Method for detecting gene mutation by combining single dNTP deletion with polymerase / ligase reaction

Abstract

The invention discloses a method for detecting gene mutation based on single dNTP deletion in combination with polymerase / ligase reaction, and relates to the technical field of molecular detection and diagnosis. According to the invention, three probes and two Partzymes are designed according to a mutant target nucleic acid sequence. DNA target nucleic acid is used as a template, specific extension based on base deletion is carried out under the action of DNA polymerase, meanwhile, a specific ligation reaction is carried out under the action of DNA ligase, two Partzyme can recognize a ligation product and assemble to form an active MNAzyme cleavage signal probe, fluorescence signals generated by cleavage are monitored in real time, and real-time quantitative detection of the target nucleic acid is achieved. The method provided by the invention is a real-time detection method, has specific extension of polymerase and specific connection of ligase, and improves the specificity of the method.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular detection and diagnosis, and in particular relates to a method for detecting gene mutations based on single dNTP deficiency combined with polymerase / ligase reaction. Background Art

[0002] Genetic mutations refer to changes that occur at the molecular level of DNA within the genome. They can be diverse, including single-base mutations, insertions, and deletions. A significant number of these mutations are closely linked to human diseases, such as cancer and various genetic disorders. For example, the JAK2 V617F mutation is caused by a single nucleotide substitution at position 1849 in exon 14 of the JAK2 gene. This substitution replaces the original G with a T. This change changes the amino acid at position 617 in the encoded JAK2 protein from valine (V) to phenylalanine (F). The JAK2 V617F mutation is present in the majority of patients with myeloproliferative cancers (MPNs). For example, in polycythemia vera (PV), this mutation is detected in over 95% of PV cases. In-depth research on genetic mutations, particularly the detection of low-frequency mutations, can provide powerful tools for liquid biopsies and minimal residual disease (MRD) detection.

[0003] Among the methods used to detect gene mutations, polymerase chain reaction (PCR) and gene sequencing are relatively common. PCR uses polymerase for amplification, resulting in high sensitivity but poor specificity. Therefore, PCR is often combined with other reactions to mitigate its limitations. For example, the amplification of refractory mutations system-polymerase chain reaction (ARMS-PCR) (Jones AV, et al., 2005) is simple and rapid, but its ability to discriminate mutation frequencies generally reaches 0.5%. Polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-SSCP) involves electrophoresis of amplified products to detect bands, a cumbersome process. Gene sequencing encompasses various types, including first-generation sequencing (such as Sanger sequencing) and second-generation sequencing. While first-generation sequencing can accurately detect DNA sequences, it can only detect approximately 10% of mutations. Second-generation sequencing can sequence thousands of genes, or even entire genomes, enabling simultaneous and parallel sequencing of multiple genes. However, second-generation sequencing is more complex in terms of data analysis, and detecting low-frequency mutations requires ultra-deep sequencing.

[0004] Therefore, it is necessary to develop a new method for detecting gene mutations (especially low-frequency mutations) with high specificity and simple operation. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting gene mutations based on single dNTP deletion combined with polymerase / ligase reaction. The present invention designs three probes and two Partzymes based on the mutant target nucleic acid sequence. Using the DNA target nucleic acid as a template, a specific extension based on base deletion is performed under the action of DNA polymerase, and a specific ligation reaction is performed under the action of DNA ligase. The two Partzymes will recognize the ligation product and assemble to form an active MNAzyme cutting signal probe, and the fluorescent signal generated by the cutting is monitored in real time to achieve real-time quantitative detection of the target nucleic acid. The present invention is a real-time detection method that combines the specific extension of the polymerase and the specific ligation of the ligase, thereby improving the specificity of the method.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] One of the purposes of the present invention is to provide a composition for detecting gene mutations based on single dNTP deletion combined with polymerase / ligase reaction, the composition comprising a probe set and an enzyme system set; the probe set comprises nucleic acid probes 1, 2, 3 and a signal probe, and the enzyme system set comprises a polymerase, a ligase and an MNAzyme; wherein:

