WO2017185767A1 - 一组检测Kras基因突变的引物和探针及其试剂盒 - Google Patents

一组检测Kras基因突变的引物和探针及其试剂盒 Download PDF

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WO2017185767A1
WO2017185767A1 PCT/CN2016/109631 CN2016109631W WO2017185767A1 WO 2017185767 A1 WO2017185767 A1 WO 2017185767A1 CN 2016109631 W CN2016109631 W CN 2016109631W WO 2017185767 A1 WO2017185767 A1 WO 2017185767A1
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cancer
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rev
mutation
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邹鸿志
牛智通
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广州市康立明生物科技有限责任公司
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Definitions

  • the invention belongs to the field of gene detection technology. More specifically, it relates to a set of primers and probes for detecting mutations in the Kras gene and kits thereof.
  • the RAS gene was first discovered in the early 1980s. First, a transforming gene isolated from human bladder cancer cell lines can mute transformation of NIH3T3 cells (mouse embryonic fibroblast cell line) from normal human tissues. The extracted DNA has no such effect.
  • the RAS gene is quite conservative in evolution and is widely found in various eukaryotes such as mammals, fruit flies, fungi, nematodes and yeast, suggesting that it has important physiological functions.
  • Kras is also known as the p21 gene due to its 21kD RAS protein.
  • the Kras gene is like a "switch" in the body: when it is normal, it can control the path that regulates cell growth; when an abnormality occurs, it causes the cells to continue to grow and prevent the cells from self-destructing. Therefore, the Kras gene plays an important regulatory role in the signaling pathways of tumor cell growth and angiogenesis.
  • the normal Kras gene can inhibit the growth of tumor cells, and once the Kras gene is mutated, it will continue to stimulate cell growth. Intracellular signal transduction disorder, disrupting the growth pattern, cell proliferation is out of control and cancerous, leading to tumorigenesis.
  • Intracellular signal transduction disorder disrupting the growth pattern, cell proliferation is out of control and cancerous, leading to tumorigenesis.
  • lupus erythematosus ( SLE) breast cancer, liver cancer, skin cancer, rheumatoid arthritis (RA), kidney cancer and some leukemia (Leukemia) have higher levels of mutations in the Kras gene.
  • the Kras gene is one of the important genetic markers for currently identified tumor-targeted therapeutics.
  • Kras protein is a key downstream regulator of EGFR (epidermal growth factor receptor) signaling pathway, involved in the regulation of cell growth and plays an important role in the carcinogenesis. Whether the mutation of Kras gene in tumor patients significantly affects the efficacy of EGFR targeted therapy. The latest clinical studies have shown that when the patient does not have a mutation in the Kras gene, taking a targeted therapeutic drug can achieve significant therapeutic effects.
  • EGFR antibody drugs such as Cetuximab and Panitumumab
  • EGFR tyrosine kinase inhibitors such as Gefitinib and Erlotinib
  • Drugs can significantly improve the efficacy of targeted therapy.
  • NCCN The US Comprehensive Cancer Network proposes that patients with cancer must undergo mutation detection of Kras gene before receiving EGFR-targeted drug therapy, and determine whether to use EGFR-targeted drugs as a clinical treatment according to the test results.
  • the Kras gene mutation test has been listed by the NCCN as a guideline for the clinical treatment of colon cancer and a guideline for clinical treatment of rectal cancer.
  • ARMs-qPCR amplification inhibition system (ARMS), also known as allele-specific PCR (ASPCR), is used to perform known mutations or gene polymorphisms. Detection.
  • the core of the method is the design of three primers, where the three primers generally comprise two forward primers and one common reverse primer; and the two forward primers differ in that the bases at the 3' end of the primer are different. That is, alleles or mutant bases and wild bases of known mutations, because Taq DNA polymerase has no corrective activity, and generally only when the 3' end of the primer is completely complementary to the template, the amplification reaction can be effectively performed.
  • two forward primers and a reverse primer can be configured as a reaction system (two reactions are required for one site), so that the high sensitivity of the real-time PCR can be combined to effectively monitor the presence or absence of amplification of the two reactions. It is possible to determine whether to mutate or determine the genotype. However, all of the above are theoretical assumptions. If other parameters of the primers meet the requirements (such as GC content, Tm value, no hairpin structure, etc.), Taq DNA polymerase starts amplification only because the bases at the 3' end are different. The efficiency is different.
  • the specificity of PCR amplification can be judged by the melting curve, and the number of bands of the reaction product can be judged. Similarly, different amplification fragments can be distinguished according to the melting temperature (melting curve).
  • the technique can be carried out by adding a double-stranded DNA binding dye to the standard PCR reagent. Without the sequence-specific probe, the high-resolution melting curve can be directly run after the PCR to complete the analysis of the sample genotype.
  • Recent studies have shown that the sensitivity of the HRM method for mutation detection can be compared to or even surpassed some current SNP/mutation analysis techniques, such as DHPLC, and its sensitivity is much higher than sequencing. This method is simple, fast, has a large throughput, low cost of use, and accurate results. However, this method also has defects such as expensive instruments and low utilization rate.
  • the technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing Kras gene mutation detection technology, and adopt a unique innovative design method to design and optimize a set of primers and probes for detecting the mutation of Kras gene, and pass the annealing temperature and use.
  • the research and optimization of the use conditions of the system and PCR program successfully established a kit for detecting mutations in the Kras gene.
  • the detection sensitivity of the Kras gene mutation is high, and the specificity and accuracy are good. Only 7 kinds of detection can be detected separately. Different types of mutations can also detect 7 types of mutations at the same time, and the application is more flexible and wider.
  • the present invention provides the following technical solutions:
  • the present invention provides a primer set comprising a primer comprising a forward primer and a reverse primer; the sequence of the forward primer is as shown in any one of SEQ ID No. 1 to SEQ ID No. 7; The sequence of the reverse primer is shown in SEQ ID No. 8.
  • the invention also provides the use of the primer set described in the preparation of a detection reagent for mutation of the Kras gene.
  • the invention also provides the use of the primer set in preparing a cancer and/or other disease detecting reagent; preferably, the cancer is selected from the group consisting of pancreatic cancer, colorectal cancer, lung cancer, ovarian cancer, thyroid cancer, bladder cancer, Breast cancer, liver cancer, skin cancer or kidney cancer; the other diseases are selected from the group consisting of lupus erythematosus, rheumatoid arthritis or leukemia.
  • the present invention also provides a probe composition, wherein the sequence of the probe is as shown in any one of SEQ ID No. 9 to SEQ ID No. 15; preferably, the 5' end of the probe is labeled with a fluorescent group. The 3' end is labeled with a corresponding quenching group.
  • the invention also provides the use of the probe composition for the preparation of a detection reagent for mutation of the Kras gene.
  • the invention also provides the use of the probe composition for preparing a cancer and/or other disease detecting reagent; preferably, the cancer is selected from the group consisting of pancreatic cancer, colorectal cancer, lung cancer, ovarian cancer, thyroid cancer, and bladder. Cancer, breast cancer, liver cancer, skin cancer or kidney cancer; the other diseases are selected from lupus erythematosus, rheumatoid arthritis or leukemia.
  • the invention also provides a detection reagent comprising the primer set and/or the probe composition.
  • the invention also provides a detection kit comprising the detection reagent.
  • the detection kit further comprises an amplification system; the amplification system comprises dNTPs, magnesium ions, reaction buffer, enzyme, nuclease-free water.
  • the amplification system in the detection kit comprises 5-20 mM dNTP, 10-40 mM magnesium ion, 1 ⁇ 10 ⁇ reaction buffer, enzyme, nuclease-free Water.
  • the final concentration of each component in the amplification system of the detection kit is:
  • the amplification procedure of the detection kit is:
  • the present invention also provides a method for detecting a mutation of a Kras gene, which is mixed with the primer set, the probe composition or the detection reagent or the detection reagent in the detection kit. Increase, the test results are obtained according to the amplification results.
