Primers and kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia.

Abstract

This invention provides primers and a kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, including LCR primers CP, SP, T1, and T2, whose nucleotide sequences are shown in SEQ ID NO:1-SEQ ID NO:4 or SEQ ID NO:5-SEQ ID NO:8. These primers have a low detection limit and strong ability to distinguish single-base mismatches.

Description

Technical Field

[0001] This invention relates to primers and a kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, belonging to the field of biosensor technology. Background Technology

[0002] Chronic myeloid leukemia (CML) is a type of hematologic malignancy (approximately 95% or more) caused by the chromosomal translocation t(9;22) forming the BCR / ABL fusion gene, which encodes a tyrosine protein kinase and leads to abnormal proliferation of granulocytes. Due to varying breakpoints, the BCR / ABL fusion can produce four transcripts: e19a2, e14a2, e1a2, and e13a2. Among these, e14a2 (b2a2) and e13a2 (b3a2) are two of the more common transcripts, expressing the classic P210 fusion protein. The BCR / ABL fusion gene is an important clinical target for the treatment of CML. Accurate detection of the BCR / ABL fusion gene is beneficial for the early diagnosis and precision treatment of CML.

[0003] Currently, the main method for detecting BCR / ABL fusion genes is real-time quantitative PCR. However, this method has many shortcomings in terms of detection speed, cost, and portability. The single detection mode also affects its accuracy for BCR / ABL detection to some extent, making it difficult to develop into an accurate and reliable diagnostic tool for BCR / ABL detection that can be routinely performed in clinical institutions at all levels.

[0004] Therefore, there is an urgent need to develop a highly accurate and reliable BCR / ABL detection method. Summary of the Invention

[0005] This invention provides primers and a kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, which can effectively solve the above-mentioned problems.

[0006] This invention is implemented as follows:

[0007] A primer for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, comprising LCR primers CP, SP, T1, and T2, the nucleotide sequences of which are shown in SEQ ID NO:1-SEQ ID NO:4 or in SEQ ID NO:5-SEQ ID NO:8.

[0008] A kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, comprising the primers described above.

[0009] In one embodiment, the kit further includes streptavidin-modified magnetic beads.

[0010] In one embodiment, the kit further includes horseradish peroxidase labeled with anti-6-carboxyfluorescein antibody, hydrogen peroxide, and TMB.

[0011] A method for amplifying the BCR / ABL fusion gene in chronic myeloid leukemia involves using the primers described above for a ligase chain reaction in the presence of the target gene.

[0012] In one embodiment, the reaction conditions for the ligase chain reaction are: 65°C for 2 min; 94°C for 1 min, 30 cycles.

[0013] In one embodiment, the reaction system for the ligase chain reaction is 5 μL CP (0.1 μM), 5 μL SP (0.1 μM), 5 μL T1 (0.1 μM), 5 μL T2 (0.1 μM), 0.2 μL Ampligase (5 U / μl), 10 μL Reaction Buffer (10×), 10 μL target gene solution, and 59.8 μL Tris-HCl buffer (pH = 7.4).

[0014] The beneficial effects of this invention are:

[0015] The primers of the present invention have the following advantages for LCR reaction of BCR / ABL fusion gene: (1) their detection limit is as low as 4.5 × 10⁻⁶. -20 M (100 μl reaction system contains 2 copies of target gene), comparable to traditional real-time quantitative PCR; (2) can distinguish single base mismatch, the signal of the completely complementary strand is more than 40 times that of the single base mismatch signal; (3) requires less primer (nM level), compared with the μM level required by traditional methods, the primer amount can be reduced by 2 to 3 orders of magnitude.

