WO2023103530A1 - 用于同时识别多种基因类型的数字扩增检测方法、检测产品和检测试剂盒 - Google Patents

用于同时识别多种基因类型的数字扩增检测方法、检测产品和检测试剂盒 Download PDF

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WO2023103530A1
WO2023103530A1 PCT/CN2022/120663 CN2022120663W WO2023103530A1 WO 2023103530 A1 WO2023103530 A1 WO 2023103530A1 CN 2022120663 W CN2022120663 W CN 2022120663W WO 2023103530 A1 WO2023103530 A1 WO 2023103530A1
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primer
detection
probe
primers
detection method
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French (fr)
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尚午
王友祥
杨志颉
张毅良
蒋正欣
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南京普济生物有限公司
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This application relates to the technical field of gene mutation detection, more specifically, it relates to a digital amplification detection method, detection product and detection kit for simultaneously identifying multiple gene mutations.
  • MLPA multiple ligation-dependent probe amplification
  • MMCA multicolor melting curve analysis
  • microfluidic technology such as Filmarray products
  • Digital PCR is a technology for absolute quantification of nucleic acid molecules, which can effectively distinguish negative signals from positive signals by dividing the reaction system. Based on this technology, scientists can obtain multiple signals with different fluorescence intensities by adjusting the concentration of different gene mutation detection probes, thereby realizing the detection of multiple gene mutations.
  • this technology is still limited by the number of primer probes in the same system: when detecting multiple gene mutations, multiple sets of primer probes need to be set, and the increase in the number of primers will increase the number of primers. The possibility of non-specific amplification products such as dimers will affect the normal combination of the probe and the sequence to be detected, reducing the accuracy and reliability of the target gene amplification detection results.
  • the present application provides a digital amplification detection method, detection products and detection kits for simultaneously identifying multiple types of gene mutations.
  • the present application provides a digital amplification detection method for simultaneously identifying multiple gene mutation types, and adopts the following technical scheme:
  • a digital amplification detection method for simultaneously identifying multiple gene mutation types comprising the following steps:
  • each target gene sequence is designed with at least two detection probes for covering the target gene sequence different regions; the bases for synthesizing the primer nucleotide sequence include four or less;
  • the small molecular compound is selected from one or more of phosphorothioate oligonucleotides, methylated oligonucleotides, peptide nucleic acids, locked nucleic acids, inosine nucleotides or rhodium metal intercalators;
  • the detection data is analyzed to obtain the site mutation types of different target gene sequences.
  • primers and probes When a variety of primers and probes are designed for PCR amplification by conventional methods, non-specific amplification (reciprocal pairing and mismatching) often occurs and non-target sequence products are generated. And in the present application, when synthesizing primer, can select four kinds of bases for use or only select three kinds of bases (missing any one in base A, base G, base C or base T), to significantly reduce Occurrence of non-specific binding upon amplification. Subsequently, a nucleotide sequence capable of forming a secondary structure is added to the 5' end of the amplified primer to reduce the probability of dimers between the primers and reduce the occurrence of non-specific amplification.
  • the scheme of this application continuously strengthens the binding strength and stability of the primer and the target gene through the special design of the primer, the addition of a nucleotide sequence that can form a secondary structure at the 5' end of the primer, and the addition of a small molecular compound. Significantly reduce the generation of non-specific amplification, enabling multiplex PCR to be realized.
  • the addition of the small molecular compound is that, especially when the nucleotide sequence of the synthetic primer contains only three bases, phosphorothioate oligonucleotides, methylated oligonucleotides, peptide nucleic acids, One or more of locked nucleic acid, inosine nucleotide or rhodium metal intercalator is used as a substitute for the missing base to improve the stability of the primer and the binding stability of the primer to the target gene.
  • the peptide nucleic acid (PNA) in it can recognize and combine with DNA or RNA sequence through Watson-Crick base pairing to form a stable double helix structure.
  • Locked nucleic acid (LNA) is a special bicyclic nucleotide derivative.
  • LNA and DNA/RNA have the same phosphate backbone in structure, so it has good recognition ability and strong affinity for DNA and RNA .
  • Inosine nucleotides or deoxyinosine can also be used as universal bases to replace missing bases. Therefore, when the small molecule compound is one or more of peptide nucleic acid, locked nucleic acid, inosine nucleotide and deoxyinosine, it can be used as a universal base to replace the missing base during primer synthesis, In order to improve the binding stability of the primer and the target gene.
  • the probe drop-off technology in this application at least two probes are set for each target gene, and there is no reference probe in the set probes; multiple different probes are combined with the target gene, and coverage of the target gene may occur different regions of the mutation to achieve comprehensive detection of the mutation site of the target gene, thereby enhancing the efficiency of probe use, and realizing the simultaneous detection of multiple mutation types in the target gene in one detection, making the reagent design more efficient and the process more convenient. The cost is lower.
  • the nucleotide sequence of the synthesized primer contains three bases and does not include base G and base C at the same time.
  • the small molecular compound is connected to the primer by additional addition;
  • S12 includes the following steps: adding a nucleotide sequence that can form a three-dimensional structure to the 5' end of the primer to obtain an initial modified primer;
  • the initial modified primers are added to the buffer, and the buffer includes small molecular compounds, and react to obtain primers for PCR amplification.
  • the buffer includes a reaction solution and a stop solution.
  • the reaction solution can be selected such as [Rh(NH 3 ) 4 (phen)][OTf] 3 (0.500g, 0.626mmol), chrysene-5,6-dione (0.162g, 0.626 mmol), 1:9H 2 O:MeCN (400mL), 1M solution of NaOH (1.5mL); the stop solution can choose 1M solution of HCl (1.5mL); "PCR-introduced loop structure as primer in DNA sequencing.” Biotechniques 25.5 (1998): 876-884.”
  • the concentration of the small molecule compound in the buffer is 4-12 ⁇ M, and the molar ratio of the small molecule compound to the initial modified primer is 0.7-1:1.
  • the small molecular compound is added to the primer after being added to the buffer.
  • Its further adding method includes: first, when preparing primers for PCR amplification, synthesize primers; then connect a section of nucleotide sequence that can form a three-dimensional structure to the 5' end of the primers to obtain initial modified primers; finally, obtain After mixing the initial modified primers with the buffer solution containing small molecular compounds, the purpose of adding small molecular compounds to the initial modified primers is realized, and finally the primers for PCR amplification are obtained.
  • the purpose of adding the small molecular compound is to enhance the binding strength between the primer and the template binding region. In this scheme, by adding an appropriate amount of small molecule compounds, the excellent effect of reducing the probability of mismatch and increasing the binding strength can be achieved.
  • the sample DNA includes cfDNA.
  • the length of the cfDNA is 150-200bp; one embodiment of the scheme of the present application can realize efficient and accurate detection of gene mutations on short-chain nucleotide fragments.
  • the length of the nucleotide sequence of the probe is 10-30 bp.
  • the nucleotide sequence of the probe is shorter, the efficiency of PCR amplification is higher and the possibility of base mismatch is significantly reduced; in addition, the shorter probe can promote fluorescein and The quenching groups are close enough to reduce the background fluorescence, thus making the detection results more accurate; at the same time, shorter probes are more suitable for detecting small fragments of cfDNA in blood.
  • the PCR reaction system does not include cytosine deoxynucleotide triphosphate.
  • the concentration of each primer for PCR amplification is 7-12 ⁇ M, and the concentration of each probe is 7-12 ⁇ M.
  • step S12 first heat to 50-80° C., and then keep it warm; then lower the temperature to 28-32° C., and keep it warm.
  • the multiple probes do not include a reference probe.
  • an embodiment of the present application provides a detection product for detecting multiple genotypes using the above detection method.
  • an embodiment of the present application provides a detection kit for detecting multiple genotypes using the above detection method.
  • the detection kit includes the primer-probe mixture; the primer-probe mixture contains the primers for PCR amplification and the probe; each target gene corresponds to at least two of the probe.
  • This application is based on digital PCR technology.
  • On the basis of conventional synthetic primers only three bases are selected first to significantly reduce non-specific binding during amplification; and nucleosides that can form three-dimensional structures are added to the 5' end of the primers.
  • the acid sequence and the introduction of small molecular compounds can significantly reduce the dimer between primers, reduce non-specific amplification, and can improve the binding specificity, binding strength and binding stability of primers to templates, and can realize multiple amplification in a single reaction system. increase.
  • probes based on the double-probe drop-off technique, at least two probes are set for each target gene, and the set probes do not contain reference probes; multiple different probes bind to the target gene, and cover Different regions of the target gene to achieve comprehensive detection of the mutation site of the target gene, so that multiple genotypes or gene mutations of the target gene can be detected simultaneously through one detection, and the detection process is more efficient
  • the length of the nucleotide sequence of the selected probe is 10-30 bp, and the nucleotide sequence of the probe is relatively short, so that the efficiency of PCR amplification is higher and the possibility of base mismatch is significantly reduced; in addition , the background fluorescence is relatively weak during detection, which makes the detection result more accurate; and the short probe is more suitable for detecting small fragments of cfDNA in blood.
  • Fig. 1 is the schematic diagram of the principle of mutation detection by the technical solution of the present application.
  • Figure 2 is a cluster analysis diagram of the KRAS mutation site of the lung cancer gene locus detected in Example 1 of the present application;
  • FIG. 3 is a cluster analysis diagram of colorectal cancer gene loci PIK3CA and BRAF mutation sites detected in Example 4 of the present application.
  • Fig. 4 is a graph showing the detection results of the cell line positive samples in Example 6 of the present application.
  • the present application designs at least two probes, multiple different probes all bind to the target gene and cover different regions of the target gene, so as to achieve comprehensive detection of the mutation site of the target gene.
  • clusters with different fluorescent colors appear at different positions on the cluster analysis graph: the abscissa and ordinate correspond to the fluorescent clusters of different mutant genes appearing on different channels; On the diagonal are clusters of fluorescence corresponding to wild-type genes.