  • the obtaining the detection result in the detecting method is comparing the amplification result of the sample to be tested with the normal sample, and the amplification result of the sample to be tested and the normal sample are significantly different.
  • the donor of the sample to be tested occurs Kras gene mutation.
  • the invention also provides a method for detecting cancer and/or other diseases, sampling the primer set, the probe composition or the detection reagent or the detection reagent in the detection kit After mixing, amplification is performed, and the detection result is obtained according to the amplification result;
  • the cancer is selected from the group consisting of pancreatic cancer, colorectal cancer, lung cancer, ovarian cancer, thyroid cancer, bladder cancer, breast cancer, liver cancer, skin cancer or kidney cancer; the other diseases are selected from the group consisting of lupus erythematosus, rheumatoid arthritis or leukemia.
  • the obtaining the detection result in the detecting method is comparing the amplification result of the sample to be tested with the normal sample, and the amplification result of the sample to be tested and the normal sample are significantly different. Or when there is a significant difference, the donor of the sample to be tested has cancer and/or other diseases;
  • the cancer is selected from the group consisting of pancreatic cancer, colorectal cancer, lung cancer, ovarian cancer, thyroid cancer, bladder cancer, breast cancer, liver cancer, skin cancer or kidney cancer; the other diseases are selected from the group consisting of lupus erythematosus, rheumatoid arthritis or leukemia.
  • the present invention discloses a set of primers and probes for detecting mutations in the Kras gene and kits therefor.
  • the forward primers are 34A-Rev-Fp10, 35A-Rev-Fp4, 38A-Rev-Fp13, 34C-Rev-Fp, 34T-Rev-Fp, 35C-Rev-Fp, 35T-Rev-Fp, reverse primers are all Kras-Rev-Rp
  • probes are 34A-Rev-Pb4, 35A-Rev-Pb2, 38A-Rev-Pb3 , 34C-Rev-Pb, 34T-Rev-Pb, 35C-Rev-Pb, 35T-Rev-Pb.
  • the detection sensitivity and accuracy of the Kras gene mutation are high and the specificity is good, and seven different mutation types can be detected separately or simultaneously.
  • Figure 1 shows the detection sensitivity results of primers and probes of the Kras gene mutant 34A
  • Figure 2 shows the results of detection sensitivity of primers and probes of the Kras gene mutant 34C
  • Figure 3 shows the detection sensitivity results of primers and probes of the Kras mutant 34T
  • Figure 4 shows the detection sensitivity results of primers and probes of the Kras gene mutant 35A
  • Figure 5 shows the results of detection sensitivity of primers and probes of the Kras gene mutant 35C
  • Figure 6 shows the results of detection sensitivity of primers and probes of the Kras mutant 35T
  • Figure 7 shows the results of detection sensitivity of primers and probes of the Kras gene mutant 38A
  • Figure 8 shows the detection specific results of primers and probes of the Kras gene mutant 34A
  • Figure 9 shows the detection specificity results of primers and probes of the Kras gene mutant 34C
  • Figure 10 shows the detection specificity results of primers and probes of the Kras gene mutant 34T
  • Figure 11 shows the detection specificity results of primers and probes of the Kras gene mutant 35A
  • Figure 12 shows the detection specificity results of primers and probes of the Kras gene mutant 35C
  • Figure 13 shows the detection specificity results of primers and probes of the Kras gene mutant 35T
  • Figure 14 shows the detection specificity results of primers and probes of the Kras gene mutant 38A.
  • the invention discloses a primer and a probe for detecting a mutation of a Kras gene and a kit thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • Another object of the present invention is to provide a kit for detecting mutations in the Kras gene.
  • a set of primers for detecting mutations in the Kras gene including forward primers and reverse primers for detecting seven Kras mutants, and 34A-Rev-Fp10 and 35A-Rev for the detection of seven Kras mutants.
  • -Fp4, 38A-Rev-Fp13, 34C-Rev-Fp, 34T-Rev-Fp, 35C-Rev-Fp, 35T-Rev-Fp, the nucleotide sequences thereof are shown in SEQ ID NO. 1 to 7, respectively;
  • the reverse primers of the seven Kras gene mutants are all Kras-Rev-Rp, and the nucleotide sequence thereof is shown in SEQ ID NO.
  • a probe for detecting Kras mutations including probes for detecting seven Kras mutants, 34A-Rev-Pb4, 35A-Rev-Pb2, 38A-Rev-Pb3, 34C-Rev-Pb, 34T- Rev-Pb, 35C-Rev-Pb, 35T-Rev-Pb, the nucleotide sequences thereof are shown in SEQ ID NO. 9 to 15, respectively.
  • each of the above probes is labeled with a fluorescent group and the 3' end is labeled with a corresponding quenching group.
  • the 5'-labeled fluorophore is FAM, HEX or VIC.
  • the quenching group labeled at the 3' end is BHQ1 or BHQ2.
  • the 5' end labeled fluorescent group is FAM.
  • the quenching group labeled at the 3' end is BHQ1.
  • a set of primers and probes for detecting mutations in the Kras gene including forward primers, reverse primers and probes; that is, the use of the above primers and probes are all within the scope of the present invention.
  • the forward primer, the reverse primer and the probe are the above-mentioned primers and probes, which are used in combination for detecting seven Kras gene mutants.
  • the present invention successfully constructs a kit for detecting a mutation of the Kras gene by a plurality of optimization experiments based on the above-mentioned detection primers and probes, and the kit is also within the scope of the present invention, and the kit includes the above Forward primer and reverse primer.
  • kit further comprises the above probe.
  • the kit further comprises a reagent for a PCR amplification system, including dNTP, magnesium ion, reaction buffer, enzyme, and nuclease-free water, when PCR amplification is performed using the kit.
  • a reagent for a PCR amplification system including dNTP, magnesium ion, reaction buffer, enzyme, and nuclease-free water, when PCR amplification is performed using the kit.
  • the system reagent comprises 5-20 mM dNTPs, 10-40 mM magnesium ions, 1 x 10 x reaction buffer, enzyme, nuclease-free water.
  • the system reagent comprises 5-15 mM dNTPs, 15 to 35 mM magnesium ions, 1 x 10 x reaction buffer, enzyme, nuclease-free water.
  • the system reagent comprises 10 mM dNTPs, 25 mM magnesium ions, 5 x reaction buffer (5 x buffer), enzyme, nuclease-free water.
  • the dNTPs are 10 mM dNTPs.
  • the enzyme is a hot start enzyme (DNA polymerase).
  • the concentration of the enzyme is preferably 5 U/ ⁇ l.
  • reaction buffer is preferably 5 x reaction buffer.
  • the magnesium ion is preferably 25 mM Mg 2+ .
  • the final reaction concentration of each component in the PCR system is as follows:
  • the PCR system is carried out according to the following ratio:
  • the PCR procedure is as follows:
  • the PCR procedure is as follows:
  • the above primers and probes and the kit thereof have good application prospects in detecting mutation of Kras gene.
  • a method for detecting mutations in the Kras gene that is, the method of using the above kit, is divided into two detection systems:
  • the detected PCR system and reaction procedure were as described above, respectively.
  • the above 7 kinds of forward primers and probes were respectively used, and the same reverse primer was added to each reaction, and 7 PCR reactions were respectively performed on the samples to be tested, and 7 different mutations were respectively determined.
  • the PCR system and the reaction procedure are the same as the detection system. The only difference is that a PCR reaction is performed on the sample to be tested.
  • the forward primer is an equal volume mixture of 7 mutant forward primers, and the probe is 7 mutants. An equal volume mixture of probes. A mutation of the Kras gene is detected in the sample to be tested.
