Gene mutation detection method and application thereof

Abstract

The invention provides a gene mutation detection method and application thereof. Particularly, the gene mutation detection method uses three novel nucleic acid probes, can realize the advantages of high sensitivity, high specificity and economy, can well realize mutation detection in low-abundance clinical samples in a wide concentration range, and has better popularization significance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gene mutation detection method and application thereof. Background Art

[0002] Preoperative diagnosis and recurrence prediction of cancer help to determine the surgical plan in a timely manner and reduce treatment costs. Liquid biopsy or puncture biopsy has become the mainstream method of tumor sampling because of its minimal invasiveness and ease of obtaining samples throughout the disease process. The tumor-specific mutations contained in the biopsy samples contain important clues to the disease and are effective indicators for tumor diagnosis and concomitant treatment. For example, the BRAF V600E mutation can be used as an indicator of benign and malignant thyroid cancer puncture nodules, and its accurate detection can assist in the judgment of benign and malignant thyroid nodules. However, due to the low genomic DNA content and low mutation abundance in biopsy samples, ultra-sensitive and highly specific performance requirements are put forward for mutation detection tools.

[0003] Nucleic acid probes have good specificity in identifying targets due to the strict principle of complementary base pairing. Among them, nucleic acid probes with extremely favorable hybridization thermodynamics (such as strand exchange probes) have high sensitivity in detecting mutations. However, such nucleic acid probes are extremely insensitive to single-base mutations because they are too stable. In order to enhance the sensitivity of nucleic acid probes in identifying mutations, weakening their binding to mutant targets is the most commonly used method. This design concept mainly relies on the fact that when the ΔG of the reaction between the probe and wild-type DNA tends to be near zero, the specificity of nucleic acid probes in identifying mutations is best. For example, in ARMS-PCR, the complementary ability with the target is weakened by introducing mismatches or hairpin structures in primers. Or the introduction of hairpins in single-stranded probes makes the hybridization of probes with mutations less favorable than that of single-stranded probes, thereby allowing higher specificity. Other hybridization probes, such as the representative toehold exchange probe, also aim to reduce the thermodynamic advantage of hybridization by introducing reverse sticky ends in strand exchange probes, thereby improving the specificity of probes. The specificity of mutation detection can be further improved by improving or experimentally optimizing the above probes. However, it will also greatly sacrifice the sensitivity of detecting mutations. Therefore, in the design of classic nucleic acid hybridization probes, there is often an optimal balance between sensitivity and specificity.

[0004] An ideal hybridization probe always needs to sacrifice sensitivity to achieve optimal specificity. Therefore, it is necessary to develop a new method for detecting low-abundance gene mutations that can simultaneously achieve high sensitivity and high specificity over a wide concentration range, fundamentally solving the problem of the balance between sensitivity and specificity of traditional hybridization probes. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a method for detecting gene mutations. The method uses three new nucleic acid probes, which can achieve the advantages of high sensitivity, high specificity and economy. It can well realize the detection of mutations in low-abundance clinical samples within a wide concentration range, and has better promotion significance.

[0006] In a first aspect of the present invention, a method for detecting gene mutations is provided, which comprises using three nucleic acid probes, wherein each end of the first probe comprises a fluorescent group modification, each end of the second probe comprises a fluorescent quenching group modification, and the 5' end or 3' end of the third probe comprises a fluorescent quenching group modification.

[0007] Preferably, the first probe and the second probe are combined into a double-stranded probe set, and the third probe contains the same nucleotide sequence as the first or second probe.

[0008] Preferably, the first probe and the second probe are complementary to each other for at least 15 bp, and the first probe and the second probe may be completely or partially complementary.

[0009] Preferably, the fluorescent group includes but is not limited to ALEX-350, FAM, VIC, TET, CAL Gold540, JOE, HEX, CAL Orange560, TAMRA, CAL Red590, ROX, CAL Red610, TEXAS RED, CAL Red635, Quasar670, CY3, CY5, CY5.5 and / or Quasar705.

[0010] Preferably, the fluorescence quenching group includes but is not limited to DABCYL, BHQ quenchers, ECLIPSE and / or TAMRA.

[0011] Preferably, the BHQ quencher includes BHQ-1 and / or BHQ-2.

[0012] Preferably, the two fluorescent groups at the ends of the first probe are the same or different. Further preferably, the two ends of the first probe include different fluorescent groups.

[0013] Preferably, the two fluorescence quenching groups at the ends of the second probe are the same or different. Further preferably, the two ends of the second probe include different fluorescence quenching groups.

[0014] Preferably, the fluorescence quenching group of the third probe is different from the fluorescence quenching group of the second probe or is the same as the fluorescence quenching group at one of the ends. Further preferably, the fluorescence quenching group of the third probe is the same as the fluorescence quenching group at one of the ends of the second probe.

[0015] Preferably, the sequence of the probe comprises the complementary sequence of the wild-type or mutant target sequence.

[0016] Preferably, the sequence of the probe has one or more mismatches compared to the complementary sequence of the wild-type or mutant target sequence.

[0017] Preferably, the mismatch is a single base transition, transversion, insertion or deletion.

[0018] Preferably, the mutation comprises a single nucleotide mutation.

