Nucleic acid composition and kit for detecting ABL kinase region of BCR-ABL fusion gene by ARMS-PCR method

Abstract

The present invention provides a nucleic acid composition and a kit for detecting the ABL kinase region of the BCR-ABL fusion gene by ARMS-PCR method; the nucleic acid composition is a primer and probe sequence designed according to 19 common mutations in the ABL kinase region of the BCR-ABL fusion gene and ABL internal reference. The kit can complete the detection in 11 wells, with simple operation, a detection sensitivity of up to 1%, and compared with other common molecular biology detection methods, it greatly reduces the detection cycle and experimental cost, while improving the experimental sensitivity and accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to a nucleic acid composition and a kit for detecting the ABL kinase region of the BCR-ABL fusion gene by the ARMS-PCR method. Background Art

[0002] Chronic myeloid leukemia (CML) is a type of malignant tumor of the hematopoietic system. More than 95% of CML cases have the BCR-ABL fusion gene, which is formed by the reciprocal translocation of the ABL gene on human chromosome 9 and the BCR gene on chromosome 22. It plays an important role in the pathogenesis and phenotype of CML. It has significantly enhanced tyrosine kinase activity and can directly participate in the transformation of cells into the CML phenotype.

[0003] The BCR-ABL fusion gene type is closely related to the phenotype. There are mainly three common types:

[0004] 1) M-bcr The main breakpoint is in the 5.8 kb region (also called b1-b5) of exons e12-e16 of the BCR gene. Most mRNA types are b2a2 or b3a2, and the fusion protein is about 210-kDa. It is seen in most CML and 1 / 3 of Ph-positive B-ALL patients.

[0005] 2) m-bcr The main breakpoint is in the 54.4 kb region between exons e2’ and e2, generating the e1a2 type of mRNA, and the fusion protein is about 190 kDa. It is mainly seen in 2 / 3 of Ph-positive B-ALL and a very small number of CML patients.

[0006] 3) μ-bcr The breakpoint is located downstream of exon e19, transcribed into the e19a2 type of mRNA, and the encoded protein size is about 230 kDa. It is mainly seen in chronic neutrophilic leukemia.

[0007] In 2001, the TKI drug imatinib was introduced as a drug for treating CML. Subsequently, cases of imatinib resistance were discovered. Point mutations in the ABL kinase region of the BCR-ABL fusion gene can weaken or even eliminate imatinib binding. Subsequently, more and more TKI drugs and resistance site mutations have been discovered. So far, more than ninety resistance mutation sites have been reported. Some mutation types are resistant to multiple TKI drugs, and the prognosis of leukemia patients is poor.

[0008] The mutation types in the ABL kinase domain of the same patient at different times may also evolve. Initially, the proportion of mutant clones may be low, but once they appear, they will increase rapidly and lead to disease progression. It is also possible that at a certain stage of the disease, the original clone type disappears and a new clone type appears. Drug-resistant mutations often occur in the blast crisis stage. Therefore, it is necessary to regularly monitor the mutation situation, which can be used to timely adjust the types and doses of TKI drugs. For some gene mutations with poor prognosis, early detection at a low level has better clinical significance. The point mutations that have been discovered mainly focus on three major categories: the ATP-binding region (p-loop), especially G250E, Y253H, E255K, etc.; the T315I mutation; the a-loop region, especially H396R. Among the mutations reported currently, the T315I mutation has the highest drug resistance level and is resistant to almost all tyrosine kinase inhibitors. Therefore, other treatment methods need to be adopted as soon as possible. Some drug-resistant mutations have a relatively low drug resistance level, and the effect can be achieved by increasing the drug dose. There are also some patients with drug-resistant mutations who can switch to second-generation, third-generation, etc. TKI drugs as alternative drugs to imatinib. Therefore, before and during treatment, integrating the quantitative results of fusion genes and the mutation results of the ABL kinase domain can provide more accurate individualized medication guidance for patients.