  • the invention provides a set of primers and probes for detecting mutation of Kras gene, and successfully establishes a kit for detecting mutation of Kras gene by optimizing the conditions of annealing temperature, using system and PCR program, and synthesizing Kras gene mutation
  • the detection sensitivity is high, and 7 different mutation types can be detected separately, and 7 mutation types can be detected simultaneously, that is, several mutations can be combined in one tube to detect mutation of Kras gene; In 7 tubes, 7 different mutation types were determined, and the application was more flexible and wider.
  • the detection sensitivity of the method is high, and one mutant can be accurately detected in 1000 wild type copies, and the detection sensitivity can reach 0.1%.
  • the method of the invention is fast, and the detection can be completed in about 75 to 90 minutes, without high instrument and testing cost, the operation is simple, the result is objectively interpreted, and it is suitable for large-scale clinical development.
  • Mutation type marker in the present invention Mutation name Amino acid change Base change 34T 12CYS G12C c.34G>T 34A 12SER G12S c.34G>A 34C 12ARG G12R c.34G>C 35T 12VAL G12V c.35G>T 35A 12ASP G12D c.35G>A 35C 12ALA G12A c.35G>C 38A 13ASP G13D c.38G>A
  • the present invention is directed to the primers for detecting the above seven mutations, sharing the same reverse primer, each using a specific forward primer.
  • the common reverse primer is:
  • the PCR template is a mutant and wild-type plasmid template, respectively, and the specific preparation process is as follows: refer to the wild type of the Kras gene and the reference sequence of 7 common mutants which are queried in the COSMIC database, and sequentially synthesize the wild type according to the reference sequence. Eight artificial nucleic acid sequences containing the mutation sites were ligated to the plasmid vector, and the plasmids carrying the wild type and the seven mutant sequences were obtained after screening. The concentration of the plasmid template is on the order of 10,000 copies. The results are shown in Table 6.
  • primers 34A-Rev-Fp10, 35A-Rev-Fp4, 38A-Rev-Fp13, 34C-Rev-Fp, 34T-Rev-Fp, 35C-Rev-Fp, 35T-Rev- The results of Fp detection made the mutant template the earliest and the difference from the wild type was the largest. Therefore, the above primers were selected as the final detection primers.
  • the probes 34A-Rev-Pb4, 35A-Rev-Pb2, 38A-Rev-Pb3, 34C-Rev-Pb, 34T-Rev-Pb, 35C-Rev-Pb, 35T-Rev- The results of the Pb assay allowed the mutant template to achieve the highest fluorescence value, so they were chosen as the final detection probe.
  • the present invention successfully assembles a kit for detecting a mutation of a Kras gene with high sensitivity and high accuracy based on the above findings, and includes the following components:
  • Reverse primer (SEQ ID NO. 8): Kras-Rev-Rp:
  • Probe sequence (5' ⁇ 3') serial number 34A 34A-Rev-Pb4 FAM-CTAGCTCCAACTACCACAAG-BHQ1 SEQ ID NO.9 35A 35A-Rev-Pb2 FAM-ATCAGCTCCAACTACCACAA-BHQ1 SEQ ID NO.10 38A 38A-Rev-Pb3 FAM-GTCTCCAGCTCCAACTACCACGC-BHQ1 SEQ ID NO.11 34C 34C-Rev-Pb FAM-TGAGCTCCAACTACCAC-BHQ1 SEQ ID NO.12 34T 34T-Rev-Pb FAM-CAAGCTCCAACTACCAC-BHQ1 SEQ ID NO.13 35C 35C-Rev-Pb FAM-TGCAGCTCCAACTACCAC-BHQ1 SEQ ID NO.14 35T 35T-Rev-Pb FAM-AACAGCTCCAACTACCAC-BHQ1 SEQ ID NO.15
  • the dNTPs required for the PCR reaction are preferably 10 mM dNTPs.
  • an enzyme preferably a Taq hot start enzyme (DNA polymerase); its concentration is preferably 5 U/ ⁇ l.
  • the reaction buffer is preferably a 5* enzyme buffer.
  • Mg 2+ preferably 25 mM Mg 2+ .
  • the PCR template is the same as the plasmid template, and the synthesized plasmid is digested to obtain a fragment of 10 4 copies, and then mixed in different ratios to obtain samples containing different concentrations of mutant and wild type: 100 % mutant, 50% mutant, 10% mutant, 5% mutant, 1% mutant, 0.1% mutant and 100% wild type sample template.
  • the template concentration is 10 4 copies.
  • the detection curves of the above-mentioned seven kinds of primers and probes for detecting mutants 34A, 34C, 34T, 35A, 35C, 35T, and 38A are shown in Figs. 8 to 14, respectively, and the seven kinds of these are also shown. Both the primer and the probe-based detection system can clearly distinguish the target detection mutant from other mutations, and each group of primers and probes have good specificity.
  • the DNA of the paraffin tissue sample was extracted using a DNA extraction kit, and the extracted DNA concentration was measured to adjust the concentration to 20 ng/ ⁇ l.
  • the following is a method for extracting DNA from QIAgen_FFPE_DNA_kit paraffin tissue:
  • Paraffin tissue samples cut the sample into 5 ⁇ 10 ⁇ m, add 1ml of xylene dewaxing, collect the precipitate after centrifugation, add 1ml of absolute ethanol, dry at room temperature or 37 degrees, add proteinase K and BufferATL, digestion and cleavage at 56 ° C Hour, incubate for 1 hour at 90 °C, add 200 ⁇ l of BufferAL and mix thoroughly by adding 200 ⁇ l of absolute ethanol. Transfer the supernatant carefully to a QIA 2 ml spin column, centrifuge at 8000 rpm for 1 min, add 500 ⁇ l of Buffer AW1, centrifuge at 8000 rpm for 1 min, carefully open the lid.
  • detection system one can distinguish specific mutations
  • Example 1 The detected PCR system and reaction procedure are shown in Tables 4 and 5, respectively, in Example 1. Seven tubes were reacted for each sample, and seven different mutations were determined. Each tube was reacted with the same reverse primer, buffer, polymerase, deoxyribonucleic acid, magnesium chloride, nuclease-free water, and template DNA. Only the forward primer and the corresponding probe.
  • the specific primer probe design method of the present invention has high accuracy and sensitivity for detecting Kras gene mutation in tumor tissues, and can detect micro-mutation templates in clinical samples; the results are reliable, and the mutations are reliable. Detection of "gold standard”: The result of direct sequencing was 100%.
  • detection system 2 only to determine whether there is a mutation, do not distinguish the type of mutation
  • One tube reaction was performed for each sample to determine whether a mutation of Kras gene occurred in the sample to be tested.
  • the same positive and negative primers, probe, buffer, polymerase, deoxyribonucleic acid, magnesium chloride, and nuclease-free were added to each tube reaction. Water and template DNA.
  • the specific primer probe design method of the present invention has high sensitivity for detecting Kras gene mutation in tumor tissues, and can detect micro-mutation templates in clinical samples; the results are reliable, and the mutation detection "gold” Standard” - the result of direct sequencing is 95%.
  • the magnetic bead capture method uses magnetic beads as a solid phase adsorption carrier and uses a specially designed reagent system and extraction process (Journal of China MedicalUniversity Magnetic Bead Method to detect and extract urine free methylated DNA; 2015, (10)).
  • the freshly collected stool sample was mixed and diluted with a buffer, and then centrifuged at 6000 rpm for 30 min to obtain a supernatant of the feces, and the supernatant was transferred to a carboxyl-coupled magnetic bead suspension containing a Kras capture fragment. After the time of reaction, the captured fragments on the magnetic beads will grab the Kras fragments in the feces and attach them to the magnetic beads. After repeated washings, the inhibitor in the capture system is sufficiently removed, and then the captured fragment is eluted from the magnetic beads to obtain a sample to be tested, and 5 ⁇ l is taken for PCR reaction.