[0019] Preferably, the single nucleotide mutation is a conversion, transversion, insertion or deletion of a single base at one or more identical or different positions in the nucleic acid sequence at the same gene locus between different individuals of the same species, and one or more nucleic acid segments of the genome are the same or different.

[0020] Preferably, the probe consists of unmodified bases.

[0021] Preferably, the base sequence of the probe may contain special modifications. These specially modified bases can help regulate the binding ability of the probe, for example, enhance the binding ability of the probe or weaken the binding ability. For example, special modified bases that can enhance the binding ability of the probe are locked nucleic acids (i.e., locked nucleic acids, abbreviated as LNA) bases or peptide nucleic acids (PNA), etc. Therefore, in one embodiment, the probe of the present invention may be composed of unmodified bases. In a preferred embodiment, the bases of the probe are modified. In a preferred embodiment, the probe of the present invention contains bases that can enhance or weaken the binding ability of the probe.

[0022] Preferably, the detection method comprises:

[0023] (1) mixing the first probe and the second probe, adding the target sequence to be detected, and detecting the fluorescence intensity;

[0024] (2) Add the third probe and detect the fluorescence intensity.

[0025] Preferably, the detection method includes mixing the first probe, the second probe and the target sequence to be detected to obtain an intermediate, and further adding the third probe to obtain a catalytic product, wherein the intermediate is a combination of the first probe and the target sequence to be detected, and the catalytic product is a combination of the first probe and the third probe, and the fluorescence intensities of the intermediate and the catalytic product are detected respectively.

[0026] Preferably, the detection method determines whether a mutation exists by detecting fluorescence intensity. Compared with the case where the wild-type target sequence is not present, the fluorescence or fluorescence intensity of the intermediates and catalytic products of the mutant target sequence is increased. When the wild-type target nucleic acid sequence is present, there is no fluorescence or the fluorescence intensity is weak, and no fluorescence response is generated.

[0027] Preferably, the detection method also includes the combination with other methods, and the other methods include but are not limited to PCR method, LCR method, TAS method, ICAN method, LAMP method, NASBA method, RCA method, TAMA method and / or UCAN method.

[0028] In a specific embodiment, the detection method further comprises relevant reagents used for PCR, and the reagents include PCR primers and assembly probes.

[0029] Preferably, the PCR primers include SEQ ID NOs: 74-75.

[0030] Preferably, the assembly probes include SEQ ID NOs: 74-78.

[0031] Preferably, the sequence of the probe includes the nucleotide sequence shown in any one of SEQ ID NOs: 3-4, 14-15, 18-19, 22-23, 26-27, 30-31, 34-35, 38-39, 42-43, 46-47, 50-51, 54-55, 58-59, 62-63, 66-67, 70-71, or a nucleotide sequence that has at least 80% identity with the nucleotide sequence shown in any one of SEQ ID NOs: 3-4, 14-15, 18-19, 22-23, 26-27, 30-31, 34-35, 38-39, 42-43, 46-47, 50-51, 54-55, 58-59, 62-63, 66-67, 70-71.

[0032] Preferably, the detection method is not used for disease diagnosis or treatment.

[0033] The second aspect of the present invention provides a probe set of probes used in the above-mentioned gene mutation detection method.

[0034] Preferably, the probe group includes the first, second and third probes mentioned above.

[0035] The third aspect of the present invention provides a kit, which comprises the above-mentioned probe set.

[0036] Preferably, the kit may also include primers or reagents required for other methods such as PCR method, LCR method, TAS method, ICAN method, LAMP method, NASBA method, RCA method, TAMA method and / or UCAN method.

[0037] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, and other specified components or steps that will not be substantially affected.

[0038] The "homology" or "identity" mentioned in the present invention refers to that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology / identity.

[0039] Beneficial effects of the present invention:

[0040] The present invention discloses a novel method for identifying catalytic mutations based on mutation-specific intermediates and catalytic products. The probe contains three new nucleic acid probes, which achieve single-base mutation detection as low as 0.1%. The novel probe invented in this patent is simple in design and does not require modification of functional nucleic acids, making DNA mutation detection more economical for clinical and scientific researchers. In addition, combined with the novel probe invented in this patent and PCR amplification technology, it was successfully applied to the detection of BRAF V600E mutations in 25 pairs of thyroid cancer postoperative tissue samples and 67 paraffin section samples (postoperative and puncture).

[0041] This method overcomes the single-recognition mutation mechanism of traditional probes and solves the problem of the mutual balance between sensitivity and specificity of traditional nucleic acid hybridization probes. Compared with existing mutation detection technologies used in clinical practice, this technology can achieve the advantages of high sensitivity, high specificity and economy in a wide range of target concentrations. It can well detect mutations in low-abundance clinical samples (such as puncture samples) in a wide concentration range, and has better promotion significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 . Schematic diagram of probe-catalyzed mutation recognition, a. Schematic diagram of traditional hybridization probe detection of mutation; b. Schematic diagram of catalytic mutation recognition of the present application; c. Schematic diagram of the principle of catalytic mutation recognition probe recognizing mutation and catalytic mutation recognition probe recognizing wild-type nucleic acid. Figure 2 .Results of verification of the mechanism of catalytic mutation recognition, a. Schematic diagram of intermediate recognition mutation; b. Heat map of theoretical yield of intermediate recognition SNV and WT versus reaction time; c. Changes of titration curves of intermediate recognition SNV and WT over time; d. Changes of discrimination factor (DF) of intermediate discrimination mutation over time; e. Schematic diagram of catalytic product recognition mutation; f. Heat map of theoretical yield of catalytic product recognition SNV and WT versus reaction time; g. Changes of titration curves of catalytic product recognition SNV and WT over time; h. Changes of discrimination factor (DF) of catalytic product discrimination mutation over time.

