A composition and kit for detecting BCR-ABL1 P210 fusion gene

Abstract

The present invention relates to the technical field of fusion gene detection, and in particular to a composition and a kit for detecting BCR-ABL1P210 fusion gene. The composition includes a pair of BCR-ABL1P210 fusion gene detection primers and a BCR-ABL1P210 fusion gene detection probe, and also includes a pair of internal reference gene ABL1 detection primers and an internal reference gene ABL1 detection probe, and both ends of the BCR-ABL1P210 fusion gene detection probe and the ABL1 detection probe are respectively labeled with a fluorescent group and a quenching group. On the basis of conventional real-time fluorescent quantitative PCR, the present invention redesigns primer probes for BCR-ABL1P210 and internal reference gene ABL1 and designs LNA modification, which can make the detection sensitivity reach 0.0002%.

Description

Technical Field

[0001] The present invention relates to the technical field of fusion gene detection, and in particular to a composition and a kit for detecting BCR-ABL1 P210 type fusion gene. Background Art

[0002] More than 95% of chronic myeloid leukemia (CML) patients have typical ph chromosomes, which are caused by the translocation of the ABL gene located at 9q34 and the BCR gene located at 22q11, forming t(9;22)(q34;q11) and BCR / ABL1 fusion gene. BCR / ABL1 fusion gene is also found in 25-30% of adult acute lymphoblastic leukemia (ALL) and 2-10% of childhood acute lymphoblastic leukemia (ALL). The breakpoints of the BCR gene are concentrated in three regions: major (major bcr, M-bcr), minor (minorbcr, m-bcr) and μ (μ-bcr), forming three major forms of BCR-ABL1 fusion gene, b3a2 type, b2a2 type and e19a2 type, which are named BCR-ABL1 p190, BCR-ABL11 p210 and BCR-ABL p230 according to the length of their encoded proteins. In chronic myeloid leukemia cases, 55% are b3a2 and 40% are b2a2. In 5% of cases, both b3a2 and b2a2 exist simultaneously due to different splicing.

[0003] BCR-ABL1 is an anti-apoptotic gene with tyrosine kinase activity. It can overactivate the Ras-Raf-MAPK signaling pathway, the PI3K-AKT-mTOR signaling pathway, and the JAK-STAT signaling pathway, leading to excessive cell proliferation and disordered cell regulation. In addition, BCR-ABL1 also inhibits DNA repair, causing genomic instability and deterioration of patients with chronic myeloid leukemia. BCR-ABL1 is formed in the pre-stage of chronic myeloid leukemia. Therefore, the detection of BCR-ABL1 has important clinical guidance significance for auxiliary diagnosis of chronic myeloid leukemia, targeted drug guidance, and minimal residual disease (MRD) monitoring.

[0004] At present, the methods for quantitative detection of BCR-ABL1 P210 fusion gene mainly include real-time fluorescence quantitative PCR and digital PCR. The real-time fluorescence quantitative PCR method can quickly detect quantitatively, but the detection sensitivity of the existing test kits does not exceed 0.01%. The digital PCR method uses BCR-ABL1 P210 fusion gene quantitative detection, and the sensitivity can reach 0.001%, but the detection cost is extremely high. Summary of the invention

[0005] Based on this, one of the objects of the present invention is to provide a composition for detecting BCR-ABL1 P210 fusion gene, comprising a pair of BCR-ABL1 P210 fusion gene detection primers and a BCR-ABL1 P210 fusion gene detection probe, wherein the upstream primer sequence of the BCR-ABL1 P210 fusion gene detection primer is 5'-GCTGACCAACTCGTGTGTGAA-3', the downstream primer sequence of the BCR-ABL1 P210 fusion gene detection primer is 5'-CACTCAGACCCTGAGGCTCA-3', the BCR-ABL1 P210 fusion gene detection probe sequence is: 5'-ACAGCATTCCGCTGACCAT-3', and the 6th to 13th bases from the 5' end of the BCR-ABL1 P210 fusion gene detection probe are LNA modification sites;

[0006] It also includes a pair of internal reference gene ABL1 detection primers and an internal reference gene ABL1 detection probe, wherein the upstream primer sequence of the internal reference gene ABL1 detection primer is 5'-GGGTCCCAAGCAACT ACATCA-3', the downstream primer sequence of the internal reference gene ABL1 detection primer is 5'-GGCCAG GACTGCTCTCACTCT-3', the sequence of the internal reference gene ABL1 detection probe is 5'-TGGC AGCTTCTTGGTG-3', and the 5th to 12th bases from the 5' end of the ABL1 detection probe are LNA modification sites;

[0007] Both ends of the BCR-ABL1 P210 fusion gene detection probe and the ABL1 detection probe are respectively labeled with a fluorescent group and a quenching group.