  • Determination system 2 (only to determine whether there is a mutation, do not distinguish the type of mutation)
  • the optimized reaction system is 25 ⁇ l, including DNA polymerase, mutant primers and probes, DNA templates, and the like.
  • the PCR detection system is shown in Table 15:
  • PCR amplification was performed on a fluorescence quantitative PCR machine. One tube reaction was performed for each sample to determine whether a mutation of Kras gene occurred in the sample to be tested. Each tube was reacted with a common forward and reverse primer, probe, buffer, polymerase, deoxyribonucleic acid, magnesium chloride, and nuclease-free. Water and template DNA.
  • the sample used for sequencing in the present embodiment is a tissue extracted nucleic acid sample
  • the nucleic acid sample used in the method of the present invention is derived from feces, wherein the amount of nucleic acid capable of being extracted is small, and thus the present invention
  • the method has the characteristics of high accuracy and sensitivity.
  • the specific primer probe designed according to the design method of the present invention has high sensitivity for detecting the mutation of Kras gene in the stool sample, and the result is reliable, and the "gold standard" for mutation detection: the coincidence rate of the result of direct sequencing is 91. %.
  • the method of the present invention is fast, simple to operate, and the result is objectively interpreted, and is suitable for large-scale clinical development.

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Abstract

一组检测Kras基因突变的引物和探针及其试剂盒。包括针对检测7种Kras基因突变型的前向引物、反向引物和探针；前向引物分别为34A-Rev-Fp10、35A-Rev-Fp4、38A-Rev-Fp13、34C-Rev-Fp、34T-Rev-Fp、35C-Rev-Fp、35T-Rev-Fp，反向引物均为Kras-Rev-Rp，探针分别为34A-Rev-Pb4、35A-Rev-Pb2、38A-Rev-Pb3、34C-Rev-Pb、34T-Rev-Pb、35C-Rev-Pb、35T-Rev-Pb。上述试剂盒对Kras基因突变的检测灵敏度和准确性高，特异性好，可分别或同时检测7种不同的突变类型。

Description

一组检测Kras基因突变的引物和探针及其试剂盒
本申请要求于2016年04月29日提交中国专利局、申请号为201610284206.0、发明名称为“一组检测Kras基因突变的引物和探针及其试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于基因检测技术领域。更具体地，涉及一组检测Kras基因突变的引物和探针及其试剂盒。
背景技术
RAS基因最早发现于上世纪八十年代初，首先从人膀胱癌细胞系中分离出的一种转化基因，可使NIH3T3细胞(小鼠胚胎成纤维细胞系)发生恶性转化，而从正常人组织中提取的DNA则无此种作用。RAS基因在进化中相当保守，广泛存在于各种真核生物如哺乳类，果蝇，真菌，线虫及酵母中，提示它有重要的生理功能。
RAS基因家族与人类肿瘤相关的基因有三种——Hras、Kras和Nras，分别定位在11、12和1号染色体上。Kras因编码21kD的RAS蛋白又名p21基因。在RAS基因中，Kras对人类癌症影响最大。Kras基因就像体内一个“开关”：当正常时能控制调控细胞生长的路径；发生异常时，则导致细胞持续生长，并阻止细胞自我毁灭。因此，Kras基因在肿瘤细胞生长以及血管生成等过程的信号传导通路中起着重要调控作用，正常的Kras基因可抑制肿瘤细胞生长，而一旦Kras基因发生突变，它就会持续刺激细胞生长，使细胞内信号传导紊乱，打乱生长规律，细胞增殖失控而癌变，从而导致肿瘤的发生。在不同的癌症当中存在不同的Kras基因突变的比例：胰腺癌90％、结肠癌50％、肺癌30％、卵巢癌15％、甲状腺癌50％、膀胱癌6％左右；另外，红斑性狼疮(SLE)、乳腺癌、肝癌、皮肤癌、类风湿性关节炎(RA)、肾癌及某些的白血病(Leukemia)等都有较高的Kras基因突变水平。也有研究表明，Kras基因突变发生在肿瘤恶变的早期，并且原发灶和转移灶的Kras基因高度保持一致。一般认为，Kras 基因状态不会因治疗而发生变化。因此，Kras基因突变的及时检测具有很大的意义，检测Kras基因突变是深入了解癌基因的情况、了解各种癌症的发展预后、放化疗疗效的重要指标。
另外，随着人类医学和药物研究水平的提高，药理遗传学(Pharmacogenetics)日渐兴起。研究表明，药物在体内的代谢、药物治疗的有效性以及副反应、甚至患者的预后都存在个体差异，受患者基因背景的影响。因此，应用药理遗传学，在基因水平上研究药物作用的个体差异，使得个性化治疗这一新一代医学概念成为可能。对药物作用相关基因的检测，可指导医生对患者准确施药，规避药物副作用，同时提高病人乃至社会的医疗费用效率。近年来推向市场的一些肿瘤靶向治疗的主流药物，都相继在其临床试验中建立和确认了相应的预测疗效的基因生物标记物。Kras基因是目前确认的肿瘤靶向治疗药物的重要基因标记物之一。Kras蛋白是EGFR(表皮生长因子受体)信号传导通路中的一个关键的下游调节因子，参与细胞生长的调节，在癌变发生过程中起着重要作用。肿瘤患者Kras基因突变与否显著影响EGFR靶向治疗药物的疗效。最新的临床研究表明，当患者本身不存在Kras基因突变，服用有关靶向治疗药物，能获得明显的治疗效果。例如非Kras基因突变的结直肠癌患者，可受益于Cetuximab和Panitumumab等EGFR抗体药物；另外对非Kras基因突变的晚期非小细胞肺癌患者使用Gefitinib和Erlotinib等EGFR酪氨酸激酶抑制剂(TKI)类药物，能明显提高靶向治疗的疗效。美国癌症综合网络(NCCN)提出肿瘤患者接受EGFR靶向药物治疗之前，必须进行Kras基因突变检测，根据检测结果决定是否使用EGFR靶向药物作为临床治疗措施。Kras基因突变检测已被NCCN列为《结肠癌临床治疗指南》与《直肠癌临床治疗指南》临床用药必检项目。