[0009] Currently, the methods for detecting mutation sites in the ABL kinase domain mainly include nested PCR and next-generation sequencing. Nested PCR was widely used in the early years mainly because two-step amplification can improve specificity. However, a major problem with it is that the opening of the first-round amplification products is prone to aerosol contamination, and the sensitivity of first-generation sequencing is not high enough. In recent years, with the increasing popularity of next-generation sequencers, the application of detecting mutation sites in the ABL kinase domain by next-generation sequencing has become more and more widespread, greatly improving the detection sensitivity. However, the experimental cost is relatively high, the experimental cycle is relatively long, and its application in clinical practice is relatively limited. Summary of the Invention

[0010] The present invention provides a nucleic acid composition for detecting the ABL kinase domain of the BCR-ABL fusion gene by ARMS-PCR, which is used to solve the defects of low detection sensitivity, high cost, and long cycle in the prior art, and broadens the application scope of clinical detection.

[0011] Based on this, the technical solution of the present invention is as follows:

[0012] A nucleic acid composition for detecting the ABL kinase region of the BCR-ABL fusion gene by ARMS-PCR method, wherein the nucleic acid composition is a primer pair and a probe set designed for 19 point mutations of F311L; F311I, T315A, F317LC, F317I, F317LA, F317L, T315I, F359I, F359C, F359V, M351T, G250E, E255K, E255V, Q252H, M244V, Y253H, H396R.

[0013] Furthermore, the primer pair and the probe set include:

[0014] T315I: The primer pair shown in SEQ ID NO: 1-2 and the probe shown in SEQ ID NO: 3;

[0015] F317L: The primer pair shown in SEQ ID NO: 1, 4 and the probe shown in SEQ ID NO: 3;

[0016] G250E: The primer pair shown in SEQ ID NO: 5-6 and the probe shown in SEQ ID NO: 7;

[0017] F359V: The primer pair shown in SEQ ID NO: 8-9 and the probe shown in SEQ ID NO: 10;

[0018] E255K: The primer pair shown in SEQ ID NO: 5, 11 and the probe shown in SEQ ID NO: 7;

[0019] Y253H: The primer pair shown in SEQ ID NO: 5, 12 and the probe shown in SEQ ID NO: 7;

[0020] H396R: The primer pair shown in SEQ ID NO: 13-14 and the probe shown in SEQ ID NO: 15;

[0021] M244V: The primer pair shown in SEQ ID NO: 5, 16 and the probe shown in SEQ ID NO: 7;

[0022] Q252H: The primer pair shown in SEQ ID NO: 5, 17 and the probe shown in SEQ ID NO: 7;

[0023] E255V: The primer pair shown in SEQ ID NO: 5, 18 and the probe shown in SEQ ID NO: 7;

[0024] F311L: primer pair shown in SEQ ID NO: 1, 19 and probe shown in SEQ ID NO: 3;

[0025] F311I: primer pair shown in SEQ ID NO: 1, 20 and probe shown in SEQ ID NO: 3;

[0026] F317LA: primer pair shown in SEQ ID NO: 1, 21 and probe shown in SEQ ID NO: 3;

[0027] F317LC: primer pair shown in SEQ ID NO: 1, 22 and probe shown in SEQ ID NO: 3;

[0028] T315A: primer pair shown in SEQ ID NO: 1, 23 and probe shown in SEQ ID NO: 24;

[0029] F317I: primer pair shown in SEQ ID NO: 1, 25 and probe shown in SEQ ID NO: 3;

[0030] M351T: primer pair shown in SEQ ID NO: 8, 26 and probe shown in SEQ ID NO: 10;

[0031] F359C: primer pair shown in SEQ ID NO: 8, 27 and probe shown in SEQ ID NO: 10;

[0032] F359I: primer pair shown in SEQ ID NO: 8, 28 and probe shown in SEQ ID NO: 10;

[0033] and internal reference: primer pair shown in SEQ ID NO: 29 - 30 and probe shown in SEQ ID NO: 31; the 5' end of the probe sequence is modified with a reporter group, and the 3' end of the probe sequence is modified with a quenching group.