[0008] On the basis of conventional real-time fluorescence quantitative PCR, the present invention redesigns primer probes for BCR-ABL1P210 and internal reference gene ABL1 and designs LNA modification. The composition can achieve a detection sensitivity of 0.002%, which is much higher than the existing detection means.

[0009] As a preferred embodiment, the fluorescent group includes but is not limited to FAM, VIC, and Cy5.

[0010] As a preferred embodiment, the quenching group includes but is not limited to MGB, Cy3, and BHQ.

[0011] As a preferred embodiment, the fluorescent label is located at the 5' end of the probe, and the quenching label is located at the 3' end of the probe.

[0012] The second object of the present invention is to protect a kit for detecting BCR-ABL1 P210 fusion gene, comprising the composition described in any one of claims 1 to 4.

[0013] As a preferred embodiment, the kit further comprises Buffer, dNTP, Taq enzyme, strong positive quality control, weak positive quality control, negative quality control, BCR-ABL1 P210 gradient standard, ABL1 gradient standard. When the kit is used for Q-PCR, the reaction system is 25 μL, the amount of Buffer added in the system is not less than 5 μL, the final concentration of dNTP is not less than 0.6×, the amount of Taq enzyme added is not less than 5 μL, the final concentration of BCR / ABL1 P210 probe is 0.25 μM or more, the final concentration of ABL1 probe is 0.30 μM or more, and the concentration of primers and probes is (1-3):1;

[0014] Among them, the contents of dATP, dCTP, and dGTP in 12.5×dNTP are all 2.5mM, and the content of dUTP is 7.5mM.

[0015] The third object of the present invention is to protect a method for detecting BCR-ABL1P210 fusion gene for non-disease diagnosis purposes, comprising the following steps:

[0016] S1, extract sample RNA;

[0017] S2, using the sample RNA, BCR-ABL1 P210 gradient standard, ABL1 gradient standard, strong positive quality control, weak positive quality control, and negative quality control in step S1 as templates, and using the fusion gene detection primers and detection probes and the internal reference gene detection primers and detection probes in the composition according to any one of claims 1 to 4 to perform fluorescent quantitative PCR amplification reactions respectively;

[0018] S3, result determination:

[0019] If the negative control product has no amplification curve, the MR of the weak positive control product is ≥3.0, and the MR of the positive control product is ≤1.5, then the experimental result is considered reliable; otherwise, the experiment is considered invalid.

[0020] If the BCR-ABL1 P210 copy number is ≥7, the ABL1 copy number is ≥32000, and (BCR-ABL1 P210 copy number / ABL1 copy number) × 100% is ≥ 0.002%, it is considered positive;

[0021] If the BCR-ABL1 P210 copy number is <7 or the Ct value is no amplification, and the ABL1 copy number is ≥32,000, it is judged as negative.

[0022] As a preferred embodiment, the Q-PCR reaction system is 25 μL, the amount of buffer added in the system is not less than 5 μL, the final concentration of dNTP is not less than 0.6×, the amount of Taq enzyme added is not less than 5 μL, the final concentration of BCR / ABL1 P210 probe is 0.25 μM or more, the final concentration of ABL1 probe is 0.30 μM or more, and the concentration of primers and probes is (1-3):1;

[0023] Among them, the contents of dATP, dCTP, and dGTP in 12.5×dNTP are all 2.5mM, and the content of dUTP is 7.5mM.

[0024] Specifically, the Q-PCR reaction system is 25 μL, wherein the amount of buffer added is 5 μL, the amount of 12.5×dNT P added is 1.2 μL, the amount of Taq enzyme added is 0.5 μL, the final concentration of BCR / ABL1 P210 primer is 0.5 μM, the final concentration of BCR / ABL1 P210 probe is 0.25 μM, the amount of ROX dye added is 0.5 μL, and the amount of template added is 10 μL; or the amount of buffer added is 5 μL, the amount of 12.5×dNT P added is 1.2 μL, the amount of Taq enzyme added is 0.5 μL, the final concentration of ABL1 primer is 0.5 μM, the final concentration of ABL1 probe is 0.3 μM, the amount of ROX dye added is 0.5 μL, and the amount of template added is 10 μL.