[0008] The probe 1 is complementary to one strand of the mutant target nucleic acid, the probes 2 and 3 are complementary to the other strand of the target nucleic acid, the probe 3 is complementary to the probe 1, and the probes 2 and 3 are separated by bases;

[0009] The MNAzyme includes Partzyme A and Partzyme B, wherein Partzyme A and Partzyme B respectively contain a target nucleic acid recognition arm, an active center and a signal probe recognition arm, wherein the target nucleic acid recognition arm is complementary to the probe 1 and its extended sequence, and the signal probe recognition arm is complementary to the signal probe.

[0010] Preferably, the 5′ terminal base of the probe 3 is a designed missing dNTP base and is phosphorylated; the probe 2 and the probe 3 are separated by 1-5 bases and cannot contain missing dNTP bases.

[0011] In addition, in the present invention, the 5′ terminal base of probe 3 cannot protrude more than that of probe 1 during design.

[0012] Preferably, the active center of the MNAzyme is a 8-17 DNAzyme or a 10-23 DNAzyme.

[0013] Preferably, the usage ratio of the probe 1, the probe 2, the probe 3, the Partzyme A and the Partzyme B is (1-3): (0.8-1.2): (0.8-1.2): (1.8-2.2): (1.8-2.2).

[0014] Preferably, the polymerase is a hot-start DNA polymerase without 5′-3′ exonuclease activity; the ligase is a thermostable DNA ligase; the signal probe contains an RNA base in the middle, one end is labeled with a quencher group, and the other end or the middle side is labeled with a fluorescent group, and the interval between the fluorescent group and the quencher group is less than 20 bp.

[0015] A second object of the present invention is to provide an application of the composition in preparing a gene mutation detection product. In the application, the method for using the gene mutation detection product comprises the following steps:

[0016] (1) The nucleic acid to be tested and the probe 1, probe 2, probe 3, Partzyme A, Partzyme B, signal probe, polymerase, and ligase in the test product are thoroughly mixed, and a ligation reaction and a polymerization reaction are performed in a real-time quantitative PCR instrument to hybridize the target nucleic acid recognition arms of Partzyme A and Partzyme B with probe 1 and its extended sequence, assemble into an MNAzyme and activate its catalytic cleavage activity, cyclically cut the signal probe hybridized with it, and release the fluorescent signal of the fluorescent group;

[0017] (2) Monitor the intensity of the fluorescence signal to obtain a real-time amplification curve, establish a standard curve based on the relationship between the cycle number and the concentration of the target nucleic acid, and calculate the content of the target nucleic acid based on the cycle number of the target nucleic acid.

[0018] Preferably, the gene mutation used for detection includes the JAK2 V617F gene mutation, and its mutation sequence is shown in SEQ ID NO.1-SEQ ID NO.2; in the step (1), the probe 1 sequence for detecting the JAK2 V617F gene mutation is shown in SEQ ID NO.5, the probe 2 sequence is shown in SEQ ID NO.6, the probe 3 sequence is shown in SEQ ID NO.7, the Partzyme A sequence is shown in SEQ ID NO.8, and the Partzyme B sequence is shown in SEQ ID NO.9.

[0019] Preferably, in step (1), the polymerase used to detect the JAK2 V617F gene mutation includes Hieff HotStart Super Specific Taq DNA Polymerase, ligase including Taq DNA ligase.

[0020] Preferably, in step (1), the polymerase / ligase reaction process for detecting the JAK2 V617F gene mutation is as follows: the mixed solution is transferred to a real-time quantitative PCR instrument, and two cycling stages are immediately performed, wherein the first stage: first denaturation at 90-99°C for 5-30s, then cooling to 58-65°C for 20-60s amplification, and 5-30 thermal cycles; the second stage: maintaining at 90-99°C for 5-30s, maintaining at 58-65°C for 20-60s, maintaining at 50°C for 60s, and repeating the thermal cycle.

