Primer pair, kit for detecting bcr-abl1 gene t315i mutation and application

Abstract

The application discloses a primer pair, a kit and application for detecting a BCR-ABL1 gene T315I mutation. The third and / or fourth bases of the 3' end of the upstream primer of the primer pair are introduced with corresponding mismatched bases, the second base of the 3' end of the primer is introduced with a dealkylation site, and the terminal base of the 3' end of the primer is modified by a locked nucleic acid. The primer pair and the kit for detecting the BCR-ABL1 gene T315I mutation can selectively amplify the T315I mutation, realize high-sensitivity detection (up to 10 copies) of the T315I mutation, have good specificity and high precision, can effectively distinguish the wild type and the mutant site, can eliminate the interference of the adjacent site T315A, are simple and rapid to operate, have a short time consumption in the whole detection process, can obtain a detection result within 90 minutes, are high in detection efficiency and low in cost.

Description

Technical Field

[0001] This invention relates to a primer pair for detecting the T315I mutation in the BCR-ABL1 gene, a corresponding kit, and its application in detecting the T315I mutation in the BCR-ABL1 gene, belonging to the field of gene detection technology in molecular biology. Background Technology

[0002] Chronic myeloid leukemia (CML) is a malignant myeloproliferative tumor that originates from pluripotent stem cells. At initial diagnosis, it is often in the chronic phase (CP). Without timely intervention, it can progress to the accelerated phase (AP) and blastic crisis (BC) within months or years, leading to extensive infiltration and proliferation of primitive cells in the bone marrow, or accompanied by extramedullary infiltration. This results in short survival, poor prognosis, and difficulty in achieving a cure. Its molecular mechanism involves the translocation of the ABL proto-oncogene on chromosome 9 to the breakpoint cluster region (BCR) on a segment of chromosome 22, forming the Philadelphia chromosome (Ph1) and producing the BCR-ABL1 fusion gene. This fusion gene encodes the P210 protein, which has abnormally elevated tyrosine protein kinase activity. This activates multiple downstream signaling pathways, including RAS, MAPK, PI3K / AKT, and STAT5, leading to the unlimited and continuous proliferation and accumulation of malignant CML cells. Ultimately, CML enters the BC phase, becoming a refractory leukemia.

[0003] Tyrosine kinase inhibitors (TKIs) target and inhibit the activity of tyrosine protein kinases, suppressing tumor cell proliferation and promoting apoptosis, ultimately leading to long-term, deep molecular remission (BCR-ABL1 fusion gene levels decreasing to negative), effectively alleviating patient symptoms and significantly improving prognosis. Imatinib (IM), the first TKI approved for the treatment of CML, can increase the 10-year overall survival rate of CML patients from less than 20% to 92%. Second-generation TKIs, such as dasatinib, niluotinib, and bosutinib, can achieve faster and deeper molecular remission. However, with the widespread clinical application of TKIs, drug resistance has gradually become a prominent issue. The mechanisms of TKI resistance are complex and diverse. Among them, point mutations in the kinase domain of BCR-ABL1 are the most common cause of secondary resistance, accounting for 30-70% of imatinib resistance. The main mechanism is that drug resistance mutations in BCR-ABL1 can affect the stability of the kinase conformation, causing it to transform from an inactive conformation to an active conformation, thereby hindering the effective binding of targeted drugs to the kinase target site, leading to TKI resistance and affecting the treatment effect.

[0004] Currently, dozens of mutation types have been identified, among which the T315I mutation is the most concerning in clinical applications. This is because, on the one hand, it has a greater risk of drug resistance and prognosis than other mutation types, and can disrupt the therapeutic effects of many first- and second-generation TKIs, hence the term "gatekeeper residue." On the other hand, the high GC content in this region makes it difficult to design primers for analysis, and the presence of nearby mutation sites (such as T315A) can interfere with the accuracy and sensitivity of T315I mutation analysis.

