当所述核酸为RNA，步骤(1)包括：先以下游引物组为反转录引物合成cDNA；然后以合成的cDNA为模板，加入上游引物组，进行多重PCR，扩增cDNA，获得多重PCR产物。根据本发明的再一具体实施例，当所述核酸为RNA，步骤(1)包括：先以上游引物组为反转录引物合成cDNA；然后以合成的cDNA为模板，加入下游引物组，进行多重PCR，扩增cDNA，获得多重PCR产物。
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)将测序结果分别与EFGR、KRAS和/或BRAF基因野生型序列比对，以确定EFGR、KRAS和/或BRAF基因突变位点。
如本发明所述的，所述“EGFR、KRAS和/或BRAF基因突变位点”或“EGFR/KRAS/BRAF基因突变位点”表示EGFR、KRAS、BRAF三个基因中的一个或多个。
第七方面，本发明提出了一种检测癌症的方法，根据本发明的实施例，所述方法包括：利用前面所述的EGFR、KRAS和/或BRAF基因检测的方法进行EFGR、KRAS和/或BRAF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，评估受检者的患癌风险。
根据本发明的实施例，上述检测癌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，所述检测结果包含以下突变中的至少之一：EGFR c.2156G>A、 EGFR c.2235_2249del15、EGFR c.2240_2257del18、EGFR c.2369C>T、EGFR c.2573T>G、EGFR c.2582T>A、EGFR c.34G>T、KRAS c.34G>T、KRAS c.34G>A、KRAS c.34G>C、KRAS c.35G>T、KRAS c.35G>A、KRAS c.35G>C、KRAS c.38G>A和BRAF c.1799T>A，则指示受检者患有癌症。需要说明的是，上述突变是根据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。
根据本发明的实施例，所述癌症包括选自结直肠癌、乳腺癌、胰腺癌、前列腺癌、非小细胞肺癌、白血病、黏蛋白腺癌、黑色素瘤、神经胶质瘤、肉瘤、卵巢癌和乳腺癌的至少之一。
本发明提供的基于下一代测序技术检测EGFR/KRAS/BRAF基因突变位点的多重PCR引物及方法的有益效果为：本发明提供了检测EGFR、KRAS和BRAF基因突变位点的多重PCR引物，包含了分别扩增这三个基因的一个或多个外显子区域的引物对，优选覆盖了EGFR 18～21号外显子、KRAS 2号外显子和BRAF 15号外显子，包括了EGFR、KRAS和BRAF基因的主要突变位点。所设计的目标序列在每个DNA样品中均得到有效的扩增，在所设计的KRAS、EGFR和BRAF基因的各个外显子中均检测到了突变，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。
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：
癌症(如肺癌、结直肠癌等)患者：来源于深圳第六人民医院，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1所示。
表1：多重PCR引物序列
Figure PCTCN2016104392-appb-000002
Figure PCTCN2016104392-appb-000003
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150bp左右，且14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EGFR 18～21号外显子、KRAS 2号外显子和BRAF 15号外显子基因突变位点。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，如上表1所示。
表1各对引物单独PCR结果如图1所示。其中，表1中14对引物分别进行扩增验证的PCR体系及PCR参数如下：
反应体系如表2所示：参照QIAGEN公司Multiplex PCR试剂盒(货号：206143)配置。
表2：
多重PCR缓冲液(Multiplex Buffer,2×) 25μl
Q溶剂(Q solution,5×) 10μl
引物 5μl
DNA 10μl
Total 50μl
循环参数：
Figure PCTCN2016104392-appb-000004
实施例1
本发明实施例1提供了一种待测DNA样品的制备方法，包括如下步骤：
从医院获取包含癌细胞的组织，采用QIAamp DNA Mini Kit(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
本发明实施例2提供了一种采用检测EGFR/KRAS/BRAF基因突变位点的多重PCR引物构建EGFR/KRAS/BRAF基因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:28所示14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两管多重PCR体系，多重PCR体系如表3所示。
表3：反应体系：
多重PCR缓冲液(Multiplex Buffer,2×) 25μl
Q溶剂(Q solution,5×) 10μl
引物 5μl
DNA 10μl
Total 50μl
各引物分两组进行等摩尔混合，引物总浓度是10微摩尔。第一组引物包括：引物编号1、2、5、6、9、10、13、14、15、16、21、22、25和26；第二组引物包括：引物编号3、4、7、8、11、12、17、18、19、20、23、24、27和28。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 PCTCN2016104392-appb-000005
PCR结束后，4℃保存PCR产物并电泳检测，在紫外下切下约150bp左右的目的片段。将第一组引物和第二组引物的PCR产物合在一起回收，得到32uL纯化后的产物，胶回收步骤采用QIAGEN公司QIAquick胶纯化试剂盒，按常规实验室操作进行。
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4所示(其中，Klenow exo-购自NEB，货号：M0212)：
表4：
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5所示(其中，Quick ligase购自NEB，M2200L)。
表5：
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SEQ ID NO：29所示：
5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC/ideoxyU/ACACTCTTTCCC TACACGACGCTCTTCCGATC*T-3’(SEQ ID NO：29)。
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6所示(具体步骤参照illumina高通量测序文库构建说明书)。
表6：
5×Q5Solution 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 PCTCN2016104392-appb-000006
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270bp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，S1～S4(分别代表不同的样本)泳道可以明显看到在250bp-300bp之间有一条带，为加上接头的片段(270bp，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EGFR、BRAS和KRAF三个基因多突变位点情况。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EGFR、BRAS和KRAF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
参照实施例2的方法，对100例肺癌癌症样品(包含癌细胞的样品)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，如表7所示：
表7：癌症样品基因突变位点检测
Figure PCTCN2016104392-appb-000007
Figure PCTCN2016104392-appb-000008
如表7所示，在测试的100例癌症样品中，三个基因的主要突变位点均有检测到。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，从突变结果中也可看出，在所设计的KRAS、EGFR和BRAF基因的各个外显子中均检测到了突变，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。
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 (14)