[0021] The application of the present invention also includes a method for detecting gene mutations based on single dNTP deletion combined with polymerase / ligase reaction, the method comprising the following steps:

[0022] (1) Design three probes, namely probe 1, probe 2, and probe 3, based on the sequence of the nucleic acid to be detected;

[0023] Among them, probe 1 is complementary to one strand of the target nucleic acid, probe 3 is complementary to probe 1, and the 5′ end of probe 3 is phosphorylated, the 3′ end of probe 3 and Partzyme B are amino-blocked, and probes 2 and 3 are separated by bases;

[0024] (2) Designing two parts of the MNAzyme: Partzyme A and Partzyme B, where Partzyme A and Partzyme B contain the target nucleic acid recognition arm, the active center, and the signal probe recognition arm, respectively;

[0025] The target nucleic acid recognition arm of Partzyme is complementary to probe 1 and its extended sequence, and the signal probe recognition arm of Partzyme is complementary to the signal probe;

[0026] (3) The nucleic acid to be tested, probe 1, probe 2, probe 3, Partzyme A, Partzyme B, signal probe, polymerase, and ligase are thoroughly mixed, and ligase reaction and polymerase reaction are carried out in a real-time quantitative PCR instrument;

[0027] The extension of probe 1 and probe 2 is carried out under the action of DNA polymerase, and the ligation reaction is to ligate probe 2 and probe 3 under the action of DNA ligase to form a long DNA chain.

[0028] The target nucleic acid recognition arms of Partzyme A and Partzyme B hybridize with probe 1 and its extended sequence, assembling into an MNAzyme and activating its catalytic cleavage activity, cyclically cleaving the signal probe hybridized with it and releasing the fluorescent signal of the fluorescent group;

[0029] (4) monitoring the fluorescence signal intensity to obtain a real-time amplification curve, establishing a standard curve based on the relationship between the cycle number and the concentration of the target nucleic acid, and calculating the target nucleic acid content based on the cycle number of the target nucleic acid;

[0030] Preferably, the active center of the MNAzyme in step (2) is preferably 8-17 DNAzyme or 10-23 DNAzyme.

[0031] Preferably, the usage ratio of probe 1, probe 2, probe 3, Partzyme A, and Partzyme B in step (3) is preferably 1:1:1:2:2.

[0032] Preferably, in step (1), probe 2 and probe 3 are separated by four bases, TTCT, and the reaction lacks a G base. The first T in the TTCT from 5' to 3' direction is the position where the target chain mutates, and the polymerization reaction stops at the first required G base.

[0033] Preferably, the signal probe in step (3) preferably contains an RNA base in the middle, is labeled with a quencher group at one end, and is labeled with a fluorescent group at the other end or in the middle, and the interval between the fluorescent group and the quencher group is 16 bp.

[0034] More preferably, the fluorescent group is FAM, VIC, ROX, CY5, HEX, Texas Red, or LightCycler Red 640.

[0035] Preferably, the polymerase in step (3) is a hot-start DNA polymerase, and the ligase is a thermostable DNA ligase.

[0036] Preferably, the DNA polymerase is obtained from a bacterium selected from the group consisting of: Thermus aquaticus (Taq), Thermusthermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermusflavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus thermophllus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcuslitoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcushorikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifexaeolieus.

[0037] More preferably, the DNA polymerase is Taq polymerase;

[0038] More preferably, the hot start DNA polymerase is Hieff HotStart Super Specific TaqDNA polymerase, thermostable DNA ligase Taq DNA ligase.

[0039] Preferably, the process of the polymerase / ligase reaction in step (3) is preferably as follows: the mixed solution is quickly transferred to a real-time quantitative PCR instrument, and two cycling stages are immediately performed, wherein the first stage: first denaturation at 94°C for 15s, then cooling to 62°C for 60s amplification, and performing 26 thermal cycles; the second stage: maintaining at 94°C for 15s, maintaining at 62°C for 30s, maintaining at 50°C for 60s, and repeating the thermal cycle.