[0025] By optimizing the PCR system, the present invention can achieve a detection sensitivity of 0.0002% for the BCR-ABL1 P210 fusion gene. By performing detection and determination according to the above method, the detection accuracy of the BCR-ABL1 P210 fusion gene is also extremely high. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the linear relationship between the logarithmic mean of the ratio of the positive sample detection values ​​in the sensitivity experiment in Example 3 of the present invention and the theoretical logarithmic value;

[0027] Figure 2 is the Buffer gradient amplification curve in Example 5 of the present invention;

[0028] Figure 3 is the DUTP gradient amplification curve in Example 5 of the present invention;

[0029] Figure 4 This is the probe concentration gradient amplification curve in Example 5 of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0031] The following embodiments are only used to illustrate the present invention, but are not used to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0032] Unless otherwise specified, the reagents used in the examples of the present invention are all commercially available. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989) compiled by Sambrook et al., or under the conditions recommended by the manufacturer.

[0033] The LNA modification involved in the present invention refers to a locked nucleic acid label, which is a synthetic nucleic acid analog containing a bridged bicyclic sugar group.

[0034] Example 1

[0035] The embodiment of the present invention provides a kit for detecting BCR-ABL1 P210 type fusion gene, which mainly includes reagent A and reagent B;

[0036] Reagent A is a mixture of BCR-ABL1 P210 fusion gene detection primers and BCR-ABL1 P210 fusion gene detection probes, and reagent B is a mixture of internal reference gene ABL1 detection primers and internal reference gene ABL1 detection probes. For specific sequence information and concentrations, see Table 1.

[0037] Also includes Buffer (Mg 2+ ), d N(U)TP, Taq enzyme;

[0038] It also includes strong positive quality control products, weak positive quality control products, negative quality control products, BCR-ABL1 P210 gradient standard products, and ABL1 gradient standard products. Please refer to Table 2 for details.

[0039] Table 1 Primer probe sequences and concentrations

[0040]

[0041]

[0042] The underlined bases are LNA-modified bases, which are the 6th to 13th bases from the 5' end in BCR-ABL1 P210 P and the 5th to 12th bases from the 5' end in ABL1 P.

[0043] Table 2 Concentrations of quality control products and standard products

[0044] Reagent name concentration Strong positive control Ratio ≥ 3.2% Weak positive control Ratio ≤ 0.1% Negative Control <![CDATA[The negative ABL1 copy number is 10 5 copies / 10μL]]> BCR / ABL1P210 Standard 1 <![CDATA[10 3 copies / 10μL]]> BCR / ABL1P210 Standard 2 <![CDATA[10 4 copies / 10μL]]> BCR / ABL1P210 Standard 3 <![CDATA[10 5 copies / 10μL]]> BCR / ABL1P210 Standard 4 <![CDATA[10 6 copies / 10μL]]> ABL1 Standard 1 <![CDATA[10 3 copies / 10μL]]> ABL1 Standard 2 <![CDATA[10 4 copies / 10μL]]> ABL1 Standard 3 <![CDATA[10 5 copies / 10μL]]> ABL1 Standard 4 <![CDATA[10 6 copies / 10μL]]>

[0045] Example 2

[0046] This embodiment provides a method for detecting the BCR-ABL1 P210 fusion gene, which is specifically performed using the kit described in Example 1. The method is as follows:

[0047] (1) Reverse transcription

[0048] Take the reverse transcription reagent out of the -20℃ refrigerator, melt it at room temperature, centrifuge it instantaneously, collect the liquid at the bottom of the tube, and place it on ice for later use. Take the corresponding amount of reverse transcription buffer (4μL / person), reverse transcriptase (1.5μL / person), reverse transcription primer (i.e., reagent A, reagent B, 2μL / person) and RNase-free water (1μL / person) to prepare a reverse transcription mixed liquid, see Table 3 for details. Vortex oscillation, centrifuge it instantaneously, and dispense 4μL per person into thin-walled 8-link reaction tubes, and place them on ice for later use.

[0049] Table 3 Reverse transcription reaction mixture system

[0050] RT reaction mixture Volume (μL) Reverse transcription buffer 4 Reverse transcriptase 1.5 Reagent A (or Reagent B) 2 RNase-free water 0.5 Total volume of reverse transcription mixture (μL) 8

[0051] Take 1000-2000 ng of the sample RNA to be tested and add it to the pre-packaged 8 μL reverse transcription reaction mixture per person, and add RNase-free water to 20 μL. For details, see Table 4.