[0034] Furthermore, the probe is modified with FAM at the 5' end and MGB at the 3' end.

[0035] Another object of the present invention is to provide the application of the above nucleic acid composition in the preparation of a kit for detecting the ABL kinase region of the BCR - ABL fusion gene.

[0036] In the above application, the kit divides the primer pair and the probe set into the following 11 tubes:

[0037] Tube 1: The primer sequences are as shown in SEQ ID NO: 1, 19, 20, 23, and the probe sequence is as shown in SEQ ID NO: 3;

[0038] Tube 2: The primer sequences are as shown in SEQ ID NO: 1, 22, 25, and the probe sequence is as shown in SEQ ID NO: 3;

[0039] Tube 3: The primer sequences are as shown in SEQ ID NO: 1, 21, 4, and the probe sequence is as shown in SEQ ID NO: 3;

[0040] Tube 4: The primer sequences are as shown in SEQ ID NO: 1 - 2, and the probe sequence is as shown in SEQ ID NO: 3;

[0041] Tube 5: The primer sequences are as shown in SEQ ID NO: 8, 28, 27, and the probe sequence is as shown in SEQ ID NO: 10;

[0042] Tube 6: The primer sequences are as shown in SEQ ID NO: 8, 9, 26, and the probe sequence is as shown in SEQ ID NO: 10;

[0043] Tube 7: The primer sequences are as shown in SEQ ID NO: 5 - 6, and the probe sequence is as shown in SEQ ID NO: 7;

[0044] Tube 8: The primer sequences used are as shown in SEQ ID NO: 5, 11, 18, 17, 16, and the probe sequence is as shown in SEQ ID NO: 7;

[0045] Tube 9: The primer sequences used are as shown in SEQ ID NO: 5, 12, and the probe sequence is as shown in SEQ ID NO: 7;

[0046] Tube 10: The primer sequences used are as shown in SEQ ID NO: 13, 14, and the probe sequence is as shown in SEQ ID NO: 15;

[0047] Tube 11: The primer sequences are as shown in SEQ ID NO: 29, 30, and the probe sequence is as shown in SEQ ID NO: 31.

[0048] Another object of the present invention is to provide a kit for detecting the ABL kinase region of the BCR - ABL fusion gene by ARMS - PCR method, which includes the nucleic acid composition as described above.

[0049] Furthermore, the kit further includes reaction reagents for fluorescence quantitative PCR amplification.

[0050] Another object of the present invention is to provide a method for detecting the ABL kinase region of the BCR-ABL fusion gene for non-diagnostic purposes, and its reaction system is 12.5 μl:

[0051] Probe qPCR Master Mix 6.25 μl

[0052] Primer-probe mix 3.25 μl

[0053] cDNA 2.5 μl

[0054] The final concentration of the primer is 300 nM, and the final concentration of the probe is 200 nM;

[0055] The primer-probe mix is the nucleic acid composition as described above.

[0056] Furthermore, the reaction conditions of the reaction system are: 95°C for 5 min; 95°C for 25 s, 64°C for 20 s, 72°C for 20 s, for 10 cycles, without collecting fluorescence; 93°C for 25 s, 60°C for 35 s, for 30 cycles, collecting fluorescence; 72°C for 20 s.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0058] The present invention designs primer and probe sequences with ABL as the internal reference according to 19 common mutations in the ABL kinase region of the BCR-ABL fusion gene, which can complete the detection of 19 common mutations and the internal reference in 11 wells. The operation is simple, and the detection sensitivity can reach 1%. Compared with other common molecular biology detection methods, the detection cycle is greatly reduced, the experimental cost is reduced, and at the same time, the experimental sensitivity and accuracy are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0060] Figure 1 It is a schematic diagram of the sensitivity detection of Example 5 of the present invention taking the E255V mutation site as an example.