[0016] The primers of this invention can be used for multimodal detection (fluorescence-colorimetric-electrochemical) of the BCR / ABL fusion gene in chronic myeloid leukemia. The key feature is that a ligase chain reaction initiated by the target nucleic acid consumes primers (CP, SP, T1, T2) to obtain a DNA double-stranded amplification product labeled with biotin and 6-carboxyfluorescein (FAM) at the beginning and end, respectively. The amplification product is immobilized onto streptavidin-modified magnetic beads (MBs) using the specific binding between biotin and streptavidin. After magnetic separation, it can be directly detected by flow cytometry, or by binding to horseradish peroxidase (6-Carboxyfluorescein antibody-labeled). Horseradish peroxidase (anti-FAM-HRP) was used to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) with hydrogen peroxide (H2O2) catalyzed by horseradish peroxidase, followed by electrochemical and colorimetric detection. This enables ultrasensitive and cross-validated detection of the target nucleic acid. The constructed multimodal biosensor platform has advantages such as high sensitivity, high reliability, simplicity, and low cost, and is expected to become a powerful tool for the early diagnosis and precision treatment of chronic myeloid leukemia. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of a multimodal sensing method based on functional magnetic bead carriers using ligase chain reaction.

[0019] Figure 2 The graph shows the linear relationship between the average fluorescence intensity (MFI) and the concentration of the target gene standard provided in Example 3 of the present invention (taking b3a2 as an example).

[0020] Figure 3 This is a linear relationship diagram between absorbance (OD) and target gene standard provided in Example 4 of the present invention (taking b3a2 as an example).

[0021] Figure 4The linear relationship between the current value and the target gene standard provided in Embodiment 5 of the present invention (taking b3a2 as an example).

[0022] Figure 5 Specificity assessment of the sensor provided in Embodiment 6 of the present invention: Comparison of current response results between the fully complementary target strand and single-base mismatched strands (T-Mut, C-Mut, and G-Mut), triple-base mismatched strands, and blank. The DNA strand concentration used was 10. -14 M (taking b3a2 as an example). Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] In the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0025] This invention provides primers for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, including LCR primers CP, SP, T1, and T2, whose nucleotide sequences are shown in SEQ ID NO:1-SEQ ID NO:4 or SEQ ID NO:5-SEQ ID NO:8. The target gene can be amplified by a ligase chain reaction using these primers.

[0026] This invention provides a kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, comprising the primers described above. After amplification of the BCR / ABL fusion gene using a ligase chain reaction with these primers, the detection of the BCR / ABL fusion gene is facilitated.

[0027] In one embodiment, the kit includes the primers described above, and also includes streptavidin-modified magnetic beads. Using this kit, a ligase chain reaction initiated with the target nucleic acid consumes the primers, yielding a DNA double-stranded amplification product labeled with biotin and 6-carboxyfluorescein (FAM) at the head and tail, respectively. The amplification product is then immobilized onto streptavidin-coated magnetic beads (MBs) using the specific binding between biotin and streptavidin. After magnetic separation, the BCR / ABL fusion gene can be directly detected by flow cytometry.

[0028] In one embodiment, the kit includes the primers described above, as well as streptavidin-modified magnetic beads, and further includes horseradish peroxidase, hydrogen peroxide, and TMB labeled with anti-6-carboxyfluorescein antibody. Using this kit, a ligase chain reaction initiated by the target nucleic acid consumes the primers to obtain a DNA double-stranded amplification product labeled with biotin and 6-carboxyfluorescein (FAM) at the head and tail, respectively. The amplification product is immobilized onto magnetic beads (MBs) coated with streptavidin using the specific binding between biotin and streptavidin. The HRP-FAM-dsDNA-MBs complex is obtained by binding to horseradish peroxidase (anti-FAM-HRP) labeled with anti-6-carboxyfluorescein antibody. Electrochemical and colorimetric detection is then performed via the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide (H2O2) catalyzed by horseradish peroxidase, thus achieving ultrasensitive and cross-validated detection of the BCR / ABL fusion gene.

[0029] This invention provides a method for amplifying the BCR / ABL fusion gene in chronic myeloid leukemia. In the presence of the target gene, the above-mentioned primers CP, SP, T1, and T2 are used for ligase chain reaction.

[0030] In one embodiment, the reaction conditions for the ligase chain reaction are: 65°C for 2 min; 94°C for 1 min, 30 cycles.

[0031] In one embodiment, the reaction system for the ligase chain reaction is 5 μL CP (0.1 μM), 5 μL SP (0.1 μM), 5 μL T1 (0.1 μM), 5 μL T2 (0.1 μM), 0.2 μL Ampligase (5 U / μl), 10 μL Reaction Buffer (10×), 10 μL target gene solution, and 59.8 μL Tris-HCl buffer (pH = 7.4).