[0040] The method for detecting gene mutations based on the lack of dGTP combined with polymerase / ligase reaction in the present invention is based on the following principle: MNAzyme is composed of two or more oligonucleotides, wherein at least two oligonucleotides are composed of multiple recognition sequences, which are called Partzyme. In the presence of target nucleic acid, two Partzymes (A and B) self-assemble to form an MNAzyme with catalytic activity. MNAzyme can simultaneously recognize target nucleic acid and signal probe, and the present invention introduces it into the polymerase / ligase reaction lacking dGTP as a real-time detection strategy. First, probes 1, 2, 3 and two Partzymes are designed. Then, through high-temperature denaturation and low-temperature annealing, the probes are extended and connected under the action of DNA polymerase and DNA ligase to achieve amplification of the nucleic acid to be tested. The Partzyme then combines with the reaction product to assemble to form an MNAzyme with an active center. The MNAzyme can cleave the signal probe to release a fluorescent signal, forming a real-time amplification curve, and realizing real-time detection (the schematic diagram is shown in FIG. 1 ). Figure 1 shown).

[0041] A third object of the present invention is to provide a gene mutation detection product, which comprises the gene mutation detection product in the composition or the application, and its product forms include detection reagents and detection kits.

[0042] Compared with the prior art, the present invention has the following technical effects:

[0043] (1) The present invention innovatively combines polymerase reaction and ligase reaction. By designing that the probes are separated by bases (such as TTCT) and the system lacks the dNTP (such as dGTP) required for the wild template, the polymerase will not extend with the wild template, and only the mutant template is allowed to complete the polymerization reaction and ligation reaction, thereby enhancing the specific recognition of single-base mutations; the present invention designs three probes (probes 1, 2, and 3), among which probes 2 and 3 are deliberately designed to be separated by bases, and combined with the phosphorylation modification of probe 3, ligation is completed only when the mutant template is present, significantly improving the ability to distinguish mutations; although the missing base may be used to block extension in the prior art, the present invention combines it with the ligation reaction and MNAzyme to achieve highly specific mutation detection, which is an inventive combination.

[0044] (2) The present invention cleverly combines polymerization reaction, ligation reaction and MNAzyme, performs specific extension under the action of DNA polymerase, and performs specific ligation reaction under the action of DNA ligase at the same time, and combines the specific recognition of MNAzyme to greatly improve the specificity of the detection method, so that it has good discrimination ability for single base differences; the highly sensitive polymerization / ligation reaction in the present invention is carried out simultaneously with MNAzyme, amplifying and detecting at the same time, and the operation is simple. Very low frequency DNA mutations in the reaction can be detected, which can be used for quantitative detection of mutant DNA; the present invention realizes dual specificity verification (extension termination + ligation verification) in a single reaction system by simultaneously utilizing polymerase extension and ligase ligation, significantly reducing false positives.

[0045] (3) The detection sensitivity of the present invention reaches 10aM and can distinguish low-frequency mutations as low as 0.05%, which is more advantageous in clinical applications (such as liquid biopsy and minimal residual lesion detection). Therefore, the present invention will provide a new approach to real-time detection with good specificity for nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the principle of detecting gene mutation based on the missing dGTP binding polymerase / ligase reaction in the present invention.

[0047] Figure 2 This is a graph of amplification curves in the presence of different enzymes in the verification experiment of Example 1 of the present invention, wherein: Figure 2 a means polymerase and ligase are present simultaneously; Figure 2 b means only ligase is present; Figure 2 c: Only polymerase is present.

[0048] Figure 3 is the sensitivity test result in Example 1 of the present invention, wherein: Figure 3 a is the relationship between △Fl and cycle number; Figure 3 b is C T The linear relationship between the value and the logarithm of the sample concentration.

[0049] Figure 4 This is the effect of different probe ratios in Example 1 of the present invention, where: a. 10:20:20:20:20; b. 20:20:20:40:40; c. 40:20:20:80:80; d. 60:20:20:120:120 (nM).

[0050] Figure 5 This is the specific amplification curve for evaluating the ability to distinguish single-base differences in Example 1 of the present invention.