[0005] Currently, several analytical techniques have been reported for detecting BCR-ABL1 resistance mutations in clinical laboratories. Among them, Sanger sequencing (SS) is the most widely used gold standard. Its main procedure involves extracting RNA from a patient's bone marrow or peripheral blood sample, reversing it to cDNA, and then using nested PCR amplification. The first round amplifies the BCR-ABL fusion gene containing the ABL kinase region, and the second round amplifies the ABL kinase region from the first round product, followed by Sanger sequencing. However, the main problem with Sanger sequencing is its low sensitivity: only 15%–20%. It cannot accurately detect samples with a mutation frequency below 15%, thus easily leading to false negatives. Samples with trace amounts of resistance mutations may be considered negative and undetectable, resulting in occult mutations.

[0006] Amplification Refractory Mutation Systems (ARMS) utilize primers designed to specifically recognize mutated bases at their 3' ends, thereby inhibiting the amplification of normal sites and reducing interference. This enables efficient amplification and specific detection of mutated sites. ARMS is characterized by its simplicity, speed, high specificity, and widespread applicability, making it highly suitable for widespread application in hospitals and the development of diagnostic kits. However, insufficient sensitivity of ARMS technology limits its clinical application.

[0007] Next-generation sequencing (NGS) was developed based on Sanger sequencing. It also uses nested PCR to construct sequencing libraries from the second round of PCR products, performs anchoring bridging, single-base extension sequencing, and data analysis. By increasing sequencing depth and repeating scans multiple times, the sensitivity of mutation detection can be improved, reaching 1%–2%, revealing complex mutation information. However, the main problems with NGS are the complex library construction technology, high cost, and long sequencing data processing time (requiring 1-2 weeks), making large-scale clinical application difficult.

[0008] Digital PCR (dPCR) is a technique that distributes samples into thousands of microdroplets to achieve highly sensitive detection and absolute quantification of analytes. The main process involves PCR amplification of the ABL kinase domain, followed by direct digital quantitative PCR detection of the PCR products. Currently, the reported dPCR technology for detecting BCR-ABL1 resistance mutations utilizes Fluidigm's BioMark microfluidic chip system. This system uses costly micro-etching technology to construct 39,960 reaction microcavities, distributing the sample and achieving single-molecule PCR amplification and absolute quantification in each microcavity based on Poisson distribution. However, Fluidigm's BioMark platform is very expensive, hindering widespread application. Compared to the BioMark microfluidic dPCR system, Bio-Rad's semi-automatic and fully automated QX200 droplet digital PCR (ddPCR) system significantly reduces analytical costs by automatically generating droplets at the oil-water interface. Moreover, ddPCR generally distinguishes between mutant and normal sites by designing different Taqman-MGB probes. Currently, it cannot solve the problem of mutual interference between combined mutations of BCR-ABL1 resistance, and it also has difficulties in the analysis of specific mutations such as T315I. Summary of the Invention

[0009] The main objective of this invention is to provide a primer pair, probe, corresponding kit, and application in detecting the T315I mutation of the BCR-ABL1 gene, so as to overcome the shortcomings of the prior art.

[0010] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0011] This invention provides a primer pair for detecting the T315I mutation in the BCR-ABL1 gene, comprising an upstream primer and a downstream primer. The third and / or fourth base at the 3' end of the upstream primer introduces a corresponding mismatched base, and the second base at the 3' end of the primer introduces an ablation site. The terminal base at the 3' end of the primer is modified with locked nucleic acid.

[0012] This invention also provides a probe for detecting the T315I mutation in the BCR-ABL1 gene, which has the sequences shown in SEQ ID No. 5 and SEQ ID No. 6.

[0013] This invention also provides a kit for detecting the T315I mutation in the BCR-ABL1 gene, comprising the aforementioned primer pair for detecting the T315I mutation in the BCR-ABL1 gene, and a probe for detecting the T315I mutation in the BCR-ABL1 gene.