  • Example 1 Digital amplification detection method and detection kit for various gene mutations related to lung cancer
  • a digital amplification detection method for simultaneously identifying multiple gene mutations for lung cancer comprising the following steps:
  • Enrolled cases Patients with pulmonary nodules who visited the Thoracic Surgery Department of Shanghai Zhongshan Hospital from January 2020 to December 2020 were studied. According to the results of chest CT and blood lung cancer tumor indicators (including KRAS), it was not possible to determine whether they were lung cancer patients. Or lung disease. All patients chose to receive surgical treatment, and 50 mL of peripheral venous blood was collected before the operation. According to the postoperative pathological results of the tumor, the samples were divided into lung cancer and lung benign disease groups (lung benign diseases include inflammatory pseudotumor, sclerosing hemangioma, tuberculosis and tuberculosis). wait).
  • lung benign diseases include inflammatory pseudotumor, sclerosing hemangioma, tuberculosis and tuberculosis). wait).
  • the cfDNA was obtained using the cfDNA extraction kit (QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA)).
  • the KRAS mutation gene and mutation site are shown in Table 1.
  • the KRAS gene 61 Detection of mutation sites.
  • KRAS gene mutations were found in the peripheral blood cfDNA of lung cancer patients, and the mutated genes included the mutation sites described in Table 1. Specific site mutations in the KRAS gene can provide references for clinical diagnosis and medication guidance for lung cancer patients, and provide more reasonable and effective individualized guidance for patients with pulmonary nodules that are difficult to diagnose with imaging and tumor indicators.
  • each target gene is designed with at least two detection probes; synthesize the nucleotides of the primers
  • the sequence includes only three bases and does not include base A and base T at the same time.
  • primers and probes according to the design principles of primers and probes (such as the literature “Bustin, Stephen A., Reinhold Mueller, and Tania Nolan.” Parameters for successful PCR primer design.” Quantitative Real-Time PCR. Humana, New York, NY, 2020.5-22.”), according to the human KRAS wild-type gene sequence published by Cosmic data, and based on the KRAS mutation site, specific primers and probes were designed, as shown in Table 2.
  • the 5' end of the mutant fluorescent probe is connected with a fluorescent reporter group FAM; the 3' end is connected with a fluorescent quencher group BHQ1; the wild-type fluorescent probe is connected with a fluorescent reporter group HEX at the 5' end; the 3' end is connected with a fluorescent Quenching group BHQ2.
  • Fluorescent reporter groups and quenchers can also be reasonably selected according to specific platforms.
  • Primer probe name Primer Probe Sequence serial number KRAS-F1 CCGCCGCGCGGCCGCCGCCT SEQ ID NO.1 KRAS-R1 CGCCGCCGCGTCCGCGC SEQ ID NO.2 KRAS-F2 CCCGCCCCTCCGGGG SEQ ID NO.3 KRAS-R2 CCGGGGCGCCGCGGG SEQ ID NO.4 KRAS-P1 TGCCGCCGCCGCTGCTGCCT SEQ ID NO.5 KRAS-P2 CGCCACCTTCGCCG SEQ ID NO.6 KRAS-P3 GTACCTCTCTCCC SEQ ID NO.7 KRAS-P4 GGAAGCAGCA SEQ ID NO.8
  • the primers are modified and ready for use, and the modification includes the following steps:
  • the components of the buffer solution are: 290mM NaCl, 4.5mM MgCl 2 , 18mM Tris (pH 7.3).
  • the buffer solution also includes 10 ⁇ M small molecule compound, the small molecule compound is a rhodium metal intercalator, and the molar ratio of the rhodium metal intercalator to the initial modified primer is 0.7:1.
  • the rhodium metal structural formula used in this example is [RhCl 3 (C10H 8 N 2 ) ⁇ (CH 3 ) 2 SO ⁇ ](CH 3 ) 2SO, and its synthesis method can be found in the literature "Caruso, Francesco, et al.” Synthesis , structure, and antitumor activity of a novel tetranuclear titanium complex.”Journal of medicinal chemistry 43.20(2000):3665-3670.”.
  • S13 will be used to detect the upstream primer of the site shown in Table 1, the downstream primer (i.e. the PCR amplification primer in S122) and be used to detect the fluorescent probe 1 and the fluorescent probe 2 of the site shown in Table 1 ( That is, the probes synthesized in S11) were dissolved together in TE solution to prepare a primer-probe mixed solution.
  • concentrations of the upstream primer, downstream primer, fluorescent probe 1 and fluorescent probe 2 in the primer-probe mixture were all 10 ⁇ M.
  • the preparation method of the primer-probe mixture is as follows: Dilute the dry powder of the four components of the upstream primer, the downstream primer, the fluorescent probe 1 and the fluorescent probe 2 with TE buffer solution to a concentration of 100 ⁇ M for each probe and primer respectively. .
  • the PCR reaction system including the sample DNA and the primer-probe mixture.
  • the PCR reaction system was prepared according to Table 3.
  • PCR Mix was purchased from NEB, and added with a final concentration of 0.1% Triton-X-100, 1U thermostable pyrophosphatase, 5 ⁇ g/ ⁇ L BSA, according to ddH 2 O, PCR mix, probes, primers, template DNA Sequentially, add the above samples into a 0.2mL PCR tube according to the addition amount of 20 ⁇ L in the reaction system in Table 3, mix the mixed system for 15 seconds by gentle vortexing, and collect the solution to the bottom of the test tube by short-time centrifugation. Load the prepared reaction systems with different proportions onto the PCR chip to form a micro-reaction unit. Put the chip into a digital PCR instrument, and perform PCR reaction according to the PCR reaction conditions in Table 4.
  • the PCR Mix does not contain the cytosine deoxynucleotide triphosphate component.
  • Figure 2 is a diagram of the detection results of clinical samples (that is, the lung cancer gene locus Cluster analysis of KRAS mutation sites), the ordinate is the FAM fluorescence channel, and the abscissa is the HEX fluorescence channel. From the analysis of the experimental results, it can be seen that the corresponding signals can be detected through the cluster analysis, corresponding to all 61 gene loci shown in Table 1.
  • the mutation abundance of the target gene group can be calculated by dividing the mutation signal (MUT) by the corresponding wild-type signal (WT). Further, the result of comparing this embodiment with the results of existing methodology (NGS) found that a total of 143 cases of clinical blood sample detection (each case has been tested by NGS) were completed through this embodiment, and the results were consistent with the results of NGS There are 133 cases of positive and 10 cases of inconsistent, the consistency rate is 93%.
  • a detection kit for simultaneously identifying multiple gene mutations for lung cancer including primers and probes for PCR amplification, TE buffer and PCR mixture.
  • the PCR mixture was purchased from NEB, and added with a final concentration of 0.1% Triton-X-100, 1U thermostable pyrophosphatase, 5 ⁇ g/ ⁇ L of BSA, and PCR mix, which did not include cytidine deoxynucleoside triphosphate Acid component.
  • the test kit also includes a positive quality control and a negative quality control.
  • the preparation method of the positive quality control product is as follows: each 200 bp of the wild-type and mutant-type sequences of each gene mutation site shown in Table 1, after synthesis, are respectively loaded into the plasmid vector pET-23d (+) (Promega). Use Qubit 3.0 for quantification, calculate the copy number concentration of the two types of plasmids, according to the copy number ratio 1:3000, 1:2000, 1:1000, 1:500, 1:200, 1:100, 1:50, 1: 10 Mix the two plasmids, and then break the plasmid mixture into fragments of about 180bp by ultrasound, quantify to 20ng/ ⁇ L, and use it as a gradient positive quality control.
  • the negative quality control product is composed of the above-mentioned plasmid containing wild type, and then cut into fragments of about 180bp by the same method, and quantified to 20ng/ ⁇ L, as the negative quality control product.
  • Example 2 Digital amplification detection method and detection kit for various gene mutations related to lung cancer
  • Digital amplification detection method for simultaneously identifying multiple gene mutations for lung cancer The difference between this example and Example 1 is that the selected small molecular compound is directly added to the primer, that is, the selected primer is Obtained after modification of locked nucleic acid. In addition, in step S122, no small molecular compound is added to the buffer.
  • the locked nucleic acid is commercially available, and may be a locked nucleic acid oligochain synthesized from Beijing Saibaisheng Gene Technology Co., Ltd.
  • a detection kit for the simultaneous identification of multiple gene mutations for lung cancer is provided.
  • Example 1 The difference between this example and Example 1 is that the primers for PCR amplification are prepared in this example, and the others are the same as Example 1.
  • Example 3 Digital amplification detection method and detection kit for various gene mutations related to lung cancer
  • the difference between this example and Example 1 is that the small molecule compound is peptide nucleic acid (purchased from Beijing Saibaisheng Gene Technology Co., Ltd.), and the peptide nucleic acid is added in step S2, specifically: in the PCR Mix, add the final Peptide nucleic acid at a concentration of 0.35 ⁇ M. And in step S122, the small molecule compound is no longer added to the buffer.
  • the small molecule compound is peptide nucleic acid (purchased from Beijing Saibaisheng Gene Technology Co., Ltd.)
  • the peptide nucleic acid is added in step S2, specifically: in the PCR Mix, add the final Peptide nucleic acid at a concentration of 0.35 ⁇ M.
  • step S122 the small molecule compound is no longer added to the buffer.
  • Example 1 The difference between this example and Example 1 is that the primers for PCR amplification are prepared in this example, and the others are the same as Example 1.
  • the method can also be used to obtain accurate detection of lung cancer-related mutation genes (such as the 61 mutation sites of the KRAS gene shown in Table 1).
  • Example 4 Digital amplification detection method and detection kit for various gene mutations related to colorectal cancer
  • a digital amplification detection method for simultaneously identifying multiple gene mutations for colorectal cancer comprising the following steps:
  • the cfDNA was obtained using the cfDNA extraction kit (QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA)).
  • PIK3CA and BRAF mutation genes and mutation sites are shown in Table 5.