[0061] Figure 2 It is a schematic diagram of the sensitivity detection of Example 5 of the present invention taking the E255K mutation site as an example.

[0062] Figure 3 It is a schematic diagram of the sensitivity detection of Example 5 of the present invention taking the T315I mutation site as an example. Detailed implementation manners

[0063] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0064] In one embodiment, upstream and downstream primers and probes are designed for 19 common mutation sites. The 19 mutation sites are: F311L (c.931T>C); F311I (c.931T>A); T315A (c.943A>G); F317LC (c.949T>C); F317I (c.949T>A); F317LA (c.951C>A); F317L (c.951C>G); T315I (c.944C>T); F359I (c.1075T>A); F359C (c.1076T>G); F359V (c.1075T>G); M351T (c.1052T>C); G250E (c.757T>C); E255K (c.763G>A); E255V (c.764A>T); Q252H (c.756G>C); M244V (c.730A>G); Y253H (c.757T>C); H396R (c.1187A>G).

[0065] The mutation sites are designed at the 3'-end of the downstream primer. According to the principle of ARMS-PCR, the mutant sequence can be completely matched and amplified, while the 3'-end of the wild type is not completely matched, and the amplification efficiency is significantly reduced. In order to improve the detection efficiency and reduce the sample consumption, the 19 common sites are divided into 10 tubes for detection, and the following 11-tube nucleic acid combination reagents are set:

[0066] Tube 1: F311L (c.931T>C), F311I (c.931T>A), T315A (c.943A>G); the primer sequences used are as shown in SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, and the probe sequence is as shown in SEQ ID NO: 3.

[0067] Tube 2: F317LC (c.949T>C); F317I (c.949T>A); the primer sequences used are as shown in SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 25, and the probe sequence is as shown in SEQ ID NO: 3.

[0068] Tube 3: F317LA (c.951C>A), F317L (c.951C>G); The primer sequences used are as shown in SEQ ID NO: 1, SEQ ID NO: 21, SEQ ID NO: 4, and the probe sequence is as shown in SEQ ID NO: 3.

[0069] Tube 4: T315I (c.944C>T); The primer sequences used are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and the probe sequence is as shown in SEQ ID NO: 3.

[0070] Tube 5: F359I (c.1075T>A), F359C (c.1076T>G); The primer sequences used are as shown in SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 27, and the probe sequence is as shown in SEQ ID NO: 10.

[0071] Tube 6: F359V (c.1075T>G), M351T (c.1052T>C); The primer sequences used are as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 26, and the probe sequence is as shown in SEQ ID NO: 10.

[0072] Tube 7: G250E (c.757T>C); The primer sequences used are as shown in SEQ ID NO: 5, SEQ ID NO: 6, and the probe sequence is as shown in SEQ ID NO: 7.

[0073] Tube 8: E255K (c.763G>A), E255V (c.764A>T), Q252H (c.756G>C), M244V (c.730A>G); The primer sequences used are as shown in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 17, SEQ ID NO: 16, and the probe sequence is as shown in SEQ ID NO: 7.

[0074] Tube 9: Y253H (c.757T>C); The primer sequences used are as shown in SEQ ID NO: 5, SEQ ID NO: 12, and the probe sequence is as shown in SEQ ID NO: 7.

[0075] Tube 10: H396R (c.1187A>G), The primer sequences used are as shown in SEQ ID NO: 13, SEQ ID NO: 14, and the probe sequence is as shown in SEQ ID NO: 15.

[0076] Tube 11 serves as the internal reference: ABL; the primer sequences used are as shown in SEQ ID NO: 29 and SEQ ID NO: 30, and the probe sequence is as shown in SEQ ID NO: 31.

[0077] All probes are modified with FAM at the 5' end and MGB at the 3' end.

[0078] The positive control is the sample after serial dilution of the second-generation sequencing mutant-positive sample according to the mutation frequency, and the negative control is the second-generation sequencing mutant-negative sample.