[0051] Figure 6This is the verification result that the method in Example 1 of the present invention can be used for quantitative determination of base mutations, wherein: Figure 6 a is the amplification curve of different ratios of JAK mut and JAK wild; Figure 6 b is the logarithm of the ratio of JAK mut to JAK wild and its C T Value relationship diagram. DETAILED DESCRIPTION

[0052] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the inventive method, steps or conditions are intended to fall within the scope of the present invention. The reagents, products, instruments, etc. used in the following examples are all commercially available, and the methods used in the examples are consistent with conventional methods unless otherwise specified.

[0053] The technical solution of the present invention is further elaborated in detail below in conjunction with embodiments.

[0054] Example 1

[0055] This example provides a method for detecting the JAK2 V617F gene mutation based on the absence of dGTP-binding polymerase / ligase reaction, wherein the nucleic acid sequence used is shown in Table 1, and specifically comprises the following steps:

[0056] Table 1 Nucleotide sequence details

[0057]

[0058]

[0059] Note: The underlined bases in JAK-mut and JAK-mut-c represent single-base differential sites. The P in probe 3 indicates phosphorylation. The single underlined base in Partzyme represents the sensor arm that hybridizes complementary to probe 1 and its extension sequence. The italicized text indicates the active region. The double underlined base represents the signal probe recognition arm. The T (FAM) in the signal probe represents a T base modified with a FAM group. BHQ1 is a quencher group modification. Note: Because "T" in NIPOST.26 represents uracil in RNA sequences and thymine in DNA sequences, the "U" in the above sequences was converted to "T" during production.

[0060] 1. Experimental Methods

[0061] 1.1 Target chain hybridization

[0062] A 10 μL hybridization system was prepared by mixing 1 μL of 10 μM JAK-mut, 1 μL of 10 μM JAK-mut-c, and 8 μL of STE buffer (100 mM NaCl, 10 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8)). The entire process was performed on ice to maintain a low temperature environment. The system was then transferred to a preheated 2720 PCR instrument (thermal cycler), denatured at 95°C for 5 minutes, hybridized at 25°C for 20 minutes, and stored at -20°C until use. Wild-type JAK and wild-type JAK-c controls were prepared using the same method. (Higher concentrations of the reaction product can be prepared and stored at -20°C for short-term storage.)

[0063] 1.2 Detection of JAK2 V617F mutation based on the missing dGTP-binding polymerase / ligase reaction

[0064] The reaction was carried out in a 10 μL system containing 1 μL Buffer (10×) (200 mM Tris-HCl, 250 mM KCl, 100 mM MgCl2, 5 mM NAD, 0.1% Triton, pH 8.3, 25°C), 0.3 μL MgCl2 (100 mM), 0.2 μL probe 1 (1 μM), 0.2 μL probe 2 (1 μM), 0.2 μL probe 3 (1 μM), 0.4 μL Part A (1 μM), 0.4 μL Part B (1 μM), 0.5 μL dCTP (100 μM), 0.5 μL dATP (100 μM), 0.5 μL dTTP (100 μM), 1 μL signal probe (2 μM), 0.15 μL Plus RNase Inhibitor(40U / μL), 0.125μL Taq DNA Ligase (40U / μL), 0.2μL Hieff Add 0.5 U / μL of HotStart Super Specific Taq DNA polymerase to the mixture, followed by 1 μL of DNA hybridized according to step 1.1 at various concentrations (including a blank run without target). Make up the remaining volume with RNase-free water. Mix thoroughly (perform all operations on ice). Transfer the mixed solution quickly to a real-time quantitative PCR instrument and immediately perform two thermal cycles. In the first stage, denaturation is performed at 94°C for 15 seconds, followed by cooling to 62°C for 60 seconds and amplification, for 26 cycles. In the second stage, a series of thermal cycles is repeated at 94°C for 15 seconds, 62°C for 30 seconds, and 50°C for 60 seconds, with fluorescence signal acquisition at 50°C.