  • Table 5 In the reaction system, through digital PCR, and the special primers and buffers used in this embodiment, PIK3CA and BRAF mutations can be achieved. Detection of a total of 41 mutation sites in 2 genes such as BRAF.
  • PIK3CA and BRAF gene mutations were found in the peripheral blood ctDNA of colorectal cancer patients, and the mutation genes contained the mutation sites described in Table 5, while no mutations were found in benign tumors.
  • Specific site mutations in PIK3CA and BRAF genes can provide references for the diagnosis and differential diagnosis of colorectal cancer, and provide more reasonable and effective individualized guidance for colorectal patients whose imaging and tumor indicators are difficult to diagnose.
  • the primers and probes for the target gene loci shown in Table 6 above were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis.
  • the primers are modified and ready for use, and the modification includes the following steps:
  • the components of the buffer solution are: 300mM NaCl, 5mM MgCl 2 , 20mM Tris (pH 7.6).
  • the components of the buffer solution also include 5 ⁇ M small molecular compound, the small molecular compound is a methylated oligonucleotide (purchased from GenScript Biotechnology Co., Ltd.), and the methylated oligonucleotide and the initial modified primer The molar ratio is 0.8:1.
  • S13 will be used to detect the upstream primer of the site shown in Table 5, the downstream primer (i.e. the PCR amplification primer in S122) and be used to detect the mutant fluorescent probe of the site shown in Table 5, the wild-type fluorescent probe
  • the needles that is, the probes synthesized in S11
  • the concentration of each upstream primer, downstream primer, and fluorescent probe in the primer-probe mixture is 10 ⁇ M.
  • the preparation method of the primer-probe mixture is as follows: Dilute the dry powder of the four components of the upstream primer, the downstream primer, the fluorescent probe 1 and the fluorescent probe 2 with TE buffer solution to a concentration of 100 ⁇ M for each probe and primer respectively. .
  • the PCR reaction system includes sample DNA and primer-probe mixture.
  • the PCR reaction system was prepared according to Table 7.
  • PCR Mix was purchased from NEB, and added with a final concentration of 0.1% Triton-X-100, 1U thermostable pyrophosphatase, 5 ⁇ g/ ⁇ L BSA, according to ddH 2 O, PCR mix, probes, primers, template DNA Sequentially, add the above samples into a 0.2 mL PCR tube according to the amount of 20 ⁇ L added in the reaction system in Table 7, mix the mixed system for 15 s with gentle vortex, and collect the solution to the bottom of the test tube by short-time centrifugation. Load the prepared reaction systems with different ratios onto the PCR chip to form a micro-reaction unit. Put the chip into a digital PCR instrument, and perform a PCR reaction according to the PCR reaction conditions in Table 8.
  • the PCR Mix does not contain deoxycytosine triphosphate (dCTP) components.
  • dCTP deoxycytosine triphosphate
  • Figure 3 is a diagram of the detection results of clinical samples (i.e. colorectal cancer gene locus PIK3CA and cluster analysis of BRAF mutation sites), the ordinate is the FAM fluorescence channel, and the abscissa is the HEX fluorescence channel. From the analysis of the experimental results, it can be seen that the corresponding signals can be detected through cluster analysis, corresponding to all 41 gene loci shown in Table 5.
  • the mutation abundance of the target gene group can be calculated by dividing the mutation signal (MUT) by the corresponding wild-type signal (WT). Using the above method, 108 cases of clinical blood samples were tested (each case was tested by NGS), of which 107 cases were consistent with NGS results, and 2 cases were inconsistent (because the panel design was different), the consistency rate was 98% .
  • the applicant also detects the gene site mutations shown in Table 5 on DNA from serum, plasma, peripheral blood, pleural effusion, body fluid or tissue, and the repeatability is good.
  • sensitive detection of colorectal cancer gene mutation sites can be realized without non-specific amplification and good specificity.
  • a variety of primers can be added for detection, which can effectively save the substrate in the reaction process.
  • a detection kit for simultaneous identification of multiple gene mutations for rectal cancer including a primer-probe mix and a PCR mix.
  • the primer probe mix includes buffer, probe in buffer, and original modified primers in buffer.
  • the components of the buffer solution are: 300 mM NaCl, 5 mM MgCl 2 , 20 mM Tris (pH 7.6), and the components of the buffer solution also include 5 ⁇ M small molecule compounds.
  • the small molecule compound is a locked nucleic acid, and the molar ratio of the locked nucleic acid to the initial modified primer is 0.8:1, and the content of each initial modified primer and each probe in the buffer is 10 ⁇ M.
  • the PCR mixture was purchased from NEB, and added with a final concentration of 0.1% Triton-X-100, 1U thermostable pyrophosphatase, 5 ⁇ g/ ⁇ L of BSA, and PCR mix, which did not include cytidine deoxynucleoside triphosphate Acid component.
  • the kit also includes positive and negative controls.
  • the preparation method of the positive quality control product is: each 200bp of the wild-type and mutant-type sequences of each gene mutation site shown in Table 5, after synthesis, were respectively loaded into the plasmid vector pET-23d(+) (Promega).
  • Use Qubit 3.0 for quantification calculate the copy number concentration of the two types of plasmids, according to the copy number ratio 1:3000, 1:2000, 1:1000, 1:500, 1:200, 1:100, 1:50, 1: 10
  • the negative quality control product is composed of the above-mentioned plasmid containing wild type, and then cut into fragments of about 180bp by the same method, and quantified to 20ng/ ⁇ L, as the negative quality control product.
  • Example 5 Digital amplification detection method and detection kit for various gene mutations related to colorectal cancer
  • step S121 the nucleotide sequence that can form a three-dimensional structure is added to the 5' end of the designed primer, which is different from Example 4.
  • the nucleotide sequence added in this example is "ACTCATCTGTGAGACTCACTATAGGAAGAGATGTCAACTCGTGCACGAGTTGACATCTCTTCTCCGAGCCGGTCGAAATATTGGAGGAAGCTCGAGCTGGAGGAAAAGTGAGTCTCACAGATGAGT (as shown in SEQ ID NO.22)" obtains the initial modified primer for amplification.
  • Example 4 After performing PCR amplification, it was found that when the nucleotide sequence capable of forming a three-dimensional structure was added to the 5' end of the designed primer, when the number of cycles was 25 times, an obvious Non-specific amplification; and when carrying out PCR amplification with the scheme of embodiment 4, when cycling at least 120 times, still no non-specific amplification occurs.
  • Example 6 Digital amplification detection method and detection kit for various gene mutations related to esophageal phosphorus carcinoma
  • a digital amplification detection method for simultaneously identifying multiple gene mutations for esophageal cancer comprising the following steps:
  • sample DNA use the purchased esophageal phosphocarcinoma cell line KYSE270 as a negative sample, use the purchased TP53 mutant cell line as a positive sample, and use a genomic DNA extraction kit (QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) )) to obtain genomic DNA.
  • QIAamp DNA Mini Kit Qiagen, Valencia, CA, USA
  • the primers and probes using the DNA sequence corresponding to the above-mentioned TP53 mutant gene as a template, use the Primer3web (version 4.1.0) online design website to design primers and probes covering the target gene loci shown in Table 9. pins, as shown in Table 10.
  • the 5' end of the mutant fluorescent probe is connected with a fluorescent reporter group FAM; the 3' end is connected with a fluorescent quencher group BHQ1; the wild-type fluorescent probe is connected with a fluorescent reporter group CY3 at the 5' end; the 3' end is connected with a fluorescent Quenching group BHQ2.
  • Fluorescent reporter groups and quenchers can also be reasonably selected according to specific platforms.
  • Primer probe name Primer Probe Sequence serial number TP53-F1 TGGGTGACAGAGTGAGACTTC SEQ ID NO.23 TP53-R1 ACCCGGCCCTCATTATTTCT SEQ ID NO.24 TP53-F2 CCAACATGGTGAAACCCCAA SEQ ID NO.25 TP53-R2 CCGAAGTGCTGGGATTACA SEQ ID NO.26 TP53-F3 GGCACCTCTAATCCCAGCTA SEQ ID NO.27 TP53-R3 CCTCAGCCTCCCGAAGTG SEQ ID NO.28 TP53-P1 ACGTTGATACCATACTGGAGGT SEQ ID NO.29 TP53-P2 AGGATTTGACTCTCA SEQ ID NO.30 TP53-P3 TACTCAGGAGGCTGAGGCAGGAGAA SEQ ID NO.31 TP53-P4 TTGCTTGGACCTGGGAGGCAGAGGT SEQ ID NO.32
  • TP53-P5 GGAGGCAGAGGTTGCAGTGAG SEQ ID NO.33
  • TP53-P6 GAGATCGCGCTATTGCACTC SEQ ID NO.34
  • the primers and probes for the target gene loci shown in Table 10 above were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis.
  • the primers are modified and ready for use, and the modification includes the following steps:
  • the components of the buffer solution are: 300mM NaCl, 5mM MgC1 2 , 20mM Tris (pH 7.6), and the components of the buffer solution also include a 5 ⁇ M small molecule compound; the small molecule compound is phosphorothioate oligonucleotide ( purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.), and the molar ratio of phosphorothioate oligonucleotides and initial modified primers was 0.6:1.
  • the 5' end sequence of the detected nucleotide sequence has a three-dimensional structure.
  • S13 will be used to detect the upstream primer of the site shown in Table 9, the downstream primer (i.e. the PCR amplification primer in S122) and be used to detect the mutant fluorescent probe of the site shown in Table 9, the wild-type fluorescent probe
  • the needles that is, the probes synthesized in S11
  • the concentration of each upstream primer, downstream primer, and fluorescent probe in the primer-probe mixture is 10 ⁇ M.
  • the preparation method of the primer-probe mixture is as follows: Dilute the dry powder of the four components of the upstream primer, the downstream primer, the fluorescent probe 1 and the fluorescent probe 2 with TE buffer solution to a concentration of 100 ⁇ M for each probe and primer respectively. .