[0043] Figure 3 .Results of high specific concentration range of catalytic mutation recognition probe for distinguishing mutations, a. Definition of new parameters: distance and relative distance (dr); b. Simulation results of target concentration and specificity at different times; c. High specific concentration window of catalytic mutation recognition probe; d. Influence of different mutation types and e. Influence of different mutation sites.

[0044] Figure 4 .Analytical performance of catalytic mutation recognition probes for detecting single nucleotide mutations, a. Projection of the relative distance (dr) between SNV and WT in three-dimensional space onto the two-dimensional plane of two detection signals; b. Two-dimensional distribution from 0% (WT) to a mixture prepared by mixing single-stranded WT with SNV at different mutation ratios (from 0.1% to 10%), each mixed sample containing a total of 100 nM synthetic DNA; c. dr values ​​at different single-stranded WT to SNV mutation ratios (from 0.1% to 10%), each sample containing a total of 100 nM synthetic DNA; d. Verification of versatility, combined with probe TER: toehold exchange reaction, chain exchange reaction.

[0045] Figure 5. Cascade PCR detection of mutation results in synthetic samples, a. Detection flow chart; b. Sensitivity of PCR-catalyzed mutation recognition probe; c. Specificity of PCR-catalyzed mutation recognition probe.

[0046] Figure 6 . Clinical validation results, a. Schematic diagram of the workflow of PCR-catalytic mutation recognition probe, Sanger sequencing and dPCR analysis of frozen surgical tissue samples and formalin-fixed paraffin-embedded (FFPE) samples; b. Two-dimensional distribution diagram of PCR-catalytic recognition probe detection of 25 pairs of frozen tissues; c. Results of catalytic recognition probe, Sanger sequencing and dPCR detection of mutations in 25 pairs of frozen postoperative tissues; d. Heat map results of catalytic recognition probe, Sanger sequencing and dPCR detection of mutations in 22 benign samples and 45 FFPE tumor samples; e. Two-dimensional distribution of FFPE tissues; f. ROC curve to determine the detection accuracy and threshold of catalytic recognition probe; g. Confusion matrix to evaluate the sensitivity and specificity of catalytic recognition probe relative to dPCR (n=117).

[0047] Figure 7 . Detection of low-abundance BRAFV600E mutations in fine needle aspiration (FNA) samples of thyroid cancer patients using catalytic mutation recognition probes. a. Schematic diagram of the workflow for detecting BRAFV600E mutations in FNA samples not confirmed by TBSRTC using PCR-catalytic mutation recognition probes; b. Distribution of gDNA in FNA samples of 52 thyroid cancer patients; c. Distribution of BRAF V600E mutation proportions in FNA samples of 52 thyroid cancer patients; d. Detection limit of BRAFV600E mutation analysis in gDNA extracted from FFPE surgical samples; e. Bar graph results of BRAF V600E mutation detection in 52 clinical FNA samples using catalytic mutation recognition probes; f. Analysis of the proportion of uncertain (grey) and confirmed (red) cases in 52 FNA samples by TBSRTC, qPCR, and catalytic mutation recognition probes; g. Confusion matrix to evaluate the clinical sensitivity and specificity of catalytic mutation recognition probes (n=52). DETAILED DESCRIPTION

[0048] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0049] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can all be obtained from commercial sources. Example 1 Design and application of catalytic mutation recognition probes

[0050] 1. Principle of catalytic mutation recognition probe

[0051] The purpose of the present invention is to provide a new method for detecting low-abundance gene mutations in a wide concentration range, aiming to solve the problem that existing mutation detection technologies cannot simultaneously achieve high sensitivity and high specificity in a wide concentration range. Figure 1 a).

[0052] The present invention increases the dimension of mutation recognition and develops a nucleic acid probe with a catalytic mutation recognition mechanism that has both mutation-specific intermediates and mutation-specific catalytic products, thereby broadening the target concentration range for mutation detection with high specificity ( Figure 1 b). The principle of catalytic mutation recognition through intermediates and catalytic products can improve the specificity of the probe in distinguishing mutations; the catalytic products gradually accumulate with the catalytic amplification reaction, which can improve the sensitivity of mutation detection ( Figure 1 c). However, it has almost no response to the wild-type target due to base mismatch ( Figure 1 c). Specifically, the catalytic mutation recognition probe includes two parts: a partially complementary (base complementarity greater than or equal to 15bp) PC double-stranded probe and an F chain. First, the SNV chain undergoes a chain displacement reaction with the PC probe to generate a high-yield double-stranded SNV-C (intermediate), at which time the green and red fluorescence are significantly enhanced. Further, an F chain with the same sequence as the P chain is added, and the F chain only has groups that quench green fluorescence. Through concentration advantage, the SNV-C double chain is gradually replaced to form an FC double chain (catalytic product), releasing the SNV. The released SNV initiates the catalytic cycle amplification reaction, resulting in a large accumulation of red fluorescence, generating a strong red fluorescence signal ( Figure 1 c). The WT sequence has a base difference, so it generates a low-yield intermediate with weak red and green fluorescence. A small amount of intermediates causes the catalytic reaction to proceed very slowly. At the same reaction time, the signal of the catalytic product of WT is weaker. By detecting the difference in the intensity of the fluorescent signal, it is possible to determine whether SNV exists. The high specificity of distinguishing high-concentration SNVs by intermediates can achieve mutation differentiation at high concentrations; the high specificity of mutation differentiation at low concentrations can be achieved by the response of the catalytic reaction to low target concentration SNVs. Combining the ability of intermediates and catalytic products to distinguish SNVs in different concentration ranges can broaden the range of high specificity for distinguishing mutations.