[0065] 1.3 Data Processing

[0066] The relationship between the number of cycles and Fluorescence (Fl) was obtained using the FAM fluorescence signal. The slow rising trend at the beginning of the amplification curve is the base signal caused by the signal probe. To deduct the base signal, the Fl in the slow rising period of the amplification curve was fitted with the cycle number to obtain a linear relationship between the base signal, and the Fl base value at different cycle numbers was calculated. The difference between the Fl value and the Fl base value was linearly fitted with the cycle number to obtain a relationship between △Fl and the cycle number. Finally, the threshold was determined, and the intersection of the threshold and the amplification curve was taken as C T Value, analysis C T The relationship between the value and the concentration of the target substance.

[0067] 1.4 Evaluation of single-base difference discrimination ability

[0068] Add a mixture of JAK2 V617F with different mutation frequencies to a total of 100 fM in a 10 μL system. The mutation frequencies are 0%, 0.05%, 0.5%, 5%, 50%, and 100%, respectively. Perform the remaining reagents, amounts, and operations according to 1.2.

[0069] 1.5 Cell culture and DNA extraction

[0070] Human erythroleukemia (HEL) cells were cultured in 1940 (BIOFIL) medium containing 10% (v / v) fetal bovine serum (Sijiqing), 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture medium was placed in a 37°C carbon dioxide incubator containing 5% CO2 for incubation. DNA was extracted from the cells using a DNA extraction kit (TIANGEN) according to the manufacturer's instructions. The extracted DNA samples were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The quantified DNA samples were stored in a -20°C refrigerator for subsequent actual sample testing.

[0071] 2. Feasibility Verification

[0072] In order to verify the feasibility of the method of the present invention, this example first tested the signals in the presence of different enzymes, such as Figure 2 As shown in the figure, the signal only occurs when DNA ligase and hot-start DNA polymerase (without 5′→3′ polymerase activity) are present at the same time. At this time, the C T The value is much smaller than the blank C T The difference between the two is clear. Neither the blank nor the sample showed any signal when only DNA ligase or hot-start DNA polymerase was present. This demonstrates that a chain reaction can only occur when both DNA ligase and hot-start DNA polymerase are present, indicating that the principle design of this method is feasible.

[0073] 3. Sensitivity test

[0074] In this example, a standard curve was determined using double-stranded DNA at different concentrations as the target sequence, and the sensitivity of the experiment was tested. Figure 3 As shown in a, as the target DNA concentration increases from 10aM to 100fM, the corresponding C T The value is decreasing, with the logarithm of the sample concentration as the horizontal axis, C T The vertical axis is plotted as Figure 3 b, Figure 3 b shows the C corresponding to each sample in the concentration range of 10aM-100fM. T The value shows a good linear relationship with the logarithm of the sample concentration (logC), and the linear equation is: C T =-52.35-4.53lgC, the correlation coefficient is R 2 =0.9997. At the same time, we tested the real-time fluorescence curve of 10 fM target DNA seven times in parallel and calculated the relative standard deviation (RSD) to be 6.92%, indicating that the method has good reproducibility.

[0075] 4. Impact of different probe ratios

[0076] Different probe ratios (Probe 1: Probe 2: Probe 3: Partzyme A: Partzyme B) can affect the reaction efficiency and sensitivity of the invention. This example tested four ratios. The specific experimental conditions are as follows:

[0077] Signal probe: 200 nM; Buffer 1×; MgCl2 3mM; dCTP, dATP, dTTP: 5μM; Plus RNase Inhibitor 0.6U / μL; Taq DNA Ligase 0.5U / μL; Hieff HotStart Super Specific Taq DNA polymerase 0.01 U / μL; Probe 1: Probe 2: Probe 3: 20 nM: Partzyme A, Partzyme B: 10:20:20:20:20 nM (group a), 20:20:20:40:40 nM (group b), 40:20:20:80:80 nM (group c), and 60:20:20:120:120 nM (group d) (the above reagent concentrations are the final concentrations in a 10 μL reaction volume). Cycle schedule: 94°C / 15 s, 62°C / 60 s, 26 cycles; 94°C / 15 s, 62°C / 30 s, 50°C / 60 s, 60 cycles. Three replicates were performed.