[0052] Table 4 Reverse transcription reaction system preparation

[0053] Components Volume (μL) Reverse transcription mixture 8 RNA to be detected 1000-2000ng RNase-free water X Total volume (μL) 20

[0054] Put the cap on the reverse transcription reaction system, vortex and centrifuge it. Turn on the PCR instrument, place the 8-tube strip containing the reaction system on the metal block, and perform the reaction according to the procedure in Table 5.

[0055] Table 5 Reverse transcription PCR program

[0056]

[0057] After the PCR reaction is completed, the product is cDNA, which can be directly used as a template for Q-PCR reaction. The product is taken out of the PCR instrument and temporarily stored in a 4°C refrigerator, or frozen at -20°C for subsequent experiments.

[0058] (2) Q-PCR

[0059] The strong positive quality control, weak positive quality control and negative quality control, BCR-ABL1 P210 qPCR reaction solution, ABL1 qPCR reaction solution, BCR-ABL1 P210 gradient standard and ABL1 gradient standard were taken out, equilibrated to room temperature, vortexed for 3-5 seconds, and centrifuged for instant use.

[0060] Preparation of Q-PCR reaction mixture: according to the number of samples to be tested, strong positive quality control, weak positive quality control, negative quality control, BCR-ABL1 P210 gradient standard and ABL1 gradient standard, see Table 6 for details.

[0061] Table 6 qPCR reaction system

[0062]

[0063]

[0064] Buffer (Mg 2+ ) and Taq enzyme were purchased from Beijing Qihengxing Biotechnology Co., Ltd. with the catalog number FS-Q3005, dNTP (12.5X) was purchased from Aikerui Biotechnology Co., Ltd. with the catalog number AG11021, 12.5X dN(U)TP mixture was prepared by mixing dATP, dCTP, dGTP (2.5 mM each) and dUTP (7.5 mM), and reverse transcriptase was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. with the catalog number RR037A.

[0065] Turn on the fluorescence quantitative PCR amplification instrument and place the PCR thin-walled reaction tube containing the reaction system on the metal module. Set the fluorescence pathway for the sample to be tested, set the target genes to BCR-ABL1 P210 and ABL1 respectively, and the pathway to FAM-MGB. At the same time, set the standard curve for BCR-ABL1 P210 and ABL1. The corresponding copy numbers of BCR-ABL1 P210 and ABL1 standards 1-4 are 2000, 20000, 200000, and 2000000, and the amplification volume is set to 25 μL. Open the program setting window in the instrument and set the program according to Table 7 below.

[0066] Table 7 Q-PCR program

[0067]

[0068]

[0069] After the PCR program is finished, observe the amplification curve and use the default value for the baseline. The threshold is manually adjusted and can be set to the inflection point of the logarithmic growth period of the fluorescence signal according to the actual situation. In addition, when making the BCR-ABL1 P210 and ABL1 standard curves, repeat twice. After adjusting the threshold and cycle number, ensure that the BCR-ABL1 P210 and ABL1 standard curves have good reproducibility twice, and ensure that the Ct value and copy number linear correlation coefficient (R 2 ) is greater than 0.98, and the linear correlation coefficient (R 2 ) is closer to 1, the more reliable the result is, and the amplification efficiency (E) is between 0.9 and 1.1. Otherwise, repeat the experiment.

[0070] (3) Result determination

[0071] a. Quality Control:

[0072] Experiments that meet the quality control standards shown in Table 8 below are considered valid experiments, otherwise, the experiment is invalid.

[0073] Table 8 Quality Control Standards

[0074] Quality Control Products Detection range Negative Control Negative, no amplification curve Weak positive control MR ≥ 3.0 (≤ 0.1%) Positive Control MR ≤ 1.5 (≥ 3.2%)

[0075] b. Interpretation of results:

[0076] After Q-PCR amplification, the BCR-ABL1 P210 and ABL1 copy numbers of the samples were calculated using the obtained BCR-ABL1 P210 and ABL1 standard curves.

[0077] The test was considered positive if the number of BCR-ABL1 P210 copies was ≥7 copies, the number of ABL1 copies was ≥32,000 copies, and (the number of BCR-ABL1 P210 copies / the number of ABL1 copies)×100% was ≥0.002%.

[0078] If the BCR-ABL1 P210 copy number is less than 7 copies or the Ct value is no amplification (Undetermined), and the ABL1 copy number is ≥32000 copies, it is judged as negative.

[0079] When the ABL1 copy number of the sample to be tested is less than 32,000 copies or the Ct value is undetermined, there may be problems with inhibitors or too low RNA addition, and retesting is required.