[0053] 2. Experimental methods

[0054] 1). Oligonucleotides and samples

[0055] The primers, assembly probes and probes used were purchased from Shanghai Bioengineering. All DNA dry powders were centrifuged at 4000rpm for 1min, dissolved with double distilled water, and stored at -20°C for later use. DNA was diluted with 1×TE buffer (1×TE(10)+0.5% Tween-20) containing 0.5% Tween-20 and 10mM MgCl2. Among them, the fluorescent or quenching modified oligonucleotide sequences with oligonucleotide length greater than 60 bases were synthesized by the Shanghai Synthesis Department of Bioengineering, and the purification method was HPLC. The remaining oligonucleotide sequences were synthesized by the Chengdu Synthesis Department of Bioengineering, and the purification method was PAGE.

[0056] 2). Sample preparation for fluorescence experiment

[0057] Preparation of buffer and DNA stock solution: Preparation of 1×TE (10) + 0.5% Tween-20: Preparation of 1×TE (containing 10mMTris-HCl, 1mM EDTA, 13.5mM MgCl2 and 0.5% Tween-20, pH 7.8-8.2) buffer: Weigh 137.23mg MgCl2·6H2O in 50mL 1×TE buffer, vortex thoroughly and dissolve. Then add 250μL Tween-20, shake well, and store in a 4℃ refrigerator for later use.

[0058] Preparation of 100μM DNA stock solution: centrifuge the DNA freeze-dried powder at 4000rpm for 2min, then add an appropriate amount of ultrapure water to dissolve, shake well and keep in a 4℃ refrigerator overnight. The next day, use a UV spectrophotometer to measure the absorbance at 260nm, calculate its actual concentration based on the theoretical molar extinction coefficient and dilute to 100μM, dispense 10μL into multiple EP tubes, and store at -20℃ in the dark for later use.

[0059] Preparation of reporter probe (catalytic recognition probe): Dilute 100 μM C ​​chain and 100 μM P chain to 10 μM. Mix 10 μM C ​​chain and 10 μM P chain at a concentration ratio of 1:1, and adjust the volume to 60 μL with 1×TE buffer. Incubate the prepared mixture at 95°C in a thermal cycler for 5 minutes, then gradually cool to 25°C at a rate of 1°C / min, and store at 4°C for later use.

[0060] 3). Catalytic recognition probes for mutation detection

[0061] When using catalytic recognition probes to detect mutations, the total volume of a reaction is 100 μL, which contains 10 μL of a specific concentration of the mutant target, 2 μL of a 1 μM PC double-stranded reporter probe, 380 nM of the F chain (unless otherwise specified), and an appropriate amount of 1×TE buffer. The mixture is quickly transferred to a Costar 96-well ELISA plate, followed by kinetic acquisition.

[0062] In the experiments exploring different stoichiometric ratios, the concentrations of F chain were 60nM, 100nM, 140nM, 200nM, and 380nM, respectively. The rest of the experimental conditions were consistent with the catalytic recognition probe mutation detection.

[0063] 4) Figure 2 Sequence used in the detection of mutations by the catalytic mutation recognition probe

[0064] Table 1 Sequences used in detecting mutations using catalytic mutation recognition probes

[0065]

[0066] 5) Figure 3 Sequences used to characterize the ability of catalytic mutation recognition probes to broaden concentration range:

[0067] Table 2 Sequences used to characterize the ability of catalytic mutation recognition probes to broaden the concentration range

[0068]

[0069]

[0070]

[0071] 4). Analytical performance evaluation of catalytic recognition probes ( Figure 4 )

[0072] Preparation of mutant targets at different concentrations: The synthesized DNA was gradiently diluted into a series of concentrations, including 1000nM, 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.62nM, 7.81nM, 3.90nM, 1.95nM, and 0.97nM.

[0073] Preparation of mixed solutions with different mutation abundances: 100 μM SNV and WT were diluted to 1 μM. Under the premise that the total concentration of SNV and WT was 100 nM, mixed solutions with mutation abundances of 0%, 0.1%, 1%, 5% and 10% were prepared for subsequent specificity experiments of catalytic recognition probes.

[0074] Sensitivity or specificity experiment: Take 10 μL of the above-mentioned sample with specific concentration or mutation abundance, mix it with 2 μL of 1 μM PC double-stranded reporter probe and 380 nM F chain (unless otherwise specified), and add 1xTE buffer to make the total volume 100 μL. Then use enzyme labeling to collect kinetic curves.