[0079] The final concentration of the primers is 300 nM, and the final concentration of the probes is 200 nM.

[0080] The above 19 common mutation types can be completed in 10 detection tubes. Some detection tubes can detect multiple mutation types simultaneously, improving the detection efficiency and reducing the sample usage. Using fluorescence quantitative PCR technology, the results are intuitive, the operation is simple, the time consumption is short, and the detection efficiency is high. In addition to the last base mismatch at the 3' end of the downstream primer, 1-2 additional mismatches are introduced to improve the detection specificity.

[0081] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art.

[0082] Example 1: Specific steps for detecting drug resistance mutation sites in the BCR-ABL kinase domain

[0083] 1. Sample RNA extraction

[0084] Automated nucleic acid extraction and purification reagents (Meiji Bio, IVD3020-TL-06) were used for RNA extraction, and the specific protocol is as follows:

[0085] 1) 3 - 5 ml of bone marrow specimen was placed in a horizontal centrifuge and centrifuged at 3400 rpm for 5 min;

[0086] 2) The upper plasma was removed, the buffy coat was aspirated, and 10 ml of red blood cell lysate was added for 4 min of red blood cell lysis;

[0087] 3) Centrifuged at 3400 rpm for 5 min, the supernatant was removed, and 10 ml of red blood cell lysate was added again for the second red blood cell lysis for 4 min;

[0088] 4) The supernatant was removed, 10 ml of PBS was added for washing once and then the supernatant was removed again;

[0089] 5) The precipitate was resuspended with 700 ul of trizol;

[0090] 6) 200 ul of chloroform was added and shaken vigorously for 15 s to mix evenly;

[0091] 7) Leave at room temperature for 3 min, centrifuge at 12,000 g at 4 °C for 15 min, and the sample is divided into three layers;

[0092] 8) Take out the kit, remove the seal bag and sealing film, and add 500 μl of the centrifuged supernatant to the 1st / 7th row;

[0093] 9) Add 50 - 100 μl of RNase Free Water to the 6th / 12th row;

[0094] 10) Turn on the automated nucleic acid extractor, place the loaded 96-well plate, and insert the 8-well magnetic sleeve;

[0095] 11) Start the program, take out the well plate and magnetic sleeve after 25 min, and transfer the product to a 1.5 ml EP tube for storage at -20 °C.

[0096] 2. Reverse transcription to obtain cDNA

[0097] Use a reverse transcription kit (Xiamen Zhishan Life Sciences Co., Ltd., LF enzyme 03) for reverse transcription. Add 15 μl of RNA to 13.5 μl of RT reaction solution 03 and 1.5 μl of RT enzyme mixture 03 for reverse transcription. Reverse transcription program: 37 °C for 15 min, 85 °C for 5 min.

[0098] 3. ARMS-PCR amplification

[0099] Using the reverse transcription product cDNA as a template, add the corresponding upstream and downstream primers and probes for fluorescence quantitative PCR.

[0100] Reaction system:

[0101] Use a 12.5 μl reaction system for ARMS-PCR amplification: 2*PCR mix ( Probe qPCRMaster Mix, Promega, A6102) 6.25 μl, primer-probe mix 3.75 μl, cDNA 2.5 μl. The final concentration of the primer is 300 μM, and the final concentration of the probe is 200 μM.

[0102] Reaction conditions: 95 °C for 5 min; 95 °C for 25 s, 64 °C for 20 s, 72 °C for 20 s, 10 cycles, no fluorescence collection; 93 °C for 25 s, 60 °C for 35 s, 30 cycles, fluorescence collection; 72 °C for 20 s.

[0103] Example 2: Mutation site interpretation

[0104] Table 1 shows the interpretation criteria for various mutant genes, and the ΔCT value is combined with the CT value for interpretation.

[0105] Table 1:

[0106]

[0107] The ΔCT value is the difference between the CT value of the ABL internal reference well and the CT value of this well. If multiple mutant wells are judged as positive, each type of mutation needs to be detected separately, and the judgment criteria are the same as those for the mixed wells.