[0014] This invention also provides a method for detecting the T315I mutation in the BCR-ABL1 gene for non-diagnostic purposes, comprising:

[0015] Provide the aforementioned kit for detecting the T315I mutation in the BCR-ABL1 gene;

[0016] Using the constructed test plasmid that may contain the T315I mutant plasmid as a template, the test plasmid that may contain the T315I mutant plasmid was subjected to PCR amplification reaction using the kit described above.

[0017] The fluorescence signal was detected to enable the detection of the T315I mutation in the BCR-ABL1 gene.

[0018] The embodiments of the present invention also provide the use of the aforementioned primer pairs or kits for detecting the T315I mutation in the BCR-ABL1 gene in the preparation of products that can detect the T315I mutation in the BCR-ABL1 gene.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0020] The primer pairs and kit provided by this invention for detecting the T315I mutation in the BCR-ABL1 gene can selectively amplify the T315I mutation, achieving highly sensitive detection of the T315I mutation (up to 10 copies); it also has good specificity and high accuracy, effectively distinguishing between wild-type and mutant sites, and eliminating interference from neighboring T315A sites; furthermore, the operation is simple and fast, the entire detection process is short, and the detection results can be obtained within 90 minutes, resulting in high detection efficiency and low cost. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram showing the distribution of T315A and T315I mutations in a sequence in a typical embodiment of the present invention;

[0023] Figure 2a and Figure 2b This is a schematic diagram illustrating the highly sensitive detection of the T315I mutation in a typical embodiment of the present invention;

[0024] Figure 3a and Figure 3b This is a schematic diagram illustrating the elimination of the interference effect of the T315A neighboring mutation on T315I in a typical embodiment of the present invention. Detailed Implementation

[0025] In view of the shortcomings of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention. It is mainly based on AMRS technology, and through the design and optimization of primers and probes, provides a highly sensitive analytical method for identifying trace mutations from a large number of wild-type background sequences and effectively eliminating interference from neighboring T315A mutations.

[0026] The following will provide a further explanation of the technical solution, its implementation process, and its principles.

[0027] One aspect of this invention provides a primer pair for detecting the T315I mutation in the BCR-ABL1 gene, comprising an upstream primer and a downstream primer, wherein the third and / or fourth base at the 3' end of the upstream primer introduces a corresponding mismatched base, the second base at the 3' end of the primer introduces an ablation site, and the terminal base at the 3' end of the primer is modified with locked nucleic acid.

[0028] Specifically, this invention reduces the binding and extension of the primer to the wild-type background sequence by introducing (i.e., adding) another mismatched base at the 3rd-4th base of the upstream primer at the 3' end, thereby achieving specific amplification and highly sensitive detection of the primer pair against the T315I mutant template. Simultaneously, LNA modification of the 3' end of the upstream primer improves primer binding stability and amplification efficiency.

[0029] Furthermore, this invention introduces an abase desaturation site (AP) at the second base at the 3' end of the upstream primer, making it correspond exactly to the mutation site of T315A, thereby eliminating interference from neighboring mutations of T315A.

[0030] In some preferred embodiments, the upstream primer has a sequence shown in at least one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, preferably SEQ ID No. 1.

[0031] In some preferred embodiments, the downstream primer has the sequence shown in SEQ ID No. 7.

[0032] Another aspect of the present invention provides a probe for detecting the T315I mutation in the BCR-ABL1 gene, having the sequences shown in SEQ ID No. 5 and SEQ ID No. 6.

[0033] Specifically, the primer pairs and probes of this invention for detecting the T315I mutation in the BCR-ABL1 gene are shown in Table 1 below:

[0034] Table 1

[0035]

[0036] Among them, AP site is the debasement site, and +T, +C are LNA modification sites.

[0037] Another aspect of the present invention provides a kit for detecting the T315I mutation in the BCR-ABL1 gene, comprising the aforementioned primer pair for detecting the T315I mutation in the BCR-ABL1 gene, and a probe for detecting the T315I mutation in the BCR-ABL1 gene.