  • the PCR reaction system includes sample DNA and primer-probe mixture.
  • the PCR reaction system was prepared according to Table 11.
  • PCR Mix was purchased from NEB, and added with a final concentration of 0.1% Triton-X-100, 1U thermostable pyrophosphatase, 5 ⁇ g/ ⁇ L BSA, according to ddH 2 O, PCR mix, probes, primers, template DNA Sequentially, add the above samples into a 0.2mL PCR tube according to the addition amount of 20 ⁇ L in the reaction system in Table 11, mix the mixed system for 15 seconds by gentle vortexing, and collect the solution to the bottom of the test tube by short centrifugation. Load the prepared reaction systems with different ratios onto the PCR chip to form a micro-reaction unit. Put the chip into a digital PCR instrument, and perform a PCR reaction according to the PCR reaction conditions in Table 12.
  • the PCR Mix does not contain deoxycytosine triphosphate (dCTP) components.
  • dCTP deoxycytosine triphosphate
  • Figure 4 is a diagram of the detection results of cell line positive samples (ie, esophageal squamous cells) Cluster analysis of cancer gene loci TP53 mutation sites), the ordinate is the FAM fluorescence channel, and the abscissa is the HEX fluorescence channel. From the analysis of the experimental results, it can be seen that the corresponding signals can be detected through cluster analysis, corresponding to all 293 gene loci shown in Table 9. The mutation abundance of the target gene group can be calculated by dividing the mutation signal (MUT) by the corresponding wild-type signal (WT).
  • MUT mutation signal
  • WT wild-type signal
  • the applicant also detects the gene site mutations shown in Table 9 on DNA from serum, plasma, peripheral blood, pleural effusion, body fluid or tissue, and the repeatability is good.
  • sensitive detection of esophageal squamous cell carcinoma gene mutation sites can be realized, without non-specific amplification, and with good specificity.
  • a variety of primers can be added for detection, which can effectively save the substrate in the reaction process.
  • a detection kit for simultaneously identifying multiple gene mutations for esophageal squamous cell carcinoma including a primer-probe mixture and a PCR mixture.
  • the primer probe mix includes buffer, probe in buffer, and original modified primers in buffer.
  • the composition of the buffer solution is: 300 mM NaCl, 5 mM MgCl 2 , 20 mM Tris (pH 7.6).
  • the components of the buffer solution also include 5 ⁇ M small molecule compound, the small molecule compound is phosphorothioate oligonucleotide, and the molar ratio of phosphorothioate oligonucleotide and initial modified primer is 0.6:1, each initial modified primer and The content of each probe in the buffer was 10 ⁇ M.
  • the PCR mixture was purchased from NEB, and added with a final concentration of 0.1% Triton-X-100, 1U of thermostable pyrophosphatase, 5 ⁇ g/ ⁇ L of BSA, and PCR mix, which did not include cytosine deoxynucleoside triphosphate Acid component.
  • the kit also includes positive and negative controls.
  • the preparation method of the positive quality control product is: each 200bp of the wild-type and mutant-type sequences of each gene mutation site shown in Table 9, after synthesis, respectively loaded into the plasmid vector pET-23d (+) (Promega).
  • Use Qubit 3.0 for quantification calculate the copy number concentration of the two types of plasmids, according to the copy number ratio 1:3000, 1:2000, 1:1000, 1:500, 1:200, 1:100, 1:50, 1: 10
  • the negative quality control product is composed of the above-mentioned plasmid containing wild type, and then cut into fragments of about 180bp by the same method, and quantified to 20ng/ ⁇ L, as the negative quality control product.

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Abstract

本申请公开了用于同时识别多种基因类型的数字扩增检测方法、检测产品和检测试剂盒，涉及基因检测技术领域；该检测方法包括：S1、提取得到样本DNA；制备引物探针混合液，包括：合成引物和探针，每个目标基因至少设计两个结合目标基因的探针，引物上修饰有小分子化合物和能够形成二级结构的核苷酸序列，得到引物探针混合液；S2、进行PCR反应；S3、对检测数据进行分析，得到不同位点的基因类型。

Description

用于同时识别多种基因类型的数字扩增检测方法、检测产品和检测试剂盒 技术领域
本申请涉及基因突变检测的技术领域，更具体地说，它涉及一种用于同时识别多种基因突变的数字扩增检测方法、检测产品和检测试剂盒。
背景技术
多重基因检测是行业内的难题，目前大多数技术只能实现有限重基因突变的检测。近些年，部分技术可以做到十多重基因突变的检测，比如多重连接依赖式探针扩增技术(multiplex ligation-dependent probe amplification，MLPA)、多色熔解曲线分析技术(multicolor melting curve analysis，MMCA)和微流控技术(如Filmarray产品)等，但是这些检测手段都存在着不同的技术问题。其中的MLPA技术，因探针设计困难费时、试剂昂贵、需要和毛细管电泳等复杂仪器配合使用等，使得其应用受限。其中的微流控技术则需要借助微流控相关的复杂仪器，而且其检测灵敏度不能满足实际应用的要求。