[0075] in Figure 4 The sequences used include:

[0076] Table 3 Figure 4 The sequence used

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] 5). Sample preparation for mutation detection ( Figure 5 )

[0086] First, the synthesized DNA templates (PCR-V600E-WT and PCR-V600E-SNV) were diluted to 100 fM. 5 μL of the synthesized DNA template, 25 μL of Taq PCR premix (2×, without dye), 4 μL of 10 μM PCR primers (V600E-FP / HRAS-FP and V600E-RP / HRAS-RP) and 16 μL of sterile water were added to a 200 μL PCR tube, with a total volume of 50 μL. The prepared PCR solution was placed on an Eppendorf Mastercycler nexus gradient amplification instrument for PCR amplification. The amplification conditions were 94°C for 5 minutes for enzyme activation, followed by 35 cycles of 94°C for 30 seconds, 52°C (BRAF V600E) / 58°C (HRAS G12V) for 30 seconds, and 72°C for 30 seconds. Finally, the extension was performed at 72°C for 5 minutes.

[0087] After amplification, take 25 μL of the amplicon and mix it with 25 μL of the assembly probe premix (V600E-H1, V600E-H2, V600E-H3, V600E-H4 and V600E-H5) at a concentration of 2 μM, and place it in the high temperature zone of Mona's multi-temperature zone workbench for 95°C heating for 5 minutes, and then quickly transfer it to 4°C to cool for 2 minutes. After cooling is completed, centrifuge briefly. Open the lid and add 50 μL of the catalytic recognition probe (V600E-C + V600E-P + 20x V600E-F) premix at a concentration of 40nM to the above PCR tube. The final concentration of the assembly probe is 500nM, and the final concentration of the catalytic recognition probe is 20nM. Then perform fluorescence detection at 37°C on an ELISA reader. The detection conditions were set as follows: the excitation / emission wavelengths of the FAM channel were 485 nm / 525 nm and the excitation / emission wavelengths of the ROX channel were 585 nm / 616 nm, and the temperature was 37°C. Figure 5 The sequence used is:

[0088] Table 4 Figure 5 The sequence used

[0089]

[0090]

[0091] 6). Clinical validation experiment ( Figure 6 )

[0092] Extraction of genomic DNA from fresh tissue samples after thyroid cancer surgery: The clinical samples involved in this experiment have been approved by the Ethics Committee of West China Hospital of Sichuan University. The genomic extraction of thyroid cancer tissues was performed using the DNeasy Blood & Tissue Kit, and the operation steps were carried out according to the kit instructions.

[0093] Extraction of genomic DNA from paraffin sections after thyroid cancer surgery: The clinical samples involved in this experiment have been approved by the Ethics Committee of West China Hospital of Sichuan University. The QIAamp DNA FFPE Tissue Kit was used to extract genomic DNA from paraffin sections after thyroid cancer surgery, and the operation steps were carried out according to the instructions of the kit.

[0094] PCR-catalytic recognition probe to detect mutations in thyroid cancer tissue samples: For mutation detection in fresh thyroid cancer tissue samples, the PCR steps and sample preparation are the same as 5). For tissue detection of paraffin sections, the number of amplification cycles is 40. After amplification, take 20μL of PCR amplicon and mix it with 30μL of 2μM assembly probe premix (V600E-H1, V600E-H2, V600E-H3, V600E-H4 and V600E-H5), and place it in the high temperature zone of Mona's multi-temperature zone workbench for heating at 95°C for 5 minutes, and then quickly transfer it to 4°C to cool for 2 minutes. After cooling is completed, perform a brief centrifugation. Among them Figure 6 The sequence used is:

[0095] Table 5 Figure 6 The sequence used

[0096]

[0097]

[0098]

[0099] 7). Clinical application experiment ( Figure 7 )

[0100] Extraction of genomic DNA from paraffin sections of thyroid cancer puncture samples: The clinical samples involved in this experiment have been approved by the Ethics Committee of West China Hospital of Sichuan University. The genomic extraction of thyroid puncture paraffin sections was performed using the QIAamp DNA FFPE Tissue Kit, and the operation steps were carried out according to the kit instructions.

[0101] PCR-catalytic recognition probe detection of mutations in thyroid cancer puncture samples: For mutation detection in thyroid cancer puncture tissue samples, the PCR steps and sample preparation are the same as 5). The number of amplification cycles is 40. After amplification, the mutation detection steps are the same as 3).

[0102] in Figure 7 The sequence used is:

[0103] Table 6 Figure 7 The sequence used

[0104]

[0105]

[0106]

[0107] 8).Clinical Methodology Cross-Validation Experiment

[0108] Sanger sequencing:

[0109] In order to analyze lung cancer cells and tissue samples using Sanger sequencing, gDNA extracted from fresh thyroid cancer tissues and paraffin sections was first subjected to PCR amplification in an Eppendorf Mastercycler nexus gradient thermal cycler. 2 μL gDNA, 25 μL Taq PCR premix (2×, without dye), 4 μL of 10 μM front and back primer mixture, and the final volume was adjusted to 50 μL with sterile water. PCR amplification used a three-step method, namely incubation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, and finally extension at 72°C for 5 min. After PCR amplification, the PCR amplicons were purified using the SanPrep column PCR product purification kit and Sanger sequencing was performed using an Applied Biosystems model 3730xL genetic analyzer.