[0038] Accordingly, another aspect of the present invention provides a method for detecting the T315I mutation in the BCR-ABL1 gene for non-diagnostic purposes, comprising:

[0039] Provide the aforementioned kit for detecting the T315I mutation in the BCR-ABL1 gene;

[0040] Using the constructed test plasmid that may contain the T315I mutant plasmid as a template, the test plasmid that may contain the T315I mutant plasmid was subjected to PCR amplification reaction using the kit described above.

[0041] The fluorescence signal was detected to enable the detection of the T315I mutation in the BCR-ABL1 gene.

[0042] In some preferred embodiments, the PCR amplification reaction uses an amplification system comprising the kit and the plasmid to be tested, wherein the concentration of the upstream primer is 0.005–1 μmol / 20 μl, the concentration of the downstream primer is 0.005–1 μmol / 20 μl, the concentration of the probe is 0.001–1 μmol / 20 μl, and the concentration of the plasmid to be tested is 10–50 ng / 20 μl.

[0043] In some preferred embodiments, the PCR amplification reaction conditions include: pre-denaturation at 95°C for 10 minutes; 10 cycles of denaturation at 95°C for 10–20 seconds, annealing and extension at 55–65°C for 15–35 seconds, and extension at 72°C for 25 seconds; and 50 cycles of denaturation at 95°C for 10–20 seconds, annealing at 58°C for 15–35 seconds, and extension at 72°C for 15–35 seconds, with fluorescence signal detected at the 50th extension cycle.

[0044] Furthermore, the detection method can detect T315I mutations with high sensitivity and effectively eliminate interference from neighboring T315A sites, achieving a detection sensitivity of 1 to 50 copies for T315I mutations in the BCR-ABL1 gene.

[0045] Another aspect of the present invention provides the use of the aforementioned primer pairs for detecting the T315I mutation in the BCR-ABL1 gene, or the kit for detecting the T315I mutation in the BCR-ABL1 gene, in the preparation of products having the function of detecting the T315I mutation in the BCR-ABL1 gene.

[0046] In summary, this invention designs specific ARMS primers based on the wild-type and corresponding mutant sequences of codon 6 of the ABL1 gene to selectively amplify the T315I mutation, achieving highly sensitive detection of the T315I mutation, effectively distinguishing between wild-type and mutant sites, and eliminating interference from the neighboring T315A site.

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described in detail below with reference to the accompanying drawings and several preferred embodiments. The following are preferred embodiments of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention. Test methods in the following embodiments that do not specify specific conditions are all performed under conventional conditions. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0048] All reagents and raw materials used in the following examples are commercially available. Test methods without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0049] Example 1: High-sensitivity detection of T315I mutation

[0050] In this embodiment, mutant plasmids and wild-type plasmids constructed by genetic engineering were used as templates. qPCR reactions were performed on wild-type, T315I, and T315A plasmid templates using the ARMS primers and blocking probes designed in Table 1, respectively.

[0051] Both wild-type and mutant plasmids involved can be prepared using conventional plasmid construction methods and PCR cloning methods.

[0052] 1) Plasmid processing and extraction:

[0053] The Kras wild-type and mutant artificial plasmids were purified using the QIAprep Spin Miniprep (Qiagen) kit as follows:

[0054] Collect 1.5 ml of bacterial culture by centrifugation at 8000 rpm. Add 250 μL of P1 buffer containing RNase A and mix well. Then add the same volume of P2 buffer and mix until the solution becomes clear (do not exceed 5 min of reaction time). Add 350 μL of N3 buffer and mix immediately. Centrifuge at 13000 rpm for 10 min. Transfer the supernatant to a QIAprep spin tube and centrifuge at 13000 rpm for 1 min. Discard the liquid. Wash once with 0.5 ml of PB, then wash twice with 0.75 ml of PE buffer containing anhydrous ethanol. Transfer the QIAprep spin tube to a new 1.5 ml centrifuge tube, add 50 μL of sterile water, let stand for 1 min, centrifuge for 1 min, and collect the filtrate. Quantify the purified plasmid using the Quant-iT PicoGreen (Invitrogen) kit.