[0032] Example 1

[0033] The exponential amplification of target nucleic acids by ligase chain reaction

[0034] To achieve high-sensitivity detection of the BCR-ABL fusion gene, this invention designed four DNA primers for ligase chain reaction, the nucleotide sequences of which are shown in Table 1 (b2a2 fusion gene) and Table 2 (b3a2 fusion gene).

[0035] The selection of target sequences for CML detection and the design of primers for LCR amplification are as follows:

[0036] (1) Selection of CML-specific target sequences

[0037] The target gene tested is the BCR / ABL fusion gene. BCR gene breakpoints are concentrated in three regions: the major bcr (M-bcr) region near exon e13 / e14, the minor bcr (m-bcr) region of the first intron, and the μ region (μ-bcr) between exons e19 and e20. ABL gene breakpoints are located in the first or second intron. Due to the different breakpoints, BCR / ABL fusion can produce four transcripts: e19a2, e14a2, e1a2, and e13a2. In chronic myeloid leukemia, the BCR gene breakpoint is often located at m-bcr, primarily producing two transcripts: e13a2 (b2a2) and e14a2 (b3a2). By searching GeneBank for the exon 13 / 14 sequence of the human BCR gene, the exon 2 sequence of the ABL gene, and the fusion gene sequences of BCR / ABL e13a2 and e14a2, and then performing sequence alignment, the fusion site and nearby sequences can be determined.

[0038] (2) LCR amplification primer design

[0039] After identifying the fusion site and the nearby sequence, the complementary strand of this sequence is cDNA, which serves as the target gene sequence for detection. Considering the optimal activity temperature range of ampligase in LCR and the half-determination temperature (Tm) of the DNA probe, the required primer lengths are determined, and four primers (CP, SP, T1, and T2) with corresponding functional group modifications are synthesized: CP is modified with biotin at the 5' end; SP is phosphorylated at the 5' end and modified with fluorescein (FAM) at the 3' end; T2 is phosphorylated at the 5' end to ensure maximum amplification efficiency and the ability to distinguish single-base mismatches.

[0040] In the presence of the target gene, CP and SP hybridize with the target gene to form a DNA double strand with a gap. The thermostable DNA ligase ampligase catalyzes the formation of phosphodiester bonds at the gap, yielding the ligation product CP-SP. After thermal denaturation, the target gene and the ligation product separate. The target gene can be used again as a template for CP and SP for ligation reactions, while the ligation product CP-SP can be used as a template for T1 and T2 to form new ligation products T1-T2 (i.e., the target gene). Therefore, starting from the second thermal cycle, the number of ligation products theoretically doubles with each thermal cycle, thereby achieving exponential amplification of the target gene and obtaining a large number of DNA double strands labeled with anchoring groups (biotin) and beacon groups (6-carboxyfluorescein) at the head and tail, respectively.

[0041] The Ampligase and Reaction Buffer used in this embodiment were purchased from Lucigen, Inc., USA, and the LCR primers (CP, SP, T1, T2) were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0042] All deoxyribonucleic acid sequences involved in the exponential amplification of the target gene nucleic acid by the ligase chain reaction, including LCR primers CP, SP, T1, T2 and the target T, are shown in Tables 1 and 2.

[0043] Table 1

[0044]

[0045] Table 2

[0046]

[0047]

[0048] Preparation of LCR reaction products: 5 μL CP (0.1 μM), 5 μL SP (0.1 μM), 5 μL T1 (0.1 μM), 5 μL T2 (0.1 μM), 0.2 μL Ampligase (5 U / μL), 10 μL Reaction Buffer (10×), 10 μL target gene solution, and 59.8 μL Tris-HCl buffer (pH = 7.4) were thoroughly mixed and placed in a PCR instrument for amplification. Reaction conditions: 65℃ for 2 min, then 94℃ for 1 min, 30 cycles. After the reaction, the product was stored at 4℃ for later use.