  1. 一种多重PCR引物，其特征在于，包括如下a)-f)引物对中的至少两对：
    a)用于扩增EGFR基因的外显子18的区域的引物对：SEQ ID NO:1和SEQ ID NO:2所示引物对和/或SEQ ID NO:3和SEQ ID NO:4所示引物对；
    b)用于扩增EGFR基因的外显子19的区域的引物对：SEQ ID NO:5和SEQ ID NO:6所示引物对和/或SEQ ID NO:7和SEQ ID NO:8所示引物对；
    c)用于扩增EGFR基因的外显子20的区域的引物对：SEQ ID NO:9和SEQ ID NO:10所示引物对、SEQ ID NO:11和SEQ ID NO:12所示引物对和SEQ ID NO:13和SEQ ID NO:14所示引物对中的一种或多种；
    d)用于扩增EGFR基因的外显子21的区域的引物对：SEQ ID NO:15和SEQ ID NO:16所示引物对、SEQ ID NO:17和SEQ ID NO:18所示引物对和SEQ ID NO:19和SEQ ID NO:20所示引物对中的一种或多种；
    e)用于扩增KRAS基因的外显子2的区域的引物对：SEQ ID NO:21和SEQ ID NO:22所示引物对和/或SEQ ID NO:23和SEQ ID NO:24所示引物对；
    f)用于扩增BRAF基因的外显子15的区域的引物对：SEQ ID NO:25和SEQ ID NO:26所示引物对和/或SEQ ID NO:27和SEQ ID NO:28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，所述多重PCR引物至少包括用于扩增EGFR、KRAS和BRAF基因中任意两个基因的引物对，所述引物对能够用于扩增所述基因的至少一个外显子区域。
  3. 如权利要求1所述的多重PCR引物，其特征在于，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:5和SEQ ID NO:6所示引物对、SEQ ID NO:9和SEQ ID NO:10所示引物对、SEQ ID NO:13和SEQ ID NO:14所示引物对、SEQ ID NO:15和SEQ ID NO:16所示引物对、SEQ ID NO:21和SEQ ID NO:22、SEQ ID NO:25和SEQ ID NO:26所示引物对；和/或
    如下引物对中的两对或多对：SEQ ID NO:3和SEQ ID NO:4所示引物对、SEQ ID NO:7和SEQ ID NO:8所示引物对、SEQ ID NO:11和SEQ ID NO:12所示引物对、SEQ ID NO:17和SEQ ID NO:18所示引物对、SEQ ID NO:19和SEQ ID NO:20所示引物对、SEQ ID NO:23和SEQ ID NO:24所示引物对、SEQ ID NO:27和SEQ ID NO:28所示引物对。
  4. 权利要求1～3任一项所述的多重PCR引物在获得和/或检测EGFR、KRAS和/或BRAF基因序列中的用途。
  5. 一种试剂盒，其包括权利要求1～3任一所述的多重PCR引物。
  6. 权利要求5的所述的试剂盒在获得和/或检测EGFR、KRAS和/或BRAF基因序列中的用途。
  7. 一种多重PCR的方法，其特征在于，利用权利要求1-3任一所述的多重PCR引物进行所述多重PCR。
  8. 一种EGFR、KRAS和/或BRAF基因检测的方法，其特征在于，包括：
    (1)利用权利要求7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EGFR、KRAS和/或BRAF基因检测结果。
  9. 如权利要求8所述的方法，其特征在于，所述核酸为DNA和/或RNA。
  10. 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。
  11. 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)将测序结果分别与EFGR、KRAS和/或BRAF基因野生型序列比对，以确定EFGR、KRAS和/或BRAF基因的突变位点。
  12. 一种评估癌症风险的方法，其特征在于，包括：
    利用权利要求8～11任一项所述的方法进行EFGR、KRAS和/或BRAF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，评估受检者的患癌风险。
  13. 根据权利要求12所述的方法，其特征在于，
    所述检测结果包含以下突变中的至少之一：EGFR c.2156G>A、EGFR c.2235_2249del15、EGFR c.2240_2257del18、EGFR c.2369C>T、EGFR c.2573T>G、EGFR c.2582T>A、EGFR c.34G>T、KRAS c.34G>T、KRAS c.34G>A、KRAS c.34G>C、KRAS c.35G>T、KRAS c.35G>A、KRAS c.35G>C、KRAS c.38G>A和BRAF c.1799T>A，则指示受检者患有癌症。
  14. 根据权利要求12所述的方法，其特征在于，所述癌症包括选自结直肠癌、乳腺癌、胰腺癌、前列腺癌、非小细胞肺癌、白血病、黏蛋白腺癌、黑色素瘤、神经胶质瘤、肉瘤、卵巢癌和乳腺癌的至少之一。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112899366A (zh) * 2021-01-06 2021-06-04 南京普济生物有限公司 一种结直肠癌基因突变位点的检测方法及其试剂盒