[0055] 2) Perform PCR amplification according to the amplification system in Table 2 below (total volume 20 μl).

[0056] Table 2

[0057]

[0058] The 2*PCR mix used in the fluorescent PCR amplification was from Roche, and the water was from Takara.

[0059] 3) The reaction conditions for PCR amplification are as follows:

[0060] Pre-denaturation at 95℃ for 10 minutes; 10 cycles of denaturation at 95℃ for 15 seconds, annealing and extension at 60℃ for 25 seconds, and extension at 72℃ for 25 seconds; 50 cycles of denaturation at 95℃ for 15 seconds, annealing at 58℃ for 25 seconds, and extension at 72℃ for 25 seconds. Sybrogreen fluorescence signal was detected during the 50-cycle extension.

[0061] 4) Detect the fluorescence signal and use the Ct value as the standard for judging the result (refer to the above).

[0062] 5) Sensitivity analysis

[0063] Mix the specific primer pair, specific blocking probe, and PCR reaction solution for the T315I plasmid. Using the T315I mutant plasmid as a template, serially dilute to 10⁻⁶ with TE buffer. 5 copies, 10 4 copies, 10 3 copies, 10 2 copies, 10 1 copies, 1 copy, with 10 5Wild-type plasmids and ddH2O were used as negative controls. Regression curves were constructed using qPCR analysis, with the copy number of the T315I mutation versus the Ct value, to determine the sensitivity and detection limit of the method. A 10-1 ratio was also used as a negative control. 5 The T315A mutant plasmid of copies was used as a control to analyze the interference of the T315A mutation on the method.

[0064] The results are as follows Figure 2a and Figure 2b As shown, this embodiment demonstrates that the method has high sensitivity, with a detection sensitivity of up to 10 copies for the T315I mutation, and that the T315A mutation does not interfere with the amplification detection of T315I.

[0065] Example 2: Elimination of the interference effect of T315I on T315A neighboring mutation

[0066] In this embodiment, mutant plasmids and wild-type plasmids constructed by genetic engineering were used as templates. The ARMS primers and blocking probes designed in Table 1 were used to perform qPCR reactions on wild-type, T315I, T315A, and T315A-I (containing both T315A and T315I mutations) plasmid templates, respectively.

[0067] Both wild-type and mutant plasmids involved can be prepared using conventional plasmid construction methods and PCR cloning methods.

[0068] 1) Plasmid processing and extraction:

[0069] Same as Example 1.

[0070] 2) Perform PCR amplification using the following amplification system (total volume 20 μl).

[0071] Same as Example 1.

[0072] 3) The reaction conditions for PCR amplification are the same as in Example 1.

[0073] 4) Detect the fluorescence signal and use the Ct value as the standard for judging the result.

[0074] 5) Sensitivity analysis

[0075] Mix the specific primer pair, specific blocking probe, and PCR reaction solution for the T315I plasmid. Using the T315A-I mutant plasmid as a template, serially dilute to 10⁻⁶ with TE buffer. 5 copies, 10 4 copies, 10 3 copies, 10 2 copies, 10 1 copies, 1 copy, with 105 Wild-type plasmids and ddH2O were used as negative controls. Regression curves were constructed using qPCR analysis, with the copy number of the T315I mutation versus the Ct value, to determine the sensitivity and detection limit of the method. A 10-1 ratio was also used as a negative control. 5 The T315A mutant plasmid of copies was used as a control to analyze the interference of the T315A mutation on the method.

[0076] The results are as follows Figure 3a and Figure 3b As shown, the method can also effectively detect T315I mutations in T315A-I plasmids containing both mutation types, with a detection sensitivity of up to 10 copies. It is not affected by neighboring T315A mutations, indicating that the method can effectively eliminate interference from mutations at adjacent sites.

[0077] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0078] Comparative Example 1

[0079] Compared with Example 1, the third and / or fourth base at the 3' end of the upstream primer used in this comparative example did not introduce a corresponding mismatched base.