[0049] Example 2

[0050] Construction of functional magnetic bead carriers

[0051] (1) Gently shake the streptavidin-modified magnetic bead stock solution (10 mg / ml) to disperse it evenly, and then take 0.4 μL into a 0.2 mL centrifuge tube.

[0052] (2) Add 100 μL of magnetic bead washing buffer and vortex to mix. Place the mixture on a small magnetic rack and wait for the magnetic beads to clump together on one side of the centrifuge tube before discarding the buffer.

[0053] (3) Repeat step (2) twice, add 50 μL of LCR reaction product to resuspend the magnetic beads and vortex to mix. Spin-assemble at room temperature for 30 min. Place the centrifuge tube on a small magnetic rack. After the magnetic beads clump together on one side of the centrifuge tube, discard the reaction solution. Repeat step (2) three times to wash the magnetic beads to obtain the FAM-dsDNA-MBs complex. Store at 4℃ for later use.

[0054] The streptavidin-modified magnetic beads (Dynabeads™ M-270 Streptavidin) used in this embodiment were purchased from Thermo Fisher Scientific.

[0055] Example 3

[0056] Flow cytometry detection

[0057] Add 100 μL of deionized water to resuspend the FAM-dsDNA-MBs complex obtained in step (4), mix well, and place it in the flow cytometer inlet. Under 488 nm laser excitation, detect the fluorescence signal of each MBs through the FL1 (FITC / FAM, 530±15 nm) channel. The parameters are set as follows: the voltage of the FL1 detector is set to 470 V, the amplifier gain is 1, the laser power is 15 mW, the sample voltage is 6.4 V, and 10,000 MBs are collected for each sample. Detection is performed on target gene standards of different concentrations. The working curve is plotted based on the relationship between the average fluorescence intensity (MFI) and the concentration of the target gene standard. Figure 2 As shown.

[0058] from Figure 2 As can be seen from this, the target DNA concentration was measured at 10... -14 M~10 -9 Within the M range, the MFI value shows a good linear relationship with the logarithm of the target chain concentration lgCtarget(M), and the detection limit is 7fM (>3σ), indicating that the constructed flow cytometry detection method has a wide linear range (6 orders of magnitude) and high sensitivity.

[0059] Example 4

[0060] Colorimetric detection

[0061] The FAM-dsDNA-MBs complex obtained in Example 2 was resuspended in 100 μL of horseradish peroxidase (Anti-FAM-HRP) labeled with 0.5 U / mL anti-6-carboxyfluorescein antibody and incubated by rotation at room temperature for 15 min. After magnetic separation, the reaction solution was discarded, and step (2) of Example 2 was repeated three times to wash the magnetic beads. The complex was resuspended in 10 μL of PBS to obtain the HRP-FAM-dsDNA-MBs complex. The complex was added to 90 μL of TMB substrate (containing H2O2) solution, mixed, and developed for 5 min. The reaction was terminated by adding 50 μL of 2M sulfuric acid solution. The solution changed from blue to yellow. The absorbance at 450 nm was measured using a microplate reader. The working curve was plotted based on the relationship between absorbance and the concentration of the target gene standard, as shown in the figure. Figure 3 As shown.

[0062] The horseradish peroxidase labeled with anti-6-carboxyfluorescein antibody used in this embodiment was purchased from Roche Pharmaceuticals Ltd. in Shanghai, and the 3,3',5,5'-tetramethylbenzidine (containing H2O2) substrate solution was purchased from Neogen Pharmaceuticals, Inc. in the United States.

[0063] from Figure 3 As can be seen from this, the target DNA concentration was measured at 10... -17 M~5×10 -15 Within the M range, the OD value showed a good linear relationship with the target chain concentration Ctarget, and the detection limit was 10aM (>3σ), indicating that the constructed colorimetric biosensor has high sensitivity.