在解决这些行业难题的过程中，一些研究者开始尝试采用数字PCR技术来辅助提高基因突变检测的准确度和信号数。数字PCR是一种核酸分子绝对定量的技术，可以通过分割反应体系的方式实现阴性信号与阳性信号的有效区分。基于这个技术，科学家们可以通过调节不同基因突变检测探针的浓度，得到不同荧光强度的多个信号，从而实现对多重基因突变的检测。但是，将该技术用于多重基因突变的检测时，其依然受到同一体系内引物探针数量的限制：当检测多重基因突变时，需要设置多组引物探针，而引物数量的增加会增加引物二聚体等非特异性扩增产物产生的可能性，并影响探针与待检测序列的正常结合，降低目标基因扩增检测结果的准确性和可靠性。
因此，亟需开发一种新的超多重基因突变的检测方法，来克服现有技术存在的难题。
发明内容
为了进一步实现超多重基因突变的准确检测，本申请提供一种用于同时识别多种基因突变类型的数字扩增检测方法、检测产品和检测试剂盒。
第一方面，本申请提供了用于同时识别多种基因突变类型的数字扩增检测方法，采用如下的技术方案：
用于同时识别多种基因突变类型的数字扩增检测方法，包括以下步骤：
S1、提取得到样本DNA，备用；
制备得到引物探针混合液，包括以下步骤：
S11、合成目标基因序列扩增和检测用的引物和探针，所述引物和探针为多个；每个所述目标基因序列至少设计有两个检测用探针，用于覆盖目标基因序列的不同区域；合成所述引物核苷酸序列的碱基包含四种或者四种以下；
S12、使得所述引物的5’端添加有能够形成二级结构的核苷酸序列，且使得所述引物上修饰有小分子化合物，即得到PCR扩增用引物；
所述小分子化合物选自硫代磷酸寡核苷酸、甲基化寡核苷酸、肽核酸、锁核酸、次黄嘌呤核苷酸或铑金属插入剂中的一种或几种；
S13、将所述PCR扩增用引物和所述探针共同溶解于核酸溶解用缓冲液中，得到引物探针混合液；
S2、配制PCR反应体系后进行PCR反应，所述PCR反应体系包括所述样本DNA和所述引物探针混合液；进行PCR反应；
S3、PCR反应结束后对检测数据进行分析，得到不同目标基因序列的位点突变类型。
以常规方法设计多种引物和探针用于PCR扩增时，常发生非特异性扩增(交互配对和错配)并产生非目标序列产物。而本申请中，在合成引物时，可以选用四种碱基或者仅仅选用三种碱基(缺失碱基A、碱基G、碱基C或者碱基T中的任意一种)，以显著降低扩增时的非特异性结合的发生。随后在扩增得到的引物的5’端加入能够形成二级结构的核苷酸序列，降低引物之间产生二聚体的 概率，减少非特异性扩增的发生。本申请的方案，通过对引物的特殊设计、结合在引物的5’端加入可形成二级结构的核苷酸序列、并加入小分子化合物，不断加强引物和目标基因的结合强度和稳定性，显著降低非特异性扩增的产生，使多重PCR得以实现。
其中的小分子化合物的添加作用在于，尤其是当合成所述引物的核苷酸序列仅仅含有三种碱基时，以硫代磷酸寡核苷酸、甲基化寡核苷酸、肽核酸、锁核酸、次黄嘌呤核苷酸或铑金属插入剂中的一种或多种作为所缺失碱基的替代，以提高引物的稳定性以及引物与目标基因的结合稳定性。其中的肽核酸(PNA)可以通过Watson-Crick碱基配对的形式识别并结合DNA或RNA序列，形成稳定的双螺旋结构。锁核酸(LNA)是一种特殊的双环状核苷酸衍生物，LNA与DNA/RNA在结构上具有相同的磷酸盐骨架，故其对DNA、RNA有很好的识别能力和强大的亲和力。次黄嘌呤核苷酸或脱氧次黄嘌呤也可以作为通用碱基，用以替代缺失碱基。因此，当小分子化合物为肽核酸、锁核酸、次黄嘌呤核苷酸以及脱氧次黄嘌呤中的一种或多种时，可以用作通用碱基，在引物合成时替代缺失的碱基，以提高引物和目的基因的结合稳定性。
而在进行探针设计时，本申请基于双探针脱落技术(参见文献“Bidshahri,Roza,et al."Quantitative detection and resolution of BRAF V600status in colorectal cancer using droplet digital PCR and a novel wild-type negative assay."The Journal of Molecular Diagnostics 18.2(2016):190-204.”所公开的方法)。现有的单探针脱落技术中，需要设计两个探针：脱落探针和参考探针，其中的脱落探针仅仅和野生型序列互补并覆盖可能发生突变的位点序列，参考探针用于和突变位点的邻近位点进行杂交并且与突变和野生型等位基因进行互补，从而实现对不同基因型的识别检测。但是，单探针脱落技术中的两个探针，仅仅有一个探针用于识别野生型基因，使得检测效率低。
因此，本申请中的探针脱落技术中，每个目标基因设置至少两个探针，且设置的探针中没有参照探针；多个不同探针均结合目标基因，且覆盖目标基因可能发生突变的不同区域，以实现对目标基因突变位点的全面检测，从而增强探针使用效率，一次检测就实现对目标基因中多种突变类型的同时检测，使得试剂设计更加高效、工艺更加简便、成本更低。
可选的，合成所述引物的核苷酸序列包含三种碱基且不同时包括碱基G和碱基C。
可选的，所述小分子化合物通过额外添加的方式和所述引物连接；S12包括以下步骤：所述引物的5’端添加可形成三维结构的核苷酸序列，得到初始修饰引物；将所述初始修饰引物加入到缓冲液中，所述缓冲液包括小分子化合物，反应，得到PCR扩增用引物。该缓冲液包括反应液和停止液，反应液可以选择如[Rh(NH 3) 4(phen)][OTf] 3(0.500g,0.626mmol)、chrysene-5,6-dione(0.162g,0.626mmol)、1:9H 2O:MeCN(400mL)、1M solution of NaOH(1.5mL)；停止液可以选择1M solution of HCl(1.5mL)；上述“引物的5’端添加可形成三维结构的核苷酸序列”为本领域常规方法，如文献“Ronaghi,Mostafa,et al."PCR-introduced loop structure as primer in DNA sequencing."Biotechniques 25.5(1998):876-884.”所公开。
可选的，所述小分子化合物在所述缓冲液中的浓度为4～12μM，所述小分子化合物和初始修饰引物的摩尔比为0.7～1:1。
通过采用上述技术方案，当选择的小分子化合物为铑金属插入剂时，小分子化合物是以加入缓冲液中后再添加至引物上。其进一步的添加方法包括：首先，在制备PCR扩增用引物时，合成引物；随后在该引物的5’端连接一段可以形成三维结构的核苷酸序列后得到初始修饰引物；最后通过将得到的初始修饰引物和含有小分子化合物的缓冲液混合后，实现在初始修饰引物上添加小分子化合物目的，最终得到PCR扩增用引物。小分子化合物的添加目的为增强引物和模板结合区的结合强度。在该方案中，通过添加适量的小分子化合物，以达到优异的降低错配概率和增加结合强度的效果。
可选的，所述样本DNA包括cfDNA。
一般来讲，所述cfDNA的长度为150-200bp；本申请的一个实施例方案能够实现对短链核苷酸片段上基因突变的高效准确的检测。
可选的，所述探针的核苷酸序列长度为10～30bp。
通过采用上述技术方案，且探针的核苷酸序列较短，使得PCR扩增的效率更高且使得碱基错配的可能性显著降低；此外，较短的探针，能够促使荧光素和淬灭基团足够接近，以减少背景荧光，进而使得检测结果更加准确；同时较短的探针更适合检测血液中的小片段cfDNA。
可选的，所述PCR反应体系中不包括三磷酸胞嘧啶脱氧核苷酸。
可选的，在所述引物探针混合液中，每条所述PCR扩增用引物的浓度为7～12μM，每条所述探针的浓度为7～12μM。
可选的，步骤S12反应时，先加热至50～80℃，随后保温；然后降温至28～32℃，保温后即可。
可选的，多个所述探针中不包含参考探针。
第二方面，本申请的一个实施例提供了采用上述检测方法进行多种基因类型检测的检测产品。
第三方面，本申请的一个实施例提供了采用上述检测方法进行多种基因类型检测的检测试剂盒。
可选的，所述检测试剂盒包括所述引物探针混合液；所述引物探针混合液内含有所述PCR扩增用引物和所述探针；每目标基因对应有至少两个所述探针。
综上所述，本申请具有以下有益效果：
1、本申请基于数字PCR技术，在常规合成引物基础上，首先仅仅选用三种碱基，以显著降低扩增时的非特异性结合；并在引物的5’端加入可形成三维结构的核苷酸序列和引入小分子化合物，能够显著降低引物之间产生二聚体，减少非特异性扩增，并能够提高引物对模板的结合特异性和结合强度、结合稳定性，可以实现单反应体系多重扩增。而在进行探针设计时，基于双探针脱落技术，每个目标基因设置至少两个探针，且设置的探针中不含有参照探针；多个不同探针均结合目标基因，且覆盖目标基因的不同区域，以实现对目标基因突变位点的全面检测，从而通过一次检测实现对目标基因多个基因类型或者基因突变的同时检测，检测过程更加高效
2、本申请中选择探针的核苷酸序列长度为10～30bp，探针的核苷酸序列较短，使得PCR扩增的效率更高且使得碱基错配的可能性显著降低；此外，检测时背景荧光比较弱，进而使得检测结果更加准确；并且短的探针更适合检测血液中的小片段cfDNA。
附图说明
图1是本申请的技术方案进行突变检测的原理示意图；
图2是本申请实施例1中检测的肺癌基因位点KRAS突变位点的聚类分析图；
图3是本申请实施例4中检测的结直肠癌基因位点PIK3CA和BRAF突变位点的聚类分析图。
图4为本申请实施例6中细胞系阳性样品检测结果图。
具体实施方式
以下结合附图和实施例对本申请作进一步详细说明。
如图1所示，本申请通过设计至少两个探针，多个不同探针均结合目标基因，且覆盖目标基因的不同区域，以实现对目标基因突变位点的全面检测。针对不同的突变型，其最终在聚类分析图上的不同位置处出现不同荧光显色的聚类：横坐标和纵坐标上对应的是不同通道上出现的不同突变型基因的荧光聚类；对角线上的是对应基因野生型的荧光聚类。
实施例1 肺癌相关的多种基因突变的数字扩增检测方法和检测试剂盒
针对肺癌的用于同时识别多种基因突变的数字扩增检测方法，包括以下步骤：
S1、提取得到样本DNA和引物探针混合液的制备
I、样本DNA的提取
入组病例：对上海中山医院胸外科2020年1月至2020年12月就诊的肺部结节患者进行研究，根据胸部CT及血液肺癌肿瘤指标(包括KRAS)检查结果，均无法确定其为肺癌还是肺良性疾病。所有患者均选择接受手术治疗，术前采外周静脉血50mL，根据肿瘤术后病理结果，将样本分为肺癌及肺良性疾病组(肺良性疾病包括炎性假瘤、硬化性血管瘤、肺结核球等)。
研究方法：应用dPCR技术研究两组样本外周血cfDNA突变情况，具体的操作步骤包括：
①从外周静脉血中采集分离20mL血浆及白细胞；
②将采集的样本在4℃条件下，1900g离心10min，得到血浆层；
③在4℃条件下，16000g离心10min，去除细胞残留；
④采用cfDNA提取试剂盒(QIAamp Circulating Nucleic Acid Kit(Qiagen，Valencia，CA，USA))获取cfDNA。