[0078] like Figure 4 As shown in the figure, when the probe ratio is 10:20:20:20:20:20, the amount of probe 1 is less than that of probe 2 and probe 3, and no more probe 1 extension products can be obtained, Partzyme A / Partzyme B cannot be effectively assembled, and no amplification curve is formed; when the probe ratio is 20:20:20:40:40, an effective amplification curve can be formed. The probe ratio is further changed to 40:20:20:80:80 or even 60:20:20:120:120, blank and sample C T The values all gradually increase, which may be due to the larger difference in the ratio of probes 2 and 3 to probe 1, and the reduced amplification efficiency. However, they can still be distinguished well, but the time is longer.

[0079] 5. Evaluation of the ability to distinguish single-base differences

[0080] In order to evaluate the ability of the present invention to distinguish single-base differences, a JAK wild type that differs from the target DNA by only one base was introduced. The specific experimental conditions are as follows:

[0081] Probe 1, Probe 2, Probe 3: 20 nM; Partzyme A, Partzyme B: 40 nM; Signaling probe: 200 nM; Buffer 1×; MgCl2 3mM; dCTP, dATP, dTTP: 5μM; Plus RNaseInhibitor 0.6U / μL; Taq DNA Ligase 0.5U / μL; Hieff HotStart SuperSpecific Taq DNA polymerase 0.01 U / μL (the above reagent concentrations are the final concentrations in a 10 μL reaction volume). Cycle schedule: 94°C / 15 s, 62°C / 60 s, 26 cycles; 94°C / 15 s, 62°C / 30 s, 50°C / 60 s, 60 cycles. Three replicates were used.

[0082] from Figure 5 It can be seen that when there is a mutant base, the cycle number of the wild-type DNA is similar to that of the blank group and is much larger than that of the mutant DNA, indicating that this method has good specificity and can be used to distinguish single-base differences in DNA sequences.

[0083] The present invention further mixed JAK mut and JAK wild in different ratios as samples, with a total amount of 100 fM, and the mutant JAK mut was 0%, 0.05%, 0.5%, 5%, 50%, and 100% respectively, and reacted. Figure 6 As shown in a, as the proportion of JAK mut increases, its C T The value is decreasing, C T The value showed a good linear relationship with the logarithm of the mutation ratio, and the regression equation was: C T =19.14-5.341gMR (MR is Mutation Ratio, R 2 =0.9948). The above results indicate that the present invention can be used for the quantitative determination of base mutations.

[0084] 5. Spike recovery of actual samples

[0085] In order to evaluate the effectiveness of the present invention on actual samples, the spike recovery method was used to measure the DNA of human erythroleukemia (HEL) cells. The results are shown in Table 2.

[0086] Table 2 Results of spike recovery experiments (n=3) of actual samples

[0087]

[0088] As shown in Table 2, the recovery rate of the present invention is between 86.7% and 98.4%, and the relative standard deviation (RSD) is between 1.17% and 8.56%, which proves that the present invention can effectively determine the cellular DNA content and is effective in detecting actual samples.

[0089] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A composition for detecting gene mutations based on single dNTP deletion combined with polymerase / ligase reaction, characterized in that: The composition comprises a probe set and an enzyme system set; the probe set comprises nucleic acid probes 1, 2, 3 and a signal probe, and the enzyme system set comprises a polymerase, a ligase and an MNAzyme; wherein: The probe 1 is complementary to one strand of the mutant target nucleic acid, the probes 2 and 3 are complementary to the other strand of the target nucleic acid, the probe 3 is complementary to the probe 1, and the probes 2 and 3 are separated by bases; The MNAzyme includes Partzyme A and Partzyme B, wherein Partzyme A and Partzyme B respectively contain a target nucleic acid recognition arm, an active center and a signal probe recognition arm, wherein the target nucleic acid recognition arm is complementary to the probe 1 and its extended sequence, and the signal probe recognition arm is complementary to the signal probe.