综上所述，Kras基因突变的检测对深入了解癌基因的情况、了解各种癌症的发展预后、放化疗疗效的评价、指导用药具有重要的意义。
目前，Kras基因突变的检测方法主要是ARMs-qPCR和HRM。(1)ARMs-qPCR：扩增阻滞突变系统(amplificationrefractorymutationsystem,ARMS)也就是常说的等位基因特异 性PCR(allele-specificPCR,ASPCR)，用于对已知突变基因或者基因多态性进行检测。该方法的核心是三条引物的设计，此处的三条引物一般包含两条正向引物和一条公用的反向引物；而这两条正向引物的区别在于引物的3’末端的碱基不同，也就是等位基因或者已知突变的突变碱基和野生碱基，因为TaqDNA聚合酶没有校正活性，且一般只有引物3’端完全和模板互补时才能有效发生扩增反应。所以可以将两条正向引物分别和反向引物配置成反应体系(一个位点需要两个反应)，这样可以结合荧光定量PCR的高灵敏度特性有效的监测两个反应的是否存在扩增，也就可以判断是否突变或确定基因型。然而上述的都是理论上的假设，假如引物其他参数都符合要求(如GC含量、Tm值、无发夹结构等)，单单因为3’末端的碱基不同，使得TaqDNA聚合酶起始扩增的效率不同，换句话说当3’末端为T时，就算不匹配，可能发生扩增的概率大于3’末端为A时的引物，这样就产生了检测特异性不高的因素，很容易造成假阳性。(2)HRM(HighResolutionMelt)：即“高分辨率熔解曲线”，是一种简单、快速、低成本的PCR扩增后检测技术，可用于高通量的突变扫描和基因分型。不同DNA的熔解温度Tm各不相同，通常片段越长、GC含量越高的片段其熔解温度就越高，因而每条DNA序列都有其特定的熔解曲线和熔解温度。如果在PCR扩增中出现非特异的扩增，那么就会出现多个熔解峰。可以通过熔解曲线判断PCR扩增的特异性，判断反应产物的条带数目。同理，也可以根据熔解温度(熔解曲线)区分不同的扩增片段。该技术在标准PCR试剂的基础上再加入双链DNA结合染料即可进行，无需序列特异性探针，在PCR结束后直接运行高分辨率熔解曲线，即可完成对样品基因型的分析。最近的研究表明，HRM方法对突变检测的灵敏度可以比拟甚至超过当前的一些SNP/突变分析技术，如DHPLC等，其灵敏度也大大高于测序。这种方法操作简便、快速、通量大、使用成本低、结果准确。但是，该方法也具有所依赖的仪器较昂贵等缺陷，普及利用率较低。
发明内容
本发明要解决的技术问题是克服现有Kras基因突变检测技术的缺陷和不足，采用独特的创新设计方法，设计优化出一组检测Kras基因突变的引物和探针，并通过对退火温度、使用体系、PCR程序等使用条件的研究优化，成功组建了一种检测Kras基因突变的试剂盒，对Kras基因突变的检测灵敏度高，而且特异性、准确性好，具体使用时既可以分别检测7种不同的突变类型，也可以同时检测7种突变类型，应用更灵活、更广泛。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种引物组，包括引物，所述引物包括正向引物和反向引物；所述正向引物的序列如SEQ ID No.1～SEQ ID No.7任一所示；所述反向引物的序列如SEQ ID No.8所示。
本发明还提供了所述的引物组在制备Kras基因突变的检测试剂中的应用。
本发明还提供了所述的引物组在制备癌症和/或其他疾病检测试剂中的应用；优选的，所述癌症选自胰腺癌、结直肠癌、肺癌、卵巢癌、甲状腺癌、膀胱癌、乳腺癌、肝癌、皮肤癌或肾癌；所述其他疾病选自红斑性狼疮、类风湿性关节炎或白血病。
本发明还提供了一种探针组合物，所述探针的序列如SEQ ID No.9～SEQ ID No.15任一所示；优选的，所述探针的5'端标记有荧光基团，3'端标记有对应的淬灭基团。
本发明还提供了所述的探针组合物在制备Kras基因突变的检测试剂中的应用。
本发明还提供了所述的探针组合物在制备癌症和/或其他疾病检测试剂中的应用；优选的，所述癌症选自胰腺癌、结直肠癌、肺癌、卵巢癌、甲状腺癌、膀胱癌、乳腺癌、肝癌、皮肤癌或肾癌；所述其他疾病选自红斑性狼疮、类风湿性关节炎或白血病。
本发明还提供了一种检测试剂，包括所述的引物组和/或所述的探针组合物。
本发明还提供了一种检测试剂盒，包括所述的检测试剂。
在本发明的一些具体实施方案中，所述的检测试剂盒还包括扩增体系；所述扩增体系包括dNTP、镁离子、反应缓冲液、酶、无核酸酶的水。
在本发明的一些具体实施方案中，所述的检测试剂盒中所述扩增体系包括5～20mM的dNTP、10～40mM的镁离子、1×～10×反应缓冲液、酶、无核酸酶的水。
在本发明的一些具体实施方案中，所述的检测试剂盒所述扩增体系中各组分的反应终浓度为：
Figure PCTCN2016109631-appb-000001
在本发明的一些具体实施方案中，所述的检测试剂盒的扩增程序为：
95℃300s；1个循环；
95℃20s，62℃30s，70℃30s；10个循环；
95℃20s，58℃60s，72℃30s；30～40个循环；
37℃30s；1个循环。
本发明还提供了一种Kras基因突变的检测方法，取样本与所述的引物组、所述的探针组合物或所述的检测试剂或所述的检测试剂盒中的检测试剂混合后扩增，根据扩增结果获得检测结果。
在本发明的一些具体实施方案中，所述的检测方法中所述获得检测结果为比较待测样本与正常样本的扩增结果，当所述待测样本与正常样本的扩增结果具有显著差异或极显著差异时，所述待测样本的供体发生 Kras基因突变。
本发明还提供了一种癌症和/或其他疾病的检测方法，取样本与所述的引物组、所述的探针组合物或所述的检测试剂或所述的检测试剂盒中的检测试剂混合后扩增，根据扩增结果获得检测结果；
所述癌症选自胰腺癌、结直肠癌、肺癌、卵巢癌、甲状腺癌、膀胱癌、乳腺癌、肝癌、皮肤癌或肾癌；所述其他疾病选自红斑性狼疮、类风湿性关节炎或白血病。
在本发明的一些具体实施方案中，所述的检测方法中所述获得检测结果为比较待测样本与正常样本的扩增结果，当所述待测样本与正常样本的扩增结果具有显著差异或极显著差异时，所述待测样本的供体患有癌症和/或其他疾病；
所述癌症选自胰腺癌、结直肠癌、肺癌、卵巢癌、甲状腺癌、膀胱癌、乳腺癌、肝癌、皮肤癌或肾癌；所述其他疾病选自红斑性狼疮、类风湿性关节炎或白血病。
本发明公开了一组检测Kras基因突变的引物和探针及其试剂盒。包括针对检测7种Kras基因突变型的前向引物、反向引物和探针；前向引物分别为34A-Rev-Fp10、35A-Rev-Fp4、38A-Rev-Fp13、34C-Rev-Fp、34T-Rev-Fp、35C-Rev-Fp、35T-Rev-Fp，反向引物均为Kras-Rev-Rp，探针分别为34A-Rev-Pb4、35A-Rev-Pb2、38A-Rev-Pb3、34C-Rev-Pb、34T-Rev-Pb、35C-Rev-Pb、35T-Rev-Pb。本发明对Kras基因突变的检测灵敏度和准确性高，特异性好，可分别或同时检测7种不同的突变类型。
附图说明
图1为Kras基因突变型34A的引物和探针的检测灵敏度结果；
图2为Kras基因突变型34C的引物和探针的检测灵敏度结果；
图3为Kras基因突变型34T的引物和探针的检测灵敏度结果；
图4为Kras基因突变型35A的引物和探针的检测灵敏度结果；
图5为Kras基因突变型35C的引物和探针的检测灵敏度结果；
图6为Kras基因突变型35T的引物和探针的检测灵敏度结果；
图7为Kras基因突变型38A的引物和探针的检测灵敏度结果；
图8为Kras基因突变型34A的引物和探针的检测特异性结果；
图9为Kras基因突变型34C的引物和探针的检测特异性结果；
图10为Kras基因突变型34T的引物和探针的检测特异性结果；
图11为Kras基因突变型35A的引物和探针的检测特异性结果；
图12为Kras基因突变型35C的引物和探针的检测特异性结果；
图13为Kras基因突变型35T的引物和探针的检测特异性结果；
图14为Kras基因突变型38A的引物和探针的检测特异性结果。
具体实施方式
本发明公开了一种检测Kras基因突变的引物和探针及其试剂盒，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明的目的是提供一组检测Kras基因突变的引物和探针。