研究结果：在入组的肺癌病人中，约48.1％发现了KRAS基因中的一种或几种突变。在所有检测到KRAS基因突变的肺癌病人中，通过卡方检验发现其与肺良性疾病患者有显著的统计学差异。
其中，对于肺癌病人，其KRAS突变基因及突变位点，如表1所示，在反应体系中，通过数字PCR技术以及本实施例的特殊引物、探针和缓冲液，可实现对KRAS基因61个突变位点的检测。
表1 本申请检测的肺癌相关目标基因的突变位点
Figure PCTCN2022120663-appb-000001
Figure PCTCN2022120663-appb-000002
研究结论：在肺癌患者的外周血cfDNA中发现KRAS基因突变，突变基因包含表1所述突变位点。KRAS基因的特定位点突变，可为肺癌患者的临床诊断和用药指导提供参考，对于影像学和肿瘤指标难以确诊的肺结节患者，提供更加合理有效的个体化指导。
II、引物探针混合液的制备
S11、合成目标基因扩增和检测用的引物和探针，所述引物和探针为多个；每个所述目标基因至少设计有两个检测用探针；合成所述引物的核苷酸序列只包括三种碱基且不同时包括碱基A和碱基T。
具体为：按照引物和探针的设计原则(如文献“Bustin,Stephen A.,Reinhold Mueller,and Tania Nolan."Parameters for successful PCR primer design."Quantitative Real-Time PCR.Humana,New York,NY,2020.5-22.”所公开的原则)，根据Cosmic数据公布的人类KRAS野生型基因序列，以KRAS的突变位点为基础，来设计特异性引物和探针，如表2所示。突变型荧光探针5’端连接有荧光报告基团FAM；3’端连接有荧光淬灭基团BHQ1；野生型荧光探针5’端连接的荧光报告基团HEX；3’端连接有荧光淬灭基团BHQ2。荧光报告基团和淬灭基团还可以根据具体的平台进行合理选择。
表2 反应管中肺癌基因突变位点检测引物探针序列
引物探针名称 引物探针序列 序列号
KRAS-F1 CCGCCGCGGCCGCCGCCT SEQ ID NO.1
KRAS-R1 CGCCGCCGCGTCCGCGC SEQ ID NO.2
KRAS-F2 CCCGCCCCTCCGGGG SEQ ID NO.3
KRAS-R2 CCGGGGCGCCGCGGG SEQ ID NO.4
KRAS-P1 TGCCGCCGCCGCTGCTGCCT SEQ ID NO.5
KRAS-P2 CGCCACCTTCGCCG SEQ ID NO.6
KRAS-P3 GTACCTCTCTCCC SEQ ID NO.7
KRAS-P4 GGAAGCAGCA SEQ ID NO.8
将上述针对表1所示目标基因位点的引物和探针设计的扩增用的初始修饰引物，送到生工生物工程(上海)股份有限公司合成。
S12、所述引物经修饰后备用，所述修饰包括以下步骤：
S121、将设计好的引物的5’端添加上核苷酸序列“CCCTGGGGGAGTATTGCGGAGGAAGGG(如SEQ ID NO.9所示)”以形成三维结构，得到扩增用的初始修饰引物。
S122、将合成得到的初始修饰引物，加入到缓冲液中加热至70℃，保温5min，然后冷却至30℃，保温25min，得到PCR扩增用引物。
其中，缓冲液的组分为：290mM NaC1，4.5mM MgC1 2，18mM Tris(pH 7.3)。缓冲液中还包括10μM的小分子化合物，小分子化合物为铑金属插入剂，且铑金属插入剂和初始修饰引物的摩尔比为0.7:1。通过X单晶衍射数据的分析，测定检测的核苷酸序列5’端序列存在三维结构。本实施例中所使用的铑金属结构式为[RhCl 3(C10H 8N 2){(CH 3) 2SO}](CH 3)2SO，其合成方法参见文献“Caruso,Francesco,et al."Synthesis,structure,and antitumor activity of a novel tetranuclear titanium complex."Journal of medicinal chemistry 43.20(2000):3665-3670.”。
S13、将用于检测表1所示位点的上游引物、下游引物(即S122中的PCR扩增用引物)和用于检测表1所示位点的荧光探针1和荧光探针2(即S11中合成得到的各探针)共同溶解于TE溶液中，制成引物探针混合溶液。上游引物、下游引物、荧光探针1和荧光探针2在引物探针混合液中的浓度均为10μM。
引物探针混合液的配制方法为：将上游引物、下游引物、荧光探针1和荧光探针2四种成分的干粉分别用TE缓冲液稀释至各探针、引物的浓度分别为100μM，备用。
S2、配制PCR反应体系后进行PCR反应，所述PCR反应体系包括所述样本DNA和所述引物探针混合液。
具体的，按照表3配制PCR反应体系。
表3 反应体系(总体积20μL)
组分 终浓度 添加量
PCR Mix / 10μL
上游引物(10μM) 0.4μM 0.8μL
下游引物(10μM) 0.4μM 0.8μL
荧光探针1(10μM) 0.2μM 0.4μL
荧光探针2(10μM) 0.2μM 0.4μL
模板DNA 1ng/μL 2μL
ddH 2O / 5.6μL
其中，PCR Mix购买自NEB，并加入终浓度0.1％的Triton-X-100，1U热稳定焦磷酸酶，5μg/μL的BSA，按照ddH 2O、PCR mix、探针、引物、模板DNA的顺序，将上述样品按照表3中反应体系中20μL的添加量加入0.2mLPCR管中，使用轻柔涡旋将混合体系混匀15s，并通过短时离心将溶液收集到试管底部。将配制好的不同比例的反应体系上样到PCR芯片上，形成微反应单元。将芯片放入数字PCR仪中，按照表4中PCR反应条件进行PCR反应。
其中，所述PCR Mix中，不包含三磷酸胞嘧啶脱氧核苷酸组分。
表4 PCR反应条件
Figure PCTCN2022120663-appb-000003
S3、PCR反应结束后对检测数据进行分析，得到不同位点的基因类型。
具体为：扩增结束后，通过电脑分析，对两个通道的有效荧光阳性点进行判读，并对结果进行分析，如图2所示，图2为临床样本检测结果图(即肺癌基因位点KRAS突变位点的聚类分析)，纵坐标为FAM荧光通道，横坐标为HEX荧光通道。由实验结果分析可看出，通过聚类分析可以检测出对应的信号，对应于表1所示全部61个基因位点。
通过将突变信号(MUT)除以对应的野生型信号(WT)可以计算出目标基因群的突变丰度。进一步地，将本实施例与现有方法学(NGS)结果的一致性比较结果发现，通过本实施例共完成143例临床血液样本检测(每例都做过NGS检测)，其中与NGS结果一致的有133例，不一致的有10例，一致率为93％。
针对肺癌的用于同时识别多种基因突变的检测试剂盒，包括PCR扩增用引物和探针、TE缓冲液和PCR混合液。
PCR混合液购买自NEB，并加入终浓度0.1％的Triton-X-100，1U热稳定焦磷酸酶，5μg/μL的BSA，还包括PCR mix，其中PCR mix不包括三磷酸胞嘧啶脱氧核苷酸组分。
检测试剂盒还包括阳性质控品和阴性质控品。
阳性质控品的制备方法为：表1所示每一基因突变位点野生型和突变型的序列各200bp，合成后，分别装入质粒载体pET-23d(+)(Promega)。使用Qubit 3.0进行定量，计算两种类型质粒的拷贝数浓度，根据拷贝数比例1:3000，1:2000，1:1000，1:500，1:200，1:100，1:50，1:10混合两种质粒，之后通过超声将质粒混合物打断为约180bp的片段，定量到20ng/μL，作为梯度阳性质控品。
阴性质控品由上述含有野生型的质粒单独构成，然后采用同样方法打断为约180bp的片段，定量到20ng/μL，作为阴性质控品。
实施例2 肺癌相关的多种基因突变的数字扩增检测方法和检测试剂盒
针对肺癌的用于同时识别多种基因突变的数字扩增检测方法：本实施例和实施例1的区别在于，所选用的小分子化合物是直接加在引物上的，即所选用的引物是经锁核酸修饰后获得的。此外在步骤S122中，其缓冲液内不再添加小分子化合物。
锁核酸通过市售获得，可以是购自北京赛百盛基因技术有限公司合成的锁核酸寡聚链。
其他同实施例1。
针对肺癌的用于同时识别多种基因突变的检测试剂盒。
本实施例和实施例1的区别在于,其中PCR扩增用引物是本实施例制备得到的，其他同实施例1。
实施例3 肺癌相关的多种基因突变的数字扩增检测方法和检测试剂盒
针对肺癌的用于同时识别多种基因突变的数字扩增检测方法
本实施例和实施例1的区别在于，小分子化合物选用的是肽核酸(购自北京赛百盛基因技术有限公司)，肽核酸是在步骤S2添加，具体为：在PCR Mix中，添加有终浓度为0.35μM的肽核酸。并且在步骤S122中，其缓冲液内不再添加小分子化合物。
其他同实施例1。
针对肺癌的用于同时识别多种基因突变的检测试剂盒
本实施例和实施例1的区别在于,其中PCR扩增用引物是本实施例制备得到的，其他同实施例1。
最终采用该方法也能够获得对肺癌相关突变基因(如表1所示的KRAS基因61个突变位点)的准确检测。
实施例4 结直肠癌相关的多种基因突变的数字扩增检测方法和检测试剂盒
针对结直肠癌的用于同时识别多种基因突变的数字扩增检测方法，包括以下步骤：
S1、提取得到样本DNA和引物探针混合液的制备
I、样本DNA的提取
入组病例：对上海中山医院肛肠外科2019年1月至2019年12月就诊的结直肠癌患者进行研究，根据CT及血液结直肠癌肿瘤指标(包括PIK3Ca和BRAF)检查结果，均无法确定其为结直肠癌还是结直肠良性疾病。所有患者均选择接受手术治疗，术前采外周静脉血50mL，根据肿瘤术后病理结果，将样本分为结直肠癌还是结直肠良性疾病。
研究方法：应用dPCR技术研究两组样本外周血cfDNA突变情况，具体的操作步骤包括：
①从外周静脉血中采集分离20mL血浆及白细胞；
②将采集的样本在4℃条件下，1900g离心10min，得到血浆层；
③在4℃条件下，16000g离心10min，去除细胞残留；
④采用cfDNA提取试剂盒(QIAamp Circulating Nucleic Acid Kit(Qiagen，Valencia，CA，USA))获取cfDNA。
研究结果：在入组的结直肠癌病人中，部分病人中检测出PIK3CA和BRAF基因中的一种或几种突变，而在肺良性疾病患者中，未发现PIK3CA和BRAF基因突变。在所有检测到PIK3CA和BRAF基因突变的结直肠癌病人中，通过卡方检验发现其与结直肠良性疾病患者有显著的统计学差异。