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105331611A (zh) * 2015-11-04 2016-02-17 深圳市瀚海基因生物科技有限公司 一种基于下一代测序技术检测egfr/kras/braf基因突变位点的多重pcr引物和方法及应用
CN105886650A (zh) * 2016-06-08 2016-08-24 广州凯普医药科技有限公司 Kras基因突变检测试剂盒
CN107630075A (zh) * 2016-07-18 2018-01-26 丰技生物科技股份有限公司 Egfr基因的突变位点的检测方法
CN107488729A (zh) * 2017-09-27 2017-12-19 辽宁科骏生物有限公司 检测egfr基因突变的引物组合物、试剂盒及方法
CN107988369B (zh) * 2017-12-28 2020-11-17 北京雅康博生物科技有限公司 一种同时检测人egfr基因45个突变位点的试剂盒
CN111518901A (zh) * 2020-04-30 2020-08-11 北京和合医学诊断技术股份有限公司 用于检测egfr基因突变的引物组及其应用方法
CN111500727A (zh) * 2020-04-30 2020-08-07 北京和合医学诊断技术股份有限公司 用于检测kras基因和braf基因突变的引物组及其应用方法
CN117106911A (zh) * 2023-08-14 2023-11-24 首都医科大学附属北京天坛医院 检测神经鞘瘤的基因集及其多重pcr-高通量测序检测试剂盒

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899438A (zh) * 2010-04-16 2010-12-01 陕西北美基因股份有限公司 一种扩增人类egfr基因的多重pcr引物及其设计方法
CN104630375A (zh) * 2015-02-16 2015-05-20 北京圣谷同创科技发展有限公司 癌症基因突变及基因扩增检测
WO2015112619A1 (en) * 2014-01-22 2015-07-30 Adam Platt Methods and systems for detecting genetic mutations
CN104818318A (zh) * 2014-07-02 2015-08-05 厦门艾德生物医药科技有限公司 一次性检测肺癌多重基因的引物、探针、检测体系和试剂盒
CN104818320A (zh) * 2014-12-03 2015-08-05 厦门艾德生物医药科技有限公司 一次性检测肺癌多重基因的引物、探针、检测体系和试剂盒
CN105331611A (zh) * 2015-11-04 2016-02-17 深圳市瀚海基因生物科技有限公司 一种基于下一代测序技术检测egfr/kras/braf基因突变位点的多重pcr引物和方法及应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012149438A1 (en) * 2011-04-28 2012-11-01 Life Technologies Corporation Methods and compositions for multiplex pcr
CN103305625B (zh) * 2013-07-08 2014-06-25 广东省人民医院 检测非小细胞肺癌驱动基因突变谱的方法及试剂盒与应用
CN104152551B (zh) * 2014-07-18 2016-08-17 普世华康江苏医疗技术有限公司 一种检测肺癌热点突变基因的组合物及其使用方法
CN104450948B (zh) * 2014-12-31 2018-03-09 北京圣谷同创科技发展有限公司 癌症检测方法、试剂盒及其应用
CN104894269B (zh) * 2015-06-09 2016-03-02 广东省人民医院 基于MassARRAY质谱平台Iplex分析的肺癌基因谱检测试剂盒

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899438A (zh) * 2010-04-16 2010-12-01 陕西北美基因股份有限公司 一种扩增人类egfr基因的多重pcr引物及其设计方法
WO2015112619A1 (en) * 2014-01-22 2015-07-30 Adam Platt Methods and systems for detecting genetic mutations
CN104818318A (zh) * 2014-07-02 2015-08-05 厦门艾德生物医药科技有限公司 一次性检测肺癌多重基因的引物、探针、检测体系和试剂盒
CN104818320A (zh) * 2014-12-03 2015-08-05 厦门艾德生物医药科技有限公司 一次性检测肺癌多重基因的引物、探针、检测体系和试剂盒
CN104630375A (zh) * 2015-02-16 2015-05-20 北京圣谷同创科技发展有限公司 癌症基因突变及基因扩增检测
CN105331611A (zh) * 2015-11-04 2016-02-17 深圳市瀚海基因生物科技有限公司 一种基于下一代测序技术检测egfr/kras/braf基因突变位点的多重pcr引物和方法及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"PCR-LDR", vol. 3, no. 31, 1 January 2012 (2012-01-01), pages 19 - 24 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112899366A (zh) * 2021-01-06 2021-06-04 南京普济生物有限公司 一种结直肠癌基因突变位点的检测方法及其试剂盒

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