本发明另一目的是提供一种检测Kras基因突变的试剂盒。
本发明上述目的通过以下技术方案实现：
一组检测Kras基因突变的引物，包括针对检测7种Kras基因突变型的前向引物和反向引物，针对检测7种Kras基因突变型的前向引物分别为34A-Rev-Fp10、35A-Rev-Fp4、38A-Rev-Fp13、34C-Rev-Fp、34T-Rev-Fp、35C-Rev-Fp、35T-Rev-Fp，其核苷酸序列分别如SEQIDNO.1～7所示；针对检测7种Kras基因突变型的反向引物均为Kras-Rev-Rp，其核苷酸序列如SEQIDNO.8所示。
一种检测Kras突变的探针，包括针对检测7种Kras基因突变型的探针，分别为34A-Rev-Pb4、35A-Rev-Pb2、38A-Rev-Pb3、34C-Rev-Pb、34T-Rev-Pb、35C-Rev-Pb、35T-Rev-Pb，其核苷酸序列分别如SEQIDNO.9～15所示。
优选地，上述每个探针的5'端标记有荧光基团，3'端标记有对应的淬灭基团。
更优选地，所述5'端标记的荧光基团为FAM、HEX或VIC。优选地，3'端标记的淬灭基团为BHQ1或BHQ2。
更优选地，所述5'端标记的荧光基团为FAM。优选地，3'端标记的淬灭基团为BHQ1。
一套检测检测Kras基因突变的引物和探针，包括前向引物、反向引物和探针；即上述引物和探针的配套使用，都在本发明的保护范围之内。所述前向引物、反向引物和探针为上述的引物和探针，依次配套使用针对检测7种Kras基因突变型。
具体地，针对检测的7种突变型、以及引物探针的配套情况如表1所示：
表1
Figure PCTCN2016109631-appb-000002
Figure PCTCN2016109631-appb-000003
另外，本发明基于上述检测引物和探针，通过大量的优化实验成功组建了一种检测Kras基因突变的试剂盒，该试剂盒也在本发明的保护范围之内，所述试剂盒包含有上述前向引物和反向引物。
进一步地，所述试剂盒还包含有上述探针。
更进一步地，所述试剂盒还包含有利用该试剂盒进行PCR扩增时，PCR扩增体系所需试剂，包括dNTP、镁离子、反应缓冲液、酶、无核酸酶的水。
优选地，所述体系试剂包括5～20mM的dNTP、10～40mM的镁离子、1×～10×反应缓冲液、酶、无核酸酶的水。
进一步优选地，所述体系试剂包括5～15mM的dNTP、15～35mM的镁离子、1×～10×反应缓冲液、酶、无核酸酶的水。
再进一步优选地，所述体系试剂包括10mM的dNTP、25mM的镁离子、5×反应缓冲液(5×buffer)、酶、无核酸酶的水。
更优选地，所述dNTPs为10mM的dNTPs。
更优选地，所述酶为热启动酶(DNA聚合酶)。
更优选地，酶的浓度优选为5U/μl。
更优选地，所述反应缓冲液优选为5×反应缓冲液。
更优选地，所述镁离子优选为25mM的Mg2+。
另外，优选地，利用上述试剂盒进行Kras突变检测时，PCR体系中各组分的反应终浓度如下：
Figure PCTCN2016109631-appb-000004
Figure PCTCN2016109631-appb-000005
更优选地，利用上述试剂盒进行Kras基因突变检测时，PCR体系按照如下比例进行：
Figure PCTCN2016109631-appb-000006
优选地，利用上述试剂盒进行Kras突变检测时，PCR程序如下：
95℃300s；1个循环；
95℃20s，62℃30s，70℃30s；10个循环；
95℃20s，58℃60s，72℃30s；30～40个循环；
37℃30s；1个循环。
更优选地，利用上述试剂盒进行Kras突变检测时，PCR程序如下：
95℃300s；1个循环；
95℃20s，62℃30s，70℃30s；10个循环；
95℃20s，58℃60s，72℃30s；35～40个循环；
37℃30s；1个循环。
本发明上述引物和探针及其试剂盒在Kras基因突变检测方面具有很好的应用前景。
一种检测Kras基因突变的方法，也即上述试剂盒的使用方法，分为两种检测体系：
(1)检测体系一：可区分具体突变型
检测的PCR体系和反应程序分别如上所述。分别利用上述7种前向引物和探针，每个反应加入相同的反向引物，针对待测样本分别进行7次PCR反应，分别测定7种不同的突变。
(2)检测体系二：仅判断有无突变，不区分突变类型
检测的PCR体系和反应程序同检测体系一，唯一不同之处在于：针对待测样品进行一次PCR反应，正向引物是7种突变的正向引物的等体积混合物，探针是7种突变的探针的等体积混合物。测定待测样本中是否发生Kras基因突变。
本发明具有以下有益效果：
本发明提供了一组检测Kras基因突变的引物和探针，并通过对退火温度、使用体系、PCR程序等使用条件的优化，成功组建了一种检测Kras基因突变的试剂盒，对Kras基因突变的检测灵敏度高，而且可以分别检测7种不同的突变类型，也可以同时检测7种突变类型，即可以将几种突变合在一个管内做，检测Kras基因突变；也可以将几种突变分别在7个管中做，分别测定7种不同的突变类型，应用更灵活、更广泛。
另外，本方法的检测灵敏度高，在1000个野生型拷贝中可以准确的检测出1个突变型，检测灵敏度可达0.1％。
本发明的方法快速，只需75～90min左右的时间即可完成检测，无需高昂的仪器和检测费用，操作简单，结果判读客观，适合于临床中大规模开展。
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1 引物和探针的设计
1、引物设计：Kras基因常见的7种突变类型如表1所示。
表1 Kras基因常见的7种突变类型
本发明中突变类型标记 突变名称 氨基酸变化 碱基变化
34T 12CYS G12C c.34G>T
34A 12SER G12S c.34G>A
34C 12ARG G12R c.34G>C
35T 12VAL G12V c.35G>T
35A 12ASP G12D c.35G>A
35C 12ALA G12A c.35G>C
38A 13ASP G13D c.38G>A
本发明针对检测上述7种突变的引物中，共用相同的反向引物，各自使用特异的前向引物。
(1)共同反向引物为：
Kras-Rev-Rp：GGCCTGCTGAAAATGACTG
(2)针对7种不同的突变类型，分别设计了多个前向引物，经过一定的优化实验后，得出表2所示的较好的引物：
表2 各突变类型的前向引物
Figure PCTCN2016109631-appb-000007
Figure PCTCN2016109631-appb-000008
Figure PCTCN2016109631-appb-000009
2、探针设计
针对7种不同的突变类型，分别设计了多个探针，经过一定的优化实验后，得出表3所示的较好的探针：
表3 针对检测各突变类型的探针
Figure PCTCN2016109631-appb-000010
Figure PCTCN2016109631-appb-000011
3、PCR体系和反应程序的优化
(1)经过实验优化后得出，利用本发明设计的引物和探针检测Kras突变时，最佳的反应体系如下表4所示：
表4 PCR反应体系
Figure PCTCN2016109631-appb-000012
(2)经过实验优化后得出，利用本发明设计的引物和探针检测Kras突变时，最佳的反应程序如下表5所示：
表5 PCR反应程序
Figure PCTCN2016109631-appb-000013
实施例2 引物和探针的进一步优化
1、前向引物优化
(1)利用上述PCR反应条件对引物进行进一步地优化。
PCR模板分别是突变型和野生型的质粒模板，其具体的制备过程如下：参考COSMIC数据库中查询到的Kras基因的野生型和7种常见突变型的参考序列，根据参考序列依次合成野生型和含有突变位点的8种人工核酸序列，将该人工序列连接到质粒载体上，经过筛选后得到分别装载了野生型和7种突变序列的质粒。质粒模板的浓度为10000拷贝数量级。结果如表6所示。
表6 引物优化结果
Figure PCTCN2016109631-appb-000015
注：“-”表示无扩增。