[0080] Tests showed that if the third and / or fourth base at the 3' end of the upstream primer does not introduce a corresponding mismatched base, it will also amplify T315-A. This reduces the Ct value difference between T315I and T315A amplifications, resulting in insufficient specificity of primer amplification and inability to effectively distinguish between T315-A and T315-I.

[0081] Comparative Example 2

[0082] Compared with Example 1, the second base at the 3' end of the upstream primer used in this comparative example did not introduce a debasement site.

[0083] Tests have shown that if the second base at the 3' end of the upstream primer does not introduce a debasement site, the amplification efficiency will differ depending on whether the primer pair contains only the T315I mutation or both T315I and T315A mutations. This means that the presence or absence of the T315A mutation will affect the amplification of the neighboring T315I mutation, thus making the T315I analysis inaccurate and failing to eliminate the interference from the neighboring T315A mutation.

[0084] Comparative Example 3

[0085] Compared with Example 1, the 3' terminal base of the upstream primer used in this comparative example was not modified with locked nucleic acid.

[0086] Tests showed that without nucleic acid modification, the primers containing artificially introduced mutations and abase decomposition sites would result in weak binding ability between the primers and the template, affecting the overall amplification efficiency and leading to insufficient detection sensitivity.

[0087] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A kit for detecting the T315I mutation in the BCR-ABL1 gene, characterized in that, This includes primer pairs for detecting the T315I mutation in the BCR-ABL1 gene, and probes for detecting the T315I mutation in the BCR-ABL1 gene. The primer pair for detecting the T315I mutation in the BCR-ABL1 gene includes an upstream primer and a downstream primer. The third base at the 3' end of the upstream primer introduces a corresponding mismatched base, and the second base at the 3' end of the primer introduces an ablation site. The terminal bases at the 3' end of the primer are modified with locked nucleic acid. The upstream primer has the sequence shown in any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, and the downstream primer has the sequence shown in SEQ ID No.

7. The probe for detecting the T315I mutation in the BCR-ABL1 gene has the sequences shown in SEQ ID No. 5 and SEQ ID No.

6.

2. The kit for detecting the T315I mutation in the BCR-ABL1 gene according to claim 1, characterized in that: The upstream primer is SEQ ID No.

1.

3. A method for detecting the T315I mutation in the BCR-ABL1 gene for non-diagnostic purposes, characterized in that, include: Provide a kit for detecting the T315I mutation in the BCR-ABL1 gene as described in claim 1 or 2; Using the constructed test plasmid that may contain the T315I mutant plasmid as a template, the test plasmid that may contain the T315I mutant plasmid was subjected to PCR amplification reaction using the kit described above. The fluorescence signal was detected to enable the detection of the T315I mutation in the BCR-ABL1 gene.

4. The detection method according to claim 3, characterized in that: The PCR amplification reaction uses an amplification system comprising the kit, the plasmid to be tested, PCR mixture, and water. The concentration of the upstream primer is 0.005–1 µmol / 20 µl, the concentration of the downstream primer is 0.005–1 µmol / 20 µl, the concentration of the probe is 0.001–1 µmol / 20 µl, and the concentration of the plasmid to be tested is 10–50 ng / 20 µl.

5. The detection method according to claim 3, characterized in that, The PCR amplification reaction conditions included: pre-denaturation at 95℃ for 10 minutes; 10 cycles of denaturation at 95℃ for 10-20 seconds, annealing and extension at 55-65℃ for 15-35 seconds, and extension at 72℃ for 25 seconds; and 50 cycles of denaturation at 95℃ for 10-20 seconds, annealing at 58℃ for 15-35 seconds, and extension at 72℃ for 15-35 seconds, with fluorescence signal detected during the 50th extension cycle.

6. The detection method according to claim 3, characterized in that: The detection method has a sensitivity of 1 to 50 copies for the T315I mutation in the BCR-ABL1 gene.

7. Use of the kit for detecting the T315I mutation in the BCR-ABL1 gene as described in claim 1 or 2 in the preparation of a product having the ability to detect the T315I mutation in the BCR-ABL1 gene.

Images