[0064] Example 5

[0065] Electrochemical detection

[0066] A magnetic glassy carbon electrode (MGCE, 3 mm diameter) was polished on chamois leather for 3 min using a suspension of 0.05 μm Al2O3 powder. It was then sonicated for 1 min each in anhydrous ethanol and ultrapure water to remove residual Al2O3 powder and other impurities, and finally dried with nitrogen. Next, the HRP-FAM-dsDNA-MBs complex obtained in Example 4 was dropped onto the surface of the magnetic glassy carbon electrode. Under the action of a magnetic field, the MBs-dsDNA-HRP complex was immobilized on the electrode surface, resulting in an MBs-modified electrode. Measurements were performed using a three-electrode system on an electrochemical workstation. The MBs-modified electrode was used as the working electrode, Ag / AgCl as the reference electrode, and a platinum wire electrode as the auxiliary electrode. Measurements were performed in a TMB (containing H2O2) solution using a time-current curve at a potential of 0.1 V and a scan time of 100 s. The working curve was plotted based on the relationship between the stable current value obtained at 100 s and the concentration of the target gene standard solution. Figure 4 As shown.

[0067] from Figure 4 It can be seen from this that at a concentration of 10 -19 M~5×10 -16 M(A diagram) and 10 -15 M~10 -12 Within the two ranges of M (Figure B), the corresponding current signal values ​​showed a good linear relationship with the logarithm of the target chain concentration, lgCtarget(M). The limit of detection was 0.045aM (equivalent to 2 copies of the target in 100μl reaction system, >3σ), indicating that the constructed electrochemical biosensor has extremely high sensitivity, comparable to traditional real-time quantitative PCR.

[0068] Example 6

[0069] Sensor specificity assessment:

[0070] The ligase chain reaction was performed according to the method in Example 1. The target gene strands added were a completely complementary strand (target), a single-base mismatch strand (T-Mut, C-Mut, and G-Mut), and a three-base mismatch strand, respectively. The concentration of the DNA strands used was 10. - 14 M (taking b3a2 as an example); then electrochemical detection was performed according to the method in Example 5. Their respective current responses were compared with the blank current response results. Experimental results are as follows... Figure 5 As shown.

[0071] from Figure 5As can be seen, the signal of the perfectly complementary strand is more than 40 times that of the single base mismatch signal, indicating that the constructed electrochemical biosensor has high specificity and can be used for the analysis of single nucleotide polymorphisms (SNPs).

[0072] Example 7

[0073] A kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia includes the primers described above, as well as streptavidin-modified magnetic beads and other necessary reagents.

[0074] Example 8

[0075] A kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia includes the primers described above, as well as streptavidin-modified magnetic beads, and further includes horseradish peroxidase labeled with anti-6-carboxyfluorescein antibody, hydrogen peroxide, TMB, and other necessary reagents.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A primer for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, characterized in that, It includes LCR primers CP, SP, T1, and T2, whose nucleotide sequences are shown in SEQ ID NO:1-SEQ ID NO:4 or in SEQ ID NO:5-SEQ ID NO:

8.

2. A kit for detecting the BCR / ABL fusion gene in chronic myeloid leukemia, characterized in that, Includes the primers described in claim 1.

3. The reagent kit according to claim 2, characterized in that, It also includes streptavidin-modified magnetic beads.

4. The reagent kit according to claim 3, characterized in that, It also includes horseradish peroxidase, hydrogen peroxide, and TMB labeled with anti-6-carboxyfluorescein antibody.

5. A method for amplifying the BCR / ABL fusion gene in chronic myeloid leukemia for non-disease diagnosis, characterized in that, In the presence of the target gene, a ligase chain reaction is performed using primers, followed by electrochemical detection; the primers include LCR primers CP, SP, T1, T2, whose nucleotide sequences are shown in SEQ ID NO:1-SEQ ID NO:4, or in SEQ ID NO:5-SEQ ID NO:

8.

6. The method according to claim 5, characterized in that, The reaction conditions for the ligase chain reaction were: 65 ℃ for 2 min; 94 ℃ for 1 min, for 30 cycles.

7. The method according to claim 5, characterized in that, The reaction system for the ligase chain reaction consisted of 5 μL of 0.1 μM CP, 5 μL of 0.1 μM SP, 5 μL of 0.1 μM T1, 5 μL of 0.1 μM T2, 0.2 μL of 5 U / μL Ampligase, 10 μL of 10× Reaction Buffer, 10 μL of target gene solution, and 59.8 μL of Tris-HCl buffer at pH 7.4.

Images