[0110] ddPCR reaction:

[0111] ddPCR was performed using the Sniper integrated ddPCR system model DQ24. Primers and TaqMan probe sequences for L858R and T790M mutations were designed based on previous studies. 11 μL of Sniper ddPCR premix, 2 μL of 5 μM primer premix, 4 μL of cfDNA sample (total amount less than 60 ng), 1 μL of 5 μM VIC-labeled TaqMan probe for WT, and 1 μL of 5 μM FAM-labeled TaqMan probe for SNV detection were mixed and the volume was adjusted to 22 μL with sterile water. Mix well, then place the consumables of the Sniper ddPCR instrument and the samples to be tested in the ddPCR detection chamber according to the instrument instructions. Set the droplet generation conditions and the fluorescence channel for signal acquisition. Liquid segmentation, amplification, and result scanning are completed step by step by setting a program. There is no need to remove samples or chips between each step, and the whole process is fully automatic. The program set the conditions as follows: 60°C, 5min for droplet generation, using the oscillation injection technology developed by Sniper to evenly spread the droplets on the detection chip, and finally divide the 20μL sample to be tested into about 20,000 droplets. Subsequently, the generated droplets activated Taq polymerase at 95°C for 15min, then amplified at 95°C for 30s and at 60°C for 1min, for a total of 40 cycles. After the thermal cycle, the droplets were scanned and imaged at 60°C using Sniper's built-in scanning system. Finally, the ddPCR test results were analyzed using SightPro software.

[0112] qPCR reaction:

[0113] ARMS-PCR analysis was performed on a qTOWER3G real-time fluorescence thermal cycler in Jena. In a typical qPCR assay, a 20 μL reaction contained 10 μL 2× TaqMan Fast qPCR master mix, 7.8 μL RNase-free water, 0.4 μL 10 μM forward primer, 0.4 μL 10 μM reverse primer, 0.4 μL 10 μM TaqMan probe labeled with FAM, and 1 μL gDNA. The PCR process was 95°C for 3 min, followed by 60 cycles of amplification at 95°C for 30 s and 60°C for 30 s using a two-step method. The fluorescence signal was then collected at 60°C by the built-in FAM channel of the qPCR. The Ct value was obtained using the above software.

[0114] 2. Catalytic mutation recognition probe detection mutation

[0115] First, the ability of the catalytic intermediates and catalytic products to recognize mutations was verified ( Figure 2 a and 2e). Theoretical modeling was performed based on thermodynamic parameters and kinetics to predict the feasibility of its catalytic mutation recognition mode. The prediction results showed that both the mutant intermediates and the catalytic products have excellent ability to distinguish mutations ( Figure 2 b and 2f). Subsequently, the specificity of the mutant intermediate and catalytic product in recognizing the mutation was verified experimentally ( Figure 2 c and 2g). The results showed that both the intermediates and the catalytic products were highly sensitive to single nucleotide variations (SNVs) and had little response to the wild type (WT) ( Figure 2 bc and 2f-g). In addition, as the reaction time increased, the SNV signal was significantly amplified, successfully improving the sensitivity ( Figure 2 g).

[0116] The ability of intermediates and catalytic products to recognize mutations was further quantitatively evaluated. By calculating the mutation discrimination factor (DF) value, it was found that the optimal concentration range for intermediates to recognize mutations was always maintained in the medium concentration range (10nM to 50nM), and was not affected by the reaction time ( Figure 2 d); the optimal concentration range of the catalytic product recognition mutation gradually moves toward low concentration (0.49nM to 10nM) as the reaction time increases ( Figure 2 h) Using intermediates and catalytic products to simultaneously identify mutations is expected to broaden the target concentration range for hybridization probes to highly specifically identify mutations.

[0117] In order to quantitatively characterize the ability of the catalytic mutation recognition probe to broaden the concentration range, the target concentration and the signals of the two detection channels were placed in a three-dimensional coordinate system, and the distance from the target (SNV or WT) detection signal to the origin was defined as parameter 1 (distance), which was used to characterize the sensitivity of the probe to detect mutations ( Figure 3 a, middle). The larger the distance, the more sensitive the probe is; the distance between the SNV and WT detection signals is defined as parameter 2 (relative distance, dr), which is used to determine the specificity of the probe in detecting mutations ( Figure 3 a, right). The larger the dr, the stronger the ability of the probe to distinguish mutations.

[0118] Next, we used the T>A mutation as a model to explore the ability of the catalytic mutation recognition probe to broaden the high-specificity concentration range. Through theoretical simulation combined with experimental verification, we found that the catalytic mutation recognition probe gradually broadens the high-specificity concentration range as the reaction time increases ( Figure 3 b). The entire target concentration range (0.49 nM to 125 nM) showed high specificity in distinguishing mutations until the reaction time was 120 min ( Figure 3 c). And for different mutation types and mutation sites, the high specific concentration range of mutation differentiation can be successfully expanded (0.49nM to 125nM), indicating that the probe has good versatility ( Figure 3 d and 3e).