其中，对于结直肠癌病人，其PIK3CA和BRAF突变基因及突变位点，如表5所示，在反应体系中，通过数字PCR，和本实施例采用的特殊引物和缓冲液，可实现PIK3CA和BRAF等2个基因总共41个突变位点的检测。
表5 实施例检测的目标基因的突变位点
Figure PCTCN2022120663-appb-000004
Figure PCTCN2022120663-appb-000005
研究结论：在结直肠癌患者的外周血ctDNA中发现PIK3CA和BRAF基因突变，突变基因包含表5所述突变位点，而在良性肿瘤中未发现其突变。PIK3CA和BRAF基因的特定位点突变，可为结直肠癌的诊断及鉴别诊断提供参考，对于影像学和肿瘤指标难以确诊的结直肠患者，提供更加合理有效的个体化指导。
II、引物探针混合液的制备
S11、合成目标基因扩增和检测用的引物和探针，所述引物和探针为多个；每个所述目标基因至少设计有两个检测用探针；合成所述引物的核苷酸序列只包括三种碱基且不同时包括碱基G和碱基C。
具体为：
引物及探针设计步骤：
1、首先在Ensemble数据库中确定包含PIK3CA和BRAF基因突变位点的cDNA序列；
2、通过BLAST确定该cDNA序列在DNA上所对应的外显子序列；
3、根据所得外显子序列，通过GenomeBrower确定所对应的完整DNA序列(包括外显子和内含子)；其中，PIK3CA和BRAF的突变位点、突变基因如表6所示。
4、按照上述引物和探针的设计原则，以上述PIK3CA和BRAF突变基因对应的DNA序列为模板，利用ION AMPLISEQ DESIGNER在线设计网站设计出覆盖表5所示目标基因位点的引物和探针，如表6所示。突变型荧光探针5’端连接有荧光报告基团FAM；3’端连接有荧光淬灭基团BHQ1；野生型荧光探针5’端连接的荧光报告基团CY3；3’端连接有荧光淬灭基团BHQ2。荧光报告基团和淬灭基团还可以根据具体的平台进行合理选择。
表6 反应管中结直肠癌基因突变位点检测引物探针序列
引物探针名称 引物探针序列 序列号
PIK3CA-F1 ACATAAAAAAATA SEQ ID NO.10
PIK3CA-R1 AAAAATTCAATTTCTAA SEQ ID NO.11
PIK3CA-F2 AAATATTTTATAAT SEQ ID NO.12
PIK3CA-R2 AACTTCAAAACAAACAAAACAAAACACA SEQ ID NO.13
PIK3CA-P1 TGAGATAACCTG SEQ ID NO.14
PIK3CA-P2 GCAGTAGAAATAATC SEQ ID NO.15
PIK3CA-P3 GATAGTAATACCACTCTGTC SEQ ID NO.16
PIK3CA-P4 TTCAGATTAGAATACCATTTCTTA SEQ ID NO.17
BRAF-F1 CACATAAACAAATTTT SEQ ID NO.18
BRAF-R1 ATAATTAAAAATTTCCA SEQ ID NO.19
BRAF-P1 ACACGGTATT SEQ ID NO.20
将上述表6所示目标基因位点的引物和探针，送到生工生物工程(上海)股份有限公司合成。
S12、所述引物经修饰后备用，所述修饰包括以下步骤：
S121、将设计好的引物的5’端添加上核苷酸序列“TCCTCATCTGTCGAGACTCACTATAGGAAGAGATGTCAACTCGTGCACGAGTTGACATCTCTTCTCCGAGCCGGTCGAAATATTGGAGGAAGCTCGAGCTGGAGGAAAAGTGAGTCTCGACAGATGAGGA(如SEQ ID NO.21所示)”，以在引物前端形成三维结构，得到扩增用的初始修饰引物。
S122、将合成得到的初始修饰引物，加入到缓冲液中加热至70℃，保温5min，然后冷却至30℃，保温25min，得到PCR扩增用引物。
其中，缓冲液的组分为：300mM NaC1，5mM MgC1 2，20mM Tris(pH 7.6)。缓冲液的组分还包括5μM的小分子化合物，小分子化合物为甲基化寡核苷酸(购自金斯瑞生物科技股份有限公司)，且甲基化寡核苷酸和初始修饰引物的摩尔比为0.8:1。通过X单晶衍射数据的分析，测定检测的核苷酸序列5’端序列存在三维结构。
S13、将用于检测表5所示位点的上游引物、下游引物(即S122中的PCR扩增用引物)和用于检测表5所示位点的突变型荧光探针、野生型荧光探针(即S11中合成得到的各探针)共同溶解于TE溶液中，制成引物探针混合溶液。各上游引物、下游引物、荧光探针在引物探针混合液中的浓度均为10μM。
引物探针混合液的配制方法为：将上游引物、下游引物、荧光探针1和荧光探针2四种成分的干粉分别用TE缓冲液稀释至各探针、引物的浓度分别为100μM，备用。
S2、配制PCR反应体系后进行PCR反应，PCR反应体系包括样本DNA和引物探针混合液。
具体的，按照表7配制PCR反应体系。
表7 反应体系(总体积20μL)
组分 终浓度 添加量
PCR Mix / 10μL
上游引物(10μM) 0.4μM 0.8μL
下游引物(10μM) 0.4μM 0.8μL
荧光探针1(10μM) 0.2μM 0.4μL
荧光探针2(10μM) 0.2μM 0.4μL
模板DNA 1ng/μL 2μL
ddH 2O / 5.6μL
其中，PCR Mix购买自NEB，并加入终浓度0.1％的Triton-X-100，1U热稳定焦磷酸酶，5μg/μL的BSA，按照ddH 2O、PCR mix、探针、引物、模板DNA的顺序，将上述样品按照表7反应体系中20μL的添加量加入0.2mLPCR管中，使用轻柔涡旋将混合体系混匀15s，并通过短时离心将溶液收集到试管底部。将配制好的不同比例的反应体系上样到PCR芯片上，形成微反应单元。将芯片放 入数字PCR仪中，按照表8中PCR反应条件进行PCR反应。
其中，所述PCR Mix中，不包含三磷酸胞嘧啶脱氧核苷酸(dCTP)组分。
表8 PCR反应条件
Figure PCTCN2022120663-appb-000006
S3、PCR反应结束后对检测数据进行分析，得到不同位点的基因类型。
具体为：
扩增结束后，通过电脑分析，对两个通道的有效荧光阳性点进行判读，并对结果进行分析，如图3所示，图3为临床样本检测结果图(即结直肠癌基因位点PIK3CA和BRAF突变位点的聚类分析)，纵坐标为FAM荧光通道，横坐标为HEX荧光通道。由实验结果分析可看出，通过聚类分析可以检测出对应的信号，对应于表5所示全部41个基因位点。
通过将突变信号(MUT)除以对应的野生型信号(WT)可以计算出目标基因群的突变丰度。采用上述方法，完成了108例临床血液样本检测(每例都做过NGS检测)，其中与NGS结果一致的有107例，不一致的有2例(因为panel设计不一样)，一致率为98％。
申请人还对血清、血浆、外周血、胸腔积液、体液或者组织来源的DNA进行表5所示基因位点突变的检测，且重复性良好。通过本实施例所述检测方法，可实现对结直肠癌基因突变位点的灵敏检测，且无非特异性扩增，特异性好。可以添加多种引物进行检测，可以有效地节省反应过程中的底物。
针对直肠癌的用于同时识别多种基因突变的检测试剂盒，包括引物探针混合液和PCR混合液。
引物探针混合液包括缓冲液、在缓冲液中的探针和在缓冲液中的初始修饰引物。缓冲液的组分为：300mM NaC1，5mM MgC1 2，20mM Tris(pH 7.6)，缓冲液的组分还包括5μM的小分子化合物。小分子化合物为锁核酸，且锁核酸和初始修饰引物的摩尔比为0.8:1，各初始修饰引物和各探针在缓冲液中的含量为10μM。
PCR混合液购买自NEB，并加入终浓度0.1％的Triton-X-100，1U热稳定焦磷酸酶，5μg/μL的BSA，还包括PCR mix，其中PCR mix不包括三磷酸胞嘧啶脱氧核苷酸组分。
试剂盒还包括阳性质控品和阴性质控品。
阳性质控品的制备方法为：表5所示每一基因突变位点野生型和突变型的序列各200bp，合成后，分别装入质粒载体pET-23d(+)(Promega)。使用Qubit 3.0进行定量，计算两种类型质粒的拷贝数浓度，根据拷贝数比例1:3000，1:2000，1:1000，1:500，1:200，1:100，1:50，1:10混合两种质粒，之后通过超声将质粒混合物打断为约180bp的片段，定量到20ng/μL，作为梯度阳性质控品。
阴性质控品由上述含有野生型的质粒单独构成，然后采用同样方法打断为约180bp的片段，定量到20ng/μL，作为阴性质控品。
实施例5 结直肠癌相关的多种基因突变的数字扩增检测方法和检测试剂盒
本实施例和实施例4的区别在于，步骤S121中，于设计好的引物5’端添加可形成三维结构的核苷酸序列和实施例4不同，本实施例中添加的核苷酸序列为“ACTCATCTGTGAGACTCACTATAGGAAGAGATGTCAACTCGTGCACGAGTTGACATCTCTTCTCCGAGCCGGTCGAAATATTGGAGGAAGCTCGAGCTGGAGGAAAAGTGAGTCTCACAGATGAGT(如SEQ ID NO.22所示)”，得到扩增用的初始修饰引物。
其他步骤同实施例4，进行PCR扩增后发现，将该可形成三维结构的核苷酸序列添加至设计好的引物的5’端时，在循环次数为25次时，已经出现了明显的非特异性扩增；而在以实施例4的方案进行PCR扩增时，循环至少120次时，仍未出现非特异性扩增。
实施例6 食管磷癌相关的多种基因突变的数字扩增检测方法和检测试剂盒
针对食管癌的用于同时识别多种基因突变的数字扩增检测方法，包括以下步骤：
S1、提取得到样本DNA和引物探针混合液的制备
I、样本DNA的提取：使用外购食管磷癌细胞系KYSE270作为阴性样品，使用外购TP53突变细胞系作为阳性样品，采用基因组DNA提取试剂盒(QIAamp DNA Mini Kit(Qiagen，Valencia，CA，USA))获取基因组DNA。
II、引物探针混合液的制备
S11、合成目标基因扩增和检测用的引物和探针，所述引物和探针为多个；每个所述目标基因至少设计有两个检测用探针；合成所述引物的核苷酸序列只包括三种碱基且不同时包括碱基G和碱基C。
具体为：
引物及探针设计步骤：
1、首先在Ensemble数据库中确定包含TP53基因突变位点的DNA序列；
2、通过BLAST确定该DNA序列在DNA上所对应的外显子序列；
3、根据所得外显子序列，通过GenomeBrower确定所对应的完整DNA序列(包括外显子和内含子)；
其中，TP53的突变位点、突变基因如表9所示。
表9 实施例检测的目标基因的突变位点
Figure PCTCN2022120663-appb-000007
Figure PCTCN2022120663-appb-000008
Figure PCTCN2022120663-appb-000009