(2)结论
由以上表6结果可以看出，引物34A-Rev-Fp10、35A-Rev-Fp4、38A-Rev-Fp13、34C-Rev-Fp、34T-Rev-Fp、35C-Rev-Fp、35T-Rev-Fp检测结果使得突变型模板扩增最早且与野生型差值最大，因此，选用上述引物作为最终检测引物。
2、探针优化
(1)同上，利用上述PCR反应条件对探针进行进一步地优化。结果如表7所示。
表7 探针优化结果
Figure PCTCN2016109631-appb-000016
注：“-”表示无扩增。
(2)结论
结论：由以上可看出，探针34A-Rev-Pb4、35A-Rev-Pb2、38A-Rev-Pb3、34C-Rev-Pb、34T-Rev-Pb、35C-Rev-Pb、35T-Rev-Pb检测结果使得突变型模板扩增达到的荧光值最高，因此选择他们做为最终检测探针。
实施例3 Kras基因突变检测试剂盒的组装
1、本发明根据上述研究结果成功组装出高灵敏性、高准确性检测Kras基因突变的试剂盒，包括如下组分：
(1)引物：前向引物如表8所示：
表8
突变类型 引物名称 引物序列(5'→3') 序列号
34A 34A-Rev-Fp10 CACTCTTGCCTACGCTACT SEQ ID NO.1
35A 35A-Rev-Fp4 CACTCTTGCCTACGTCAT SEQ ID NO.2
38A 38A-Rev-Fp13 ACGGACTCTTGCCTACGT SEQ ID NO.3
34C 34C-Rev-Fp CACTCTTGCCTACGCCATG SEQ ID NO.4
34T 34T-Rev-Fp CACTCTTGCCTACGCGACA SEQ ID NO.5
35C 35C-Rev-Fp CACTCTTGCCTACGCCTG SEQ ID NO.6
35T 35T-Rev-Fp GCACTCTTGCCTACGTCAA SEQ ID NO.7
反向引物(SEQIDNO.8)：Kras-Rev-Rp：
GGCCTGCTGAAAATGACTG。
(2)探针，如表9所示：
表9
突变类型 探针名称 探针序列(5'→3') 序列号
34A 34A-Rev-Pb4 FAM-CTAGCTCCAACTACCACAAG-BHQ1 SEQ ID NO.9
35A 35A-Rev-Pb2 FAM-ATCAGCTCCAACTACCACAA-BHQ1 SEQ ID NO.10
38A 38A-Rev-Pb3 FAM-GTCTCCAGCTCCAACTACCACGC-BHQ1 SEQ ID NO.11
34C 34C-Rev-Pb FAM-TGAGCTCCAACTACCAC-BHQ1 SEQ ID NO.12
34T 34T-Rev-Pb FAM-CAAGCTCCAACTACCAC-BHQ1 SEQ ID NO.13
35C 35C-Rev-Pb FAM-TGCAGCTCCAACTACCAC-BHQ1 SEQ ID NO.14
35T 35T-Rev-Pb FAM-AACAGCTCCAACTACCAC-BHQ1 SEQ ID NO.15
(3)PCR反应所需要的dNTPs，优选为10mM的dNTPs。
(4)酶；优选为Taq热启动酶(DNA聚合酶)；其浓度优选为5U/μl。
(5)反应缓冲液，优选为5*酶缓冲液。
(6)Mg2+，优选为25mM的Mg2+。
(7)无核酸酶的水。
2、上述试剂盒使用时PCR反应体系和反应程序分别如实施例1中的表4和表5所示。
实施例4 引物和探针的灵敏度检测
1、PCR模板同上为质粒模板，将合成的质粒进行酶切并得到104拷贝数量级的酶切片段，然后对两者进行不同比例的混合，得到含有不同浓度突变型和野生型的样品：100％突变型，50％突变型，10％突变型，5％突变型，1％突变型，0.1％突变型和100％野生型样品模板。
使用上述反应体系和程序进行检测。
2、突变型34A、34C、34T、35A、35C、35T、38A的检测曲线图分别如附图1～7所示。数据如表10和表11所示。
表10 灵敏度检测结果
Figure PCTCN2016109631-appb-000017
表11 灵敏度检测结果
Figure PCTCN2016109631-appb-000018
注：“-”表示无扩增。
结论：由以上结果可以看出，每种突变型的检测体系均可达到0.1％(千分之一)的检测灵敏度。
实施例5 引物和探针的准确性检测(Kras基因不同突变类型之间的特异性测试)
1、分别利用突变型34A、34C、34T、35A、35C、35T、38A的引物和探针组成的检测体系，分别检测7种不同突变型的模板以及野生型模板，模板浓度为104拷贝数量级。
2、各体系检测各个模板的Ct值如表12所示，从测试的结果看，以这7种引物和探针为基础构建的检测体系，均能够明显地将目标检测突变型和其它突变型区分开来，并且检测其他突变型的Ct值与模板突变型Ct值的差值都在10以上，说明本申请中使用的引物和探针有很好的特异性。
表12
Figure PCTCN2016109631-appb-000019
注：“-”表示无扩增。
3、另外，上述7种引物和探针的体系检测突变型34A、34C、34T、35A、35C、35T、38A的检测曲线图分别如附图8～14所示，也表明了以这7种引物和探针为基础构建的检测体系，均能够明显地将目标检测突变型和其它突变型区分开来，各组引物和探针都有很好的特异性。
实施例6 临床样本(石蜡组织样本)检测
1、样本
测定20例结直肠癌患者临床样本(石蜡组织样本)，其中10例Kras基因突变，10例正常。
2、样本的处理方法：
利用DNA提取试剂盒提取石蜡组织样本的DNA，测定提取的DNA浓度，调整浓度至20ng/μl。以下是QIAgen_FFPE_DNA_kit石蜡组织提取DNA的试剂盒的方法：
石蜡组织样本，将样本切成5～10μm，加入1ml的二甲苯脱蜡，离心后收集沉淀，加入1ml的无水乙醇，室温或者37度晾干，加入蛋白酶K和BufferATL，56℃消化裂解1小时，90摄氏度孵化1小时，加入200μlBufferAL混匀再加入200μl的无水乙醇充分混匀，将上清小心转移到QIA2ml离心柱中，8000rpm离心1min，加入500μl的BufferAW1，8000rpm离心1min，小心打开盖子加入500μlBufferAW2，8000rpm离心 1min，空管离心14000rpm3min，加入100μlBufferATE于膜中央，温育5min，14000rpm离心1min。取1μl测定OD值，将提取的样本DNA稀释到20ng/μl，取1μl进行PCR反应。
3、检测体系一：可区分具体突变型
(1)检测的PCR体系和反应程序分别如实施例1中的表4和表5所示。每个样本进行7管反应，分别测定7种不同的突变，每管反应加入相同的反向引物，缓冲液，聚合酶，脱氧核糖核酸，氯化镁，无核酸酶的水以及模板DNA，所不同的只有前向引物和所对应的探针。
(2)测定结果如表13所示
表13 20例临床样本的检测结果
Figure PCTCN2016109631-appb-000020
注：“Mut”表示突变“Wt”表示野生型。
结果显示，在20例样本中检测出10例样本为Kras基因突变型，其 中2例样本发生c.35G>T突变，1例发生c.34G>A突变，1例c.34G>C突变，3例发生c.35G>A突变，1例发生c.34G>A突变，1例发生c.35G>T突变，2例发生c.38G>A突变。其中4号样本，同时发生c.35G>T和c.34G>T突变。其余10例样本均为野生型。
上述样本均经过测序验证，结果显示，测序结果和使用本发明方法测定的结果完全一致，一致性(准确性)为100％。
综上所述结果表明，本发明的特异性的引物探针设计方法对检测肿瘤组织中的Kras基因突变有高准确性和灵敏度，可检测临床样本中的微量突变模板；其结果可靠，与突变检测“金标准”：直接测序发的结果符合率为100％。
4、检测体系二：仅判断有无突变，不区分突变类型
(1)检测的PCR体系和反应程序分别如实施例1中的表4和表5所示，唯一不同之处在于：PCR体系中，正向引物换成7种突变的正向引物的等体积混合物，探针换成7种突变的探针的等体积混合物。