[0119] 3. Analyze performance

[0120] In order to characterize the analytical performance of the catalytic mutation recognition probe in mutation detection, the sensitivity and specificity of the nucleic acid probe for detecting mutant targets were first evaluated. The nucleic acid probe was used to detect synthetic single-stranded targets at different concentrations, and its limit of detection (LOD) was estimated to be 0.24nM (P<0.001), which is more sensitive than the detection limit of most traditional nucleic acid hybridization probes (LOD=1.98nM). The specificity of the probe in analyzing low variant allele frequencies (VAFs) mutations was also determined. Since the concentration of the real sample is unknown, the dR value in this case is equivalent to the projection on the plane of the two fluorescence channels and the concentration, displayed in a three-dimensional space, where the detection signal of the wild type (WT) is lower and the detection signal of the single nucleotide variation (SNV) is higher ( Figure 4 a). By analyzing DNA mixtures with VAF values ​​ranging from 0.1% to 10%, it was found that the detection signal of 0.1% mutation was significantly different from that of 0% (WT), indicating that the method of the present application is more effective in distinguishing SNVs with VAF values ​​as low as 0.1% ( Figure 4 b and 4c).

[0121] In order to further investigate the universality of the catalytic mutation recognition probe, corresponding probes were designed for 17 biologically important SNVs and their ability to distinguish mutations was explored. The results showed that the probe successfully distinguished 17 mutations, and its ability to distinguish mutations was enhanced compared with the traditional toehold exchange probe, with the enhancement factor ranging from 2.1 to 31.4 (average 14) ( Figure 4 d).

[0122] The probe consists only of nucleic acids, so it has good compatibility with nucleic acid detection methods. To demonstrate its usefulness in biotechnology applications, the catalytic mutation recognition probe was next combined with PCR to analyze SNVs in synthetic DNA templates. Before cascade PCR amplification, it was cascaded with the previously developed mismatch-mediated assembly probe (PANDA) to convert dsDNA into ssDNA. Then, the two were combined with PCR, and the workflow is as follows: Figure 5 By cascading the above nucleic acid detection and amplification methods, BRAF V600E mutations as low as 1aM were successfully detected from synthetic samples ( Figure 5 b) and BRAF V600E mutation with a mutation abundance of 0.1% ( Figure 5 c), this result shows the excellent detection sensitivity and specificity of the new technology of the present application. This detection performance can meet the detection needs of mutations in real thyroid cancer samples.

[0123] 4. Clinical Validation

[0124] In order to confirm the ability of the probe detection technology of this application in detecting clinical samples, it was then used to analyze SNVs in real clinical samples. The clinically important BRAF V600E was selected as the target. BRAF V600E is a conventional auxiliary diagnostic indicator for preoperative diagnosis and prognosis of thyroid cancer.

[0125] A total of 117 clinical postoperative tissue samples from thyroid patients were analyzed, including 25 pairs of surgical frozen tissue samples (tumor and adjacent tissue) and 67 formalin-fixed paraffin-embedded (FFPE) postoperative tissue section samples. After genomic DNA was extracted from fresh tissue using QIAGEN's kit, genomic DNA containing BRAF V600E was PCR amplified, DNA assembled, and analyzed using a catalytic mutation recognition probe ( Figure 6a). For comparison, the same genomic DNA was tested using standard Sanger sequencing and digital PCR. In 25 adjacent tissue samples, the catalytic mutation recognition probe did not find any BRAF V600E mutations, which was consistent with the results of Sanger sequencing and digital PCR. In 25 tumor tissue samples, the catalytic mutation recognition probe detected BRAF V600E positive in 22 tumor tissue samples ( Figure 6 b). Comparison with Sanger sequencing revealed that the sequencing results of two cases (tumor tissues #4 and #5) were inconsistent with the results of the catalytic mutation recognition probe test ( Figure 6 c). The two cases were further confirmed using digital PCR testing, which revealed 18 and 15 BRAF V600E-positive droplets, respectively ( Figure 6 c). The detection results of the probe of the present application showed 100% consistency with digital PCR, demonstrating the excellent mutation detection capability of this technology.

[0126] Given the excellent analytical performance of the catalytic mutation recognition probe, the next challenge is to use the catalytic mutation recognition probe to analyze low-abundance BRAF V600E mutations in FFPE samples. The detection flow chart is as follows: Figure 6 a. Twenty-two samples (26-47#) of benign thyroid nodules were collected and analyzed to establish a baseline and serve as a negative control for the detection of BRAF V600E. BRAF V600E mutations were analyzed in 45 FFPE samples of thyroid cancer using three methods: catalytic mutation recognition probes, Sanger sequencing, and digital PCR. Figure 6 d and Figure 6 The results in e show that the catalytic mutation recognition probe and digital PCR showed 100% consistency. However, Sanger sequencing only successfully completed the sequencing of 6 samples. It is speculated that this may be due to the breakage of genomic DNA caused by heating at 90°C for a long time (1 hour) during the extraction of genomic DNA.

[0127] Based on digital PCR, the receiver operating characteristic (ROC) curve was drawn, and its area under the line (AUC) value was 1, which has a good diagnostic value ( Figure 6 f). The clinical sensitivity and specificity of the catalytic mutation recognition probe for detecting mutations were determined to be 100% (n=117) ( Figure 6 g).

[0128] 5. Clinical Application

[0129] Thyroid nodular lesions are a common problem for clinicians and a diagnostic challenge for pathologists to accurately diagnose malignancy to ensure that patients receive appropriate treatment. Fine needle aspiration (FNA) is the most commonly used method to help establish the diagnosis of malignancy preoperatively. However, approximately 25% of thyroid nodules are classified as malignant with uncertainty at the time of diagnosis by the Bethesda Thyroid Fine Needle Aspiration Cytology Reporting System (TBSRTC). Current molecular testing for BRAF V600E mutations shows greater potential and has been used to confirm the diagnosis in suspicious cases. However, the small FNA sample size and low abundance of BRAF V600E mutations make it challenging to analyze BRAF V600E mutations using conventional PCR or hybridization-based techniques.