Figure PCTCN2022120663-appb-000010
4、按照上述引物和探针的设计原则，以上述TP53突变基因对应的DNA序列为模板，利用Primer3web(version 4.1.0)在线设计网站设计出覆盖表9所示目标基因位点的引物和探针，如表10所示。突变型荧光探针5’端连接有荧光报告基团FAM；3’端连接有荧光淬灭基团BHQ1；野生型荧光探针5’端连接的荧光报告基团CY3；3’端连接有荧光淬灭基团BHQ2。荧光报告基团和淬灭基团还可以根据具体的平台进行合理选择。
表10 反应管中结直肠癌基因突变位点检测引物探针序列
引物探针名称 引物探针序列 序列号
TP53-F1 TGGGTGACAGAGTGAGACTTC SEQ ID NO.23
TP53-R1 ACCCGGCCCTCATTATTTCT SEQ ID NO.24
TP53-F2 CCAACATGGTGAAACCCCAA SEQ ID NO.25
TP53-R2 CCGAAGTGCTGGGATTACA SEQ ID NO.26
TP53-F3 GGCACCTCTAATCCCAGCTA SEQ ID NO.27
TP53-R3 CCTCAGCCTCCCGAAGTG SEQ ID NO.28
TP53-P1 ACGTTGATACCATACTGGAGGT SEQ ID NO.29
TP53-P2 AGGATTTGACTCTCA SEQ ID NO.30
TP53-P3 TACTCAGGAGGCTGAGGCAGGAGAA SEQ ID NO.31
TP53-P4 TTGCTTGGACCTGGGAGGCAGAGGT SEQ ID NO.32
TP53-P5 GGAGGCAGAGGTTGCAGTGAG SEQ ID NO.33
TP53-P6 GAGATCGCGCTATTGCACTC SEQ ID NO.34
将上述表10所示目标基因位点的引物和探针，送到生工生物工程(上海)股份有限公司合成。
S12、所述引物经修饰后备用，所述修饰包括以下步骤：
S121、将设计好的引物的5’端添加上核苷酸序列“AATGCCAACAATGACCGCAGGTACCCAGATTCAAGGAGAACAGAAGCAGTCAGTCATGGCCCCCTGGTGTTTTTGAATAATTTGGGTAAAAAGAAGGGACGGACTGAAGATGGAGGTACGAGATTGTTGGCAC(如SEQ ID NO.35所示)”，以在引物前端形成三维结构，得到扩增用的初始修饰引物。
S122、将合成得到的初始修饰引物，加入到缓冲液中加热至70℃，保温5min，然后冷却至30℃，保温25min，得到PCR扩增用引物。
其中，缓冲液的组分为：300mM NaC1，5mM MgC1 2，20mM Tris(pH 7.6)，缓冲液的组分中还包括5μM的小分子化合物；该小分子化合物为硫代磷酸寡核苷酸(购自西格玛奥德里奇(上海)贸易有限公司)，且硫代磷酸寡核苷酸和初始修饰引物的摩尔比为0.6:1。通过X单晶衍射数据的分析，测定检测的核苷酸序列5’端序列存在三维结构。
S13、将用于检测表9所示位点的上游引物、下游引物(即S122中的PCR扩增用引物)和用于检测表9所示位点的突变型荧光探针、野生型荧光探针(即S11中合成得到的各探针)共同溶解于TE溶液中，制成引物探针混合溶液。各上游引物、下游引物、荧光探针在引物探针混合液中的浓度均为10μM。
引物探针混合液的配制方法为：将上游引物、下游引物、荧光探针1和荧光探针2四种成分的干粉分别用TE缓冲液稀释至各探针、引物的浓度分别为100μM，备用。
S2、配制PCR反应体系后进行PCR反应，PCR反应体系包括样本DNA和引物探针混合液。
具体的，按照表11配制PCR反应体系。
表11 反应体系(总体积20μL)
组分 终浓度 添加量
PCR Mix / 10μL
上游引物(10μM) 0.4μM 0.8μL
下游引物(10μM) 0.4μM 0.8μL
荧光探针1(10μM) 0.2μM 0.4μL
荧光探针2(10μM) 0.2μM 0.4μL
模板DNA 1ng/μL 2μL
ddH 2O / 5.6μL
其中，PCR Mix购买自NEB，并加入终浓度0.1％的Triton-X-100，1U热稳定焦磷酸酶，5μg/μL的BSA，按照ddH 2O、PCR mix、探针、引物、模板DNA的顺序，将上述样品按照表11反应体系中20μL的添加量加入0.2mLPCR管中，使用轻柔涡旋将混合体系混匀15s，并通过短时离心将溶液收集到试管底部。将配制好的不同比例的反应体系上样到PCR芯片上，形成微反应单元。将芯片放入数字PCR仪中，按照表12中PCR反应条件进行PCR反应。
其中，所述PCR Mix中，不包含三磷酸胞嘧啶脱氧核苷酸(dCTP)组分。
表12 PCR反应条件
Figure PCTCN2022120663-appb-000011
S3、PCR反应结束后对检测数据进行分析，得到不同位点的基因类型。
具体为：扩增结束后，通过电脑分析，对两个通道的有效荧光阳性点进行判读，并对结果进行分析，如图4所示，图4为细胞系阳性样品检测结果图(即食管鳞癌基因位点TP53突变位点的聚类分析)，纵坐标为FAM荧光通道，横坐标为HEX荧光通道。由实验结果分析可看出，通过聚类分析可以检测出对应的信号，对应于表9所示全部293个基因位点。通过将突变信号(MUT)除以对应的野生型信号(WT)可以计算出目标基因群的突变丰度。
申请人还对血清、血浆、外周血、胸腔积液、体液或者组织来源的DNA进行表9所示基因位点突变的检测，且重复性良好。通过本实施例所述检测方法，可实现对食管鳞癌基因突变位点的灵敏检测，且无非特异性扩增，特异性好。可以添加多种引物进行检测，可以有效地节省反应过程中的底物。
针对食管鳞癌的用于同时识别多种基因突变的检测试剂盒,包括引物探针混合液和PCR混合液。
引物探针混合液包括缓冲液、在缓冲液中的探针和在缓冲液中的初始修饰引物。缓冲液的组分为：300mM NaC1，5mM MgC1 2，20mM Tris(pH 7.6)。缓冲液的组分还包括5μM的小分子化合物，小分子化合物为硫代磷酸寡核苷酸，且硫代磷酸寡核苷酸和初始修饰引物的摩尔比为0.6:1，各初始修饰引物和各探针在缓冲液中的含量为10μM。
PCR混合液购买自NEB，并加入终浓度0.1％的Triton-X-100，1U热稳定焦磷酸酶，5μg/μL的BSA，还包括PCR mix，其中PCR mix不包括三磷酸胞嘧啶脱氧核苷酸组分。
试剂盒还包括阳性质控品和阴性质控品。
阳性质控品的制备方法为：表9所示每一基因突变位点野生型和突变型的序列各200bp，合成后，分别装入质粒载体pET-23d(+)(Promega)。使用Qubit 3.0进行定量，计算两种类型质粒的拷贝数浓度，根据拷贝数比例1:3000，1:2000，1:1000，1:500，1:200，1:100，1:50，1:10混合两种质粒，之后通过超声将质粒混合物打断为约180bp的片段，定量到20ng/μL，作为梯度阳性质控品。
阴性质控品由上述含有野生型的质粒单独构成，然后采用同样方法打断为约180bp的片段，定量到20ng/μL，作为阴性质控品。
本具体实施例仅仅是对本申请的解释，其并不是对本申请的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本申请的权利要求范围内都受到专利法的保护。

Claims (10)

  1. 用于同时识别多种基因类型的数字扩增检测方法，其特征在于，包括以下步骤：
    S1、提取得到样本DNA；
    制备得到引物探针混合液，包括以下步骤：
    S11、合成目标基因扩增和检测用的引物和探针，所述引物和探针为多个；每个所述目标基因至少设计有两个检测用探针，用于覆盖目标基因的不同区域；合成所述引物核苷酸序列的碱基包含四种或者四种以下；
    S12、使得所述引物的5’端添加有能够形成二级结构的核苷酸序列，且使得所述引物上修饰有小分子化合物，得到PCR扩增用引物；
    所述小分子化合物选自硫代磷酸寡核苷酸、甲基化寡核苷酸、肽核酸、锁核酸、次黄嘌呤核苷酸或铑金属插入剂中的一种或几种；
    S13、将所述PCR扩增用引物和所述探针共同溶解于核酸溶解用缓冲液中，得到引物探针混合液；
    S2、配制PCR反应体系后进行PCR反应，所述PCR反应体系包括所述样本DNA和所述引物探针混合液；进行PCR反应；
    S3、PCR反应结束后对检测数据进行分析，得到不同位点的基因类型。
  2. 根据权利要求1所述的检测方法，其特征在于，合成所述引物的核苷酸序列包含三种碱基且不同时包含碱基C或者碱基G。
  3. 根据权利要求1所述的检测方法，其特征在于，所述小分子化合物通过额外添加的方式和所述引物连接；S12包括以下步骤：所述引物的5’端添加可形成三维结构的核苷酸序列，得到初始修饰引物；将所述初始修饰引物加入到缓冲液中，所述缓冲液包括小分子化合物，反应得到PCR扩增用引物。
  4. 根据权利要求3所述的检测方法，其特征在于，所述小分子化合物在所述缓冲液中的浓度为4～12μM，所述小分子化合物和初始修饰引物的摩尔比为0.7～1:1。
  5. 根据权利要求1所述的检测方法，其特征在于，所述探针的核苷酸序列长度为10～30bp。
  6. 根据权利要求1所述的检测方法，其特征在于，所述样本DNA包括cfDNA。
  7. 根据权利要求1所述的检测方法，其特征在于，在所述引物探针混合液中，每条所述PCR扩增用引物的浓度为7～12μM，每条所述探针的浓度为7～12μM。
  8. 根据权利要求1所述的检测方法，其特征在于，步骤S12反应时，先加热至50～80℃，随后保温；然后降温至28～32℃，保温后即可。
  9. 采用权利要求1-8任一所述检测方法进行多种基因类型检测的检测产品。
  10. 采用权利要求1-8任一所述检测方法进行多种基因类型检测的检测试剂盒。
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CN112342275A (zh) * 2020-11-26 2021-02-09 厦门大学 一种检测目的核酸是否含有突变的方法和试剂盒
CN112899365A (zh) * 2021-01-06 2021-06-04 南京普济生物有限公司 一种肺癌基因突变位点的检测方法及其试剂盒
CN114196740A (zh) * 2021-12-09 2022-03-18 南京普济生物有限公司 用于同时识别多种基因类型的数字扩增检测方法、检测产品和检测试剂盒

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