每个样本进行1管反应，测定待测的样本中是否发生Kras基因突变，每管反应加入相同的正反向引物，探针，缓冲液，聚合酶，脱氧核糖核酸，氯化镁，无核酸酶的水以及模板DNA。
(2)测定结果如表14所示
表14 20例临床样本的检测结果
Figure PCTCN2016109631-appb-000021
Figure PCTCN2016109631-appb-000022
注：“Mut”表示突变“Wt”表示野生型。
结果显示，在20例样本中检测出9例样本为Kras基因突变型。上述样本均经过测序验证，结果显示，其中12号样本的测序结果显示其为突变型，而本发明方法显示其为野生型，其余样本的测序结果和使用本方法测定的结果完全一致，一致性(准确性)为95％。
综上所述结果表明，本发明的特异性的引物探针设计方法对检测肿瘤组织中的Kras基因突变有高灵敏度，可检测临床样本中的微量突变模板；其结果可靠，与突变检测“金标准”——直接测序的结果符合率为95％。
实施例7 临床样本(粪便样本)检测
1、样本
48例粪便样本Kras基因片段的测定。
2、样本处理方法
磁珠捕获方法是采用磁珠作为固相吸附载体并使用特定设计的试剂体系及提取流程(JournalofChinaMedicalUniversity应用磁珠法检测并提取尿液游离甲基化DNA；2015,(10))。
将新鲜收取的粪便样本使用缓冲液进行混匀和稀释，然后在6000rpm下离心30min获得粪便的上清，将该上清转移到含有Kras捕获片段的羧基偶联的磁珠悬液中，经过一定时间的反应后磁珠上的捕获片段会将粪便中的Kras片段抓取，并依附在磁珠上。经过多次的洗涤后，充分的去掉捕获体系中的抑制物，然后将捕获片段从磁珠上洗脱下来，得到待测样品，取5μl进行PCR反应。
3、测定体系二(仅判断有无突变，不区分突变类型)
(1)经过优化后的反应体系为25μl，包括DNA聚合酶，突变型引物及探针，DNA模板等。所述的PCR检测体系如表15所示：
表15
Figure PCTCN2016109631-appb-000023
(2)在荧光定量PCR仪上进行PCR扩增。每个样本进行1管反应，测定待测的样本中是否发生Kras基因突变，每管反应加入共同的正反向引物，探针，缓冲液，聚合酶，脱氧核糖核酸，氯化镁，无核酸酶的水以及模板DNA。
反应条件如表16所示：
表16
Figure PCTCN2016109631-appb-000024
4、测定结果如表17所示：
表17
Figure PCTCN2016109631-appb-000025
结果显示，按照本发明所述方法，在48例样本中检测出9例样本为Kras基因突变型。其中结直肠癌样本中为8例，正常样本中为1例。
上述结果均与样本的组织来源测序结果进行验证，结果显示，样本中按照本方法的测试结果显示有1例野生型样本测定结果为突变型，2例突变型未检出，被判断为野生型，正常组中有一例突变型样本本方法未检测到，其余样本的测序结果和使用本方法测定的结果完全一致，一致性为91％。
另外，考虑到本实施例中测序所用的样本是组织提取的核酸样本，而本发明方法所用的核酸样本是来自粪便中的，其中能够提取到的人的核酸量很少，因此本发明所述方法具有高准确性和灵敏度的特点。
上述结果表明，根据本发明设计方法设计的特异性的引物探针对检测粪便样本中的Kras基因突变有高灵敏度，其结果可靠，与突变检测“金标准”：直接测序发的结果符合率为91％。而且，本发明所述的方法快速，操作简单，结果判读客观，适合于临床中大规模开展。
以上对本发明所提供的检测Kras基因突变的引物和探针及其试剂盒进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2016109631-appb-000026
Figure PCTCN2016109631-appb-000027
Figure PCTCN2016109631-appb-000028
Figure PCTCN2016109631-appb-000030
Figure PCTCN2016109631-appb-000031
Figure PCTCN2016109631-appb-000032
Figure PCTCN2016109631-appb-000033
Figure PCTCN2016109631-appb-000034
Figure PCTCN2016109631-appb-000035
Figure PCTCN2016109631-appb-000036
Figure PCTCN2016109631-appb-000037
Figure PCTCN2016109631-appb-000038

Claims (16)

  1. 一种引物组，其特征在于，包括引物，所述引物包括正向引物和反向引物；所述正向引物的序列如SEQ ID No.1～SEQ ID No.7任一所示；所述反向引物的序列如SEQ ID No.8所示。
  2. 根据权利要求1所述的引物组在制备Kras基因突变的检测试剂中的应用。
  3. 根据权利要求1所述的引物组在制备癌症和/或其他疾病检测试剂中的应用；优选的，所述癌症选自胰腺癌、结直肠癌、肺癌、卵巢癌、甲状腺癌、膀胱癌、乳腺癌、肝癌、皮肤癌或肾癌；所述其他疾病选自红斑性狼疮、类风湿性关节炎或白血病。
  4. 一种探针组合物，其特征在于，所述探针的序列如SEQ ID No.9～SEQ ID No.15任一所示；优选的，所述探针的5'端标记有荧光基团，3'端标记有对应的淬灭基团。
  5. 根据权利要求4所述的探针组合物在制备Kras基因突变的检测试剂中的应用。
  6. 根据权利要求4所述的探针组合物在制备癌症和/或其他疾病检测试剂中的应用；优选的，所述癌症选自胰腺癌、结直肠癌、肺癌、卵巢癌、甲状腺癌、膀胱癌、乳腺癌、肝癌、皮肤癌或肾癌；所述其他疾病选自红斑性狼疮、类风湿性关节炎或白血病。
  7. 一种检测试剂，其特征在于，包括如权利要求1所述的引物组和/或如权利要求4所述的探针组合物。
  8. 一种检测试剂盒，其特征在于，包括如权利要求7所述的检测试剂。
  9. 根据权利要求8所述的检测试剂盒，其特征在于，还包括扩增体系；所述扩增体系包括dNTP、镁离子、反应缓冲液、酶、无核酸酶的水。
  10. 根据权利要求9所述的检测试剂盒，其特征在于，所述扩增体系包括5～20mM的dNTP、10～40mM的镁离子、1×～10×反应缓冲液、酶、无核酸酶的水。
  11. 根据权利要求8所述检测试剂盒，其特征在于，所述扩增体系中 各组分的反应终浓度为：
    Figure PCTCN2016109631-appb-100001
  12. 根据权利要求8至11任一项所述检测试剂盒，其特征在于，扩增程序为：
    95℃300s；1个循环；
    95℃20s，62℃30s，70℃30s；10个循环；
    95℃20s，58℃60s，72℃30s；30～40个循环；
    37℃30s；1个循环。
  13. 一种Kras基因突变的检测方法，其特征在于，取样本与如权利要求1所述的引物组、如权利要求4所述的探针组合物或如权利要求7所述的检测试剂或如权利要求8至12任一项所述的检测试剂盒中的检测试剂混合后扩增，根据扩增结果获得检测结果。
  14. 根据权利要求13所述的检测方法，其特征在于，所述获得检测结果为比较待测样本与正常样本的扩增结果，当所述待测样本与正常样本的扩增结果具有显著差异或极显著差异时，所述待测样本的供体发生Kras基因突变。
  15. 一种癌症和/或其他疾病的检测方法，其特征在于，取样本与如权利要求1所述的引物组、如权利要求4所述的探针组合物或如权利要求7所述的检测试剂或如权利要求8至12任一项所述的检测试剂盒中的检测试剂混合后扩增，根据扩增结果获得检测结果；
    所述癌症选自胰腺癌、结直肠癌、肺癌、卵巢癌、甲状腺癌、膀胱癌、乳腺癌、肝癌、皮肤癌或肾癌；所述其他疾病选自红斑性狼疮、类风湿性关节炎或白血病。
  16. 根据权利要求15所述的检测方法，其特征在于，所述获得检测结果为比较待测样本与正常样本的扩增结果，当所述待测样本与正常样本的扩增结果具有显著差异或极显著差异时，所述待测样本的供体呈阳性。
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