2. The composition for detecting gene mutations based on single dNTP deletion combined with polymerase / ligase reaction according to claim 1, characterized in that: The 5′ terminal base of the probe 3 is a designed missing dNTP base and is phosphorylated; the probe 2 and the probe 3 are separated by 1-5 bases and cannot contain missing dNTP bases.

3. The composition for detecting gene mutations based on single dNTP deletion combined with polymerase / ligase reaction according to claim 2, characterized in that: The active center of the MNAzyme is an 8-17 DNAzyme or a 10-23 DNAzyme.

4. The composition for detecting gene mutations based on single dNTP deletion combined with polymerase / ligase reaction according to claim 3, characterized in that: The usage ratio of the probe 1, the probe 2, the probe 3, the Partzyme A and the Partzyme B is (1-3): (0.8-1.2): (0.8-1.2): (1.8-2.2): (1.8-2.2).

5. The composition for detecting gene mutations based on single dNTP deletion combined with polymerase / ligase reaction according to claim 4, characterized in that: The polymerase is a hot-start DNA polymerase without 5′-3′ exonuclease activity; the ligase is a heat-resistant DNA ligase; the signal probe contains an RNA base in the middle, a quenching group is marked on one end, and a fluorescent group is marked on the other end or the middle side, and the interval between the fluorescent group and the quenching group is less than 20 bp.

6. Use of the composition according to any one of claims 1 to 5 in preparing a gene mutation detection product, characterized in that: In the application, the method for using the gene mutation detection product includes the following steps: (1) The nucleic acid to be tested and the probe 1, probe 2, probe 3, Partzyme A, Partzyme B, signal probe, polymerase, and ligase in the test product are thoroughly mixed, and a ligation reaction and a polymerization reaction are performed in a real-time quantitative PCR instrument to hybridize the target nucleic acid recognition arms of Partzyme A and Partzyme B with probe 1 and its extended sequence, assemble into an MNAzyme and activate its catalytic cleavage activity, cyclically cut the signal probe hybridized with it, and release the fluorescent signal of the fluorescent group; (2) Monitor the intensity of the fluorescence signal to obtain a real-time amplification curve, establish a standard curve based on the relationship between the cycle number and the concentration of the target nucleic acid, and calculate the content of the target nucleic acid based on the cycle number of the target nucleic acid.

7. The use according to claim 6, characterized in that The gene mutation used for detection includes the JAK2V617F gene mutation, whose mutation sequence is shown in SEQ ID NO.1-SEQ ID NO.2; in the step (1), the probe 1 sequence for detecting the JAK2 V617F gene mutation is shown in SEQ ID NO.5, the probe 2 sequence is shown in SEQ ID NO.6, the probe 3 sequence is shown in SEQ ID NO.7, the Partzyme A sequence is shown in SEQ ID NO.8, and the Partzyme B sequence is shown in SEQ ID NO.

9.

8. The use according to claim 7, characterized in that In the step (1), the polymerase used to detect the JAK2V617F gene mutation includes HotStart Super Specific Taq DNA Polymerase, ligase including Taq DNA ligase.

9. The use according to claim 8, characterized in that In step (1), the polymerase / ligase reaction process for detecting the JAK2V617F gene mutation is as follows: the mixed solution is transferred to a real-time quantitative PCR instrument, and two cycling stages are immediately performed, wherein the first stage: first denaturation at 90-99°C for 5-30s, then cooling to 58-65°C for 20-60s amplification, and 5-30 thermal cycles; the second stage: maintaining at 90-99°C for 5-30s, maintaining at 58-65°C for 20-60s, maintaining at 50°C for 60s, and repeating the thermal cycle.

10. A gene mutation detection product, characterized in that: It comprises the composition according to any one of claims 1-5 or the gene mutation detection product for use according to any one of claims 6-10, and its product forms include detection reagents and detection kits.

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