[0080] Example 3 Accuracy Experiment

[0081] Blood samples from 10 normal subjects and 10 positive samples were selected and RNA was extracted. The samples were tested and determined according to the method described in Example 2, where the samples from normal subjects were numbered N1 to N20, and the samples from patients were numbered M1 to M10. The results are shown in Table 9 below.

[0082] Table 9 Accuracy experimental results

[0083]

[0084]

[0085] As shown in Table 9, the test results are consistent with the actual situation when the test method of Example 2 is used, which shows that the test results of the present invention are accurate and reliable.

[0086] Example 4 Sensitivity Experiment

[0087] The BCR-ABL1 P210 positive samples were gradiently diluted with negative samples, and the dilution gradients were 100%, 10%, 1%, 0.1%, 0.01%, 0.0032% and 0.002%, numbered E1 to E7, respectively. The first 4 gradients were repeated 3 times, and the last 3 gradients were repeated 5 times. The average value was taken. The results are shown in Table 10.

[0088] Table 10 Sensitivity test results

[0089]

[0090]

[0091] The correlation coefficient and linear range were calculated based on the test data shown in Table 10. The results are as follows: Figure 1 As shown, R 2 >0.98, acceptable, and the minimum detection sensitivity is 0.0002%.

[0092] Example 5 Study on quantitative PCR system

[0093] The reagents used in this example are the same as those in Table 5, specifically Buffer (Mg 2+ ) and Taq enzyme were purchased from Beijing Qihengxing Biotechnology Co., Ltd. with the catalog number FS-Q3005, dNTP (12.5X) was purchased from Aikerui Biotechnology Co., Ltd. with the catalog number AG11021, and reverse transcriptase was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. with the catalog number RR037A. The following variables were studied:

[0094] (1) Buffer gradient test: Change the buffer and ddH in the Q-PCR system in Table 6 of Example 2 2O addition amount, ensure the system remains unchanged, prepare different Buffer (Mg 2+ ) Gradient(Buffer(Mg 2+ ) was added in the system with the addition amount of 3 μL, 4 μL, 5 μL and 6 μL, respectively, and Q-PCR was performed on BCR / ABL1 P210 standards 1-4 and ABL1 standards 1-4. The results are shown in Tables 11 and Figure 2 As shown:

[0095] Table 11 Effects of different buffer addition amounts on Q-PCR

[0096]

[0097]

[0098] From Table 11 and Figure 2 It can be seen that Buffer (Mg 2+ ) When the input volume is above 5 μL, the effect on the system does not change much, therefore, the optimal input volume is 5 μL.

[0099] (2) dN(U)TP gradient test: Changing the dUTP and ddH in the Q-PCR reaction system in Table 6 of Example 2 2 O addition amount, ensure that the system remains unchanged, and do a gradient test on d N(U)TP in the reaction system. The dUTP gradient is 1.2μL, 1.6μL, 2.0μL and 2.4μL, and the concentration in the total system is 0.6X, 0.8X, 1X and 1.2X, respectively. BCR / ABL1P210 standard products 1-4 and ABL1 standard products 1-4 are used. The results are shown in Tables 12 and 12. Figure 3 As shown:

[0100] Table 12 Effects of different dN(U)TP concentrations on Q-PCR

[0101]

[0102] From Table 12 and Figure 3 It can be seen that when the input volume of DUTP is above 1.2 μL, its effect on the system does not change much. Therefore, the optimal input volume is 1.2 μL and the final concentration is 0.6X.

[0103] (3) Primer-probe gradient test: Change the primers, probes and ddH in the Q-PCR system in Table 6 of Example 2. 2O addition amount, ensure that the system remains unchanged, prepare systems with different primer probe concentrations, where the final concentration of the primers is 0.5μM, set a concentration gradient for the BCR / ABL1P210 and ABL1 probes, and the probe concentrations are 0.15μM, 0.20μM, 0.25μM, and 0.30μM, respectively, using BCR / ABL1P210 standard products 1-4 and ABL1 standard products 1-4. The results are as shown in Tables 13 and Figure 4 As shown:

[0104] Table 13 Effect of different primer probe concentrations on Q-PCR

[0105]

[0106] From Table 13 and Figure 4 It can be seen that when the final concentration of BCR / ABL1 P210 probe is above 0.25 μM and the final concentration of ABL1 probe is above 0.30 μM, the effect on the system does not change much. Therefore, the optimal final concentration of BCR / ABL1P210 probe is 0.25 μM, and the final concentration