[0108] Example 3: Next-generation sequencing process

[0109] Experimental reagents: Aiji Taikang Custom Panel (IGMU253V1) kit

[0110] Experimental steps:

[0111] 1. The first-round multiplex reaction is carried out in 2 reaction tubes, and the multiplex primers used are primer pool T1 and primer pool T2 respectively. The system is shown in Table 2.

[0112] Table 2:

[0113]

[0114] 1) Multiplex PCR reaction conditions:

[0115] Hot lid at 105 °C, volume 12 μl;

[0116] 95 °C for 3 min 30 s;

[0117] 98 °C for 20 s, 60 °C for 2 min, 28 cycles;

[0118] 72 °C for 5 min.

[0119] 2) After the reaction is completed, combine 2 samples of the same sample, add 26 μl of water, add 0.8X of purified magnetic beads, 40 μl, mix well, incubate at room temperature for 5 min, and place the sample on the magnetic stand for 3 min.

[0120] 3) Remove the supernatant, add 180 μl of 80% ethanol, and let stand for 30 s;

[0121] 4) Remove the supernatant, add 180 μl of 80% ethanol, and let stand for 30 s;

[0122] 5) Thoroughly remove the supernatant, let stand at room temperature for 3 min until the surface of the magnetic beads becomes matte;

[0123] 6) Remove the PCR tube, add 20 μl of nuclease-free water, mix well, and incubate at room temperature for 2 min;

[0124] 7) Place the PCR tube back on the magnetic stand and let stand for 3 min;

[0125] 8) Pipette 8.1 ul of the supernatant for subsequent reactions.

[0126] 4. Second-round adapter sequence PCR reaction

[0127] The reaction system is shown in Table 3.

[0128] Table 3:

[0129]

[0130] 1) PCR reaction conditions

[0131] Hot lid at 105 °C, volume 18 ul;

[0132] 95 °C for 3 min 30 s;

[0133] 98 °C for 20 s, 58 °C for 1 min, 72 °C for 30 s, 9 cycles;

[0134] 72 °C for 5 min.

[0135] 2) After the reaction, add 32 ul of ddH2O and 40 ul of 0.8X purification magnetic beads, mix well, let stand at room temperature for 5 min, and place the PCR tube on the magnetic rack for 3 min;

[0136] 3) Remove the supernatant, add 180 ul of 80% ethanol, and let stand for 30 s;

[0137] 4) Remove the supernatant, add 180 ul of 80% ethanol, and let stand for 30 s;

[0138] 5) Thoroughly remove the supernatant and air-dry for 3 min until the surface of the magnetic beads is matte;

[0139] 6) Remove the PCR tube, add 52 ul of nuclease-free water, mix well, and let stand at room temperature for 2 min;

[0140] 7) Place the PCR tube on the magnetic rack and let stand for 3 min;

[0141] 8) Pipette 48 ul of the supernatant into a new EP tube.

[0142] Perform concentration and length quality inspections, sequence on an Illumina NextSeq 550AR sequencer, and analyze the data after sequencing.

[0143] Example 4: Detection of negative samples

[0144] Verify the accuracy of the negative samples detected by the primers and probes. Specifically, collect 5 mL of peripheral blood specimens from 10 healthy individuals and perform the detection according to the steps of Example 1. The detection results are shown in Table 4.

[0145] Table 4:

[0146]

[0147]

[0148] Example 5: Comparative Detection of ARMS-PCR Results and Next-Generation Sequencing Results in Clinical Samples

[0149] Collect 152 bone marrow or peripheral blood specimens from patients with chronic myeloid leukemia and detect the mutation sites in the kinase region by ARMS-PCR and next-generation sequencing. The results are shown in Table 5.