[0130] To further demonstrate the clinical potential of the catalytic mutation recognition probe in detecting low-abundance mutations in thyroid nodule FNA samples, 52 FNA samples were collected, all of which had not been confirmed by thyroid cytopathology at West China Hospital of Sichuan University ( Figure 7 a). First, the gDNA content and VAFs of BRAF V600E in FNA samples were quantified. The results showed that the distribution of gDNA content and VAFs in FNA samples was 2ng-34ng (mean = 11.15ng) and 0.12%-37% (mean = 9.35%), respectively. ( Figure 7 b and 7c).

[0131] To demonstrate the potential of the catalytic mutation recognition probe to detect low-abundance gDNA and low-VAFs BRAF V600E, four assays (Sanger sequencing, qPCR, dPCR, and catalytic mutation recognition probe) were evaluated for their detection limits ( Figure 7 d). The gDNA extracted from FFPE samples was serially diluted, and then the BRAF V600E mutation was detected using four detection methods. The results showed that Sanger sequencing could only detect BRAF V600E from 50ng gDNA, while the detection limit of qPCR was slightly higher than that of Sanger sequencing, at 5.53ng. Both dPCR and catalytic mutation recognition probes could detect BRAF V600E mutations as low as 0.2ng gDNA. This result suggests that the catalytic mutation recognition probe has the potential to analyze low-abundance and low-concentration BRAF V600E in FNA samples in an economical manner.

[0132] Taking advantage of the excellent analytical performance of the catalytic mutation recognition probe, it was used to analyze BRAFV600E in 52 FNA samples, all of which were identified by thyroid cytopathology and qPCR in our hospital ( Figure 7a). Of the 52 FNA samples, 45 were positive for BRAF V600E identified by the catalytic mutation recognition probe ( Figure 7 e). Compared with the test results of TBSRTC, qPCR successfully identified 46.1% (24 / 52) of malignant tumors, while the catalytic mutation recognition probe successfully identified 86.5% (45 / 52) of malignant tumors ( Figure 7 f). Compared with the clinical results, the clinical sensitivity and specificity of the catalytic mutation recognition probe (n=52) were 90.0% and 100%, respectively ( Figure 7 g). The results showed that the catalytic mutation recognition probe significantly reduced the number of uncertain cases and also had the potential to provide a more sensitive and accurate mutation analysis technique for preoperative diagnosis of thyroid puncture samples.

[0133] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily conceive of changes or substitutions within the technical scope disclosed by the present invention, and such changes or substitutions shall be within the scope of protection of the present invention.

Claims

1. A method for detecting gene mutation, characterized in that: The detection method includes using three probes, wherein the ends of the first probes are each modified with a fluorescent group, the ends of the second probes are each modified with a fluorescent quenching group, and the 5' end or 3' end of the third probe is modified with a fluorescent quenching group.

2. The detection method according to claim 1, characterized in that: The first probe and the second probe are combined into a double-stranded probe set, the first probe and the second probe are complementary to each other by at least 15 bp, and the third probe contains the same nucleotide sequence as the first or second probe.

3. The detection method according to any one of claims 1-2, characterized in that: The fluorescent groups include ALEX-350, FAM, VIC, TET, CAL Gold540, JOE, HEX, CAL Orange560, TAMRA, CAL Red590, ROX, CAL Red610, TEXAS RED, CAL Red635, Quasar670, CY3, CY5, CY5.5 and / or Quasar705; The fluorescence quenching group includes DABCYL, BHQ quencher, ECLIPSE and / or TAMRA.

4. The detection method according to any one of claims 1 to 3, characterized in that: The two fluorescent groups at the ends of the first probe are the same or different, the two fluorescent quenching groups at the ends of the second probe are the same or different, and the fluorescent quenching group of the third probe is different from the fluorescent quenching group of the second probe or is the same as the fluorescent quenching group at at least one of the ends.

5. The detection method according to any one of claims 1 to 4, characterized in that: The sequence of the probe comprises the complementary sequence of the wild-type or mutant target sequence, or the sequence of the probe has one or more mismatches compared to the complementary sequence of the wild-type or mutant target nucleic acid sequence.

6. The detection method according to any one of claims 1 to 5, characterized in that: The mutations include single nucleotide mutations.

7. The detection method according to any one of claims 1 to 6, characterized in that: The detection method comprises: (1) mixing the first probe and the second probe, adding the target sequence to be detected, and detecting the fluorescence intensity; (2) Add the third probe and detect the fluorescence intensity.

8. The detection method according to any one of claims 1 to 7, characterized in that: The detection method also includes the combination with other methods, and the other methods include PCR method, LCR method, TAS method, ICAN method, LAMP method, NASBA method, RCA method, TAMA method and / or UCAN method.

9. A probe set for use in the gene mutation detection method according to any one of claims 1 to 8, characterized in that: The probe group includes the first, second and third probes.

10. A kit, characterized in that: The kit comprises the probe set according to claim 9.

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