[0150] Table 5:

[0151]

[0152] It was found that 11 cases and 127 cases were judged as positive and negative respectively by both ARMS-PCR and next-generation sequencing. Among them, the mutation types judged as positive were all the same, mainly including single mutations and compound mutations such as T315I, E255K, F317L, and H396R. The coincidence rate was 91%. There were 11 specimens with positive NGS results but negative ARMS-PCR results. Among them, the mutation frequencies of 9 cases were less than 1%, and 2 cases were L324P mutations, which were not within the detection range of ARMS-PCR. The NGS results of 3 specimens were negative, but the ARMS-PCR results were positive.

[0153] The coincidence rate between the ARMS-PCR mutation detection results and the next-generation sequencing results is relatively high, averaging over 90%. Within the detection sites and sensitivity range of ARMS-PCR mutations, the coincidence rate is greater than 98%.

[0154] Example 6: Sensitivity Detection of Mutation Sites

[0155] Figures 1-3 Sensitivity detection schematic diagrams are respectively provided taking the mutation sites of E255V, E255K, and T315I as examples. Figure 1 Taking the E255V mutation sample with a 60% mutation rate as a reference product for serial dilution, a total of 8 gradients are diluted, and the mutation can still be effectively detected at the lowest dilution of 0.94%. Figure 2 、 Figure 3 Taking the T315I and E255K mutation samples with a 30% mutation rate as reference products for serial dilution, the mutation can still be effectively detected at the lowest dilution of 0.47%. The laboratory sets the sensitivity of this experiment at 1%.

[0156] The above detection method and system can quickly and simply screen out 19 common mutation sites, with relatively low costs, and the sensitivity can reach 1% according to the verification method of Example 6.

[0157] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A kit for detecting the ABL kinase region of the BCR-ABL fusion gene by ARMS-PCR method, characterized in that, The kit is used for detecting 19 point mutations including F311L, F311I, T315A, F317LC, F317I, F317LA, F317L, T315I, F359I, F359C, F359V, M351T, G250E, E255K, E255V, Q252H, M244V, Y253H, H396R, and includes the following 11 tubes of nucleic acid combination reagents: Tube 1: The primer sequences are as shown in SEQ ID NO: 1, 19, 20, 23, and the probe sequence is as shown in SEQ ID NO: 3; Tube 2: The primer sequences are as shown in SEQ ID NO: 1, 22, 25, and the probe sequence is as shown in SEQ ID NO: 3; Tube 3: The primer sequences are as shown in SEQ ID NO: 1, 21, 4, and the probe sequence is as shown in SEQ ID NO: 3; Tube 4: The primer sequences are as shown in SEQ ID NO: 1-2, and the probe sequence is as shown in SEQ ID NO: 3; Tube 5: The primer sequences are as shown in SEQ ID NO: 8, 28, 27, and the probe sequence is as shown in SEQ ID NO: 10; Tube 6: The primer sequences are as shown in SEQ ID NO: 8, 9, 26, and the probe sequence is as shown in SEQ ID NO: 10; Tube 7: The primer sequences are as shown in SEQ ID NO: 5-6, and the probe sequence is as shown in SEQ ID NO: 7; Tube 8: The primer sequences used are as shown in SEQ ID NO: 5, 11, 18, 17, 16, and the probe sequence is as shown in SEQ ID NO: 7; Tube 9: The primer sequences used are as shown in SEQ ID NO: 5, 12, and the probe sequence is as shown in SEQ ID NO: 7; Tube 10: The primer sequences used are as shown in SEQ ID NO: 13, 14, and the probe sequence is as shown in SEQ ID NO: 15; Tube 11: The primer sequences are as shown in SEQ ID NO: 29, 30, and the probe sequence is as shown in SEQ ID NO:

31.

2. The kit according to claim 1, wherein The probe is modified with FAM at the 5' end and MGB at the 3' end.

3. The kit according to claim 1, wherein It also includes reaction reagents for fluorescence quantitative PCR amplification.

4. Use of the kit according to any one of claims 1-3 in the preparation of a product for detecting the ABL kinase region of the BCR-ABL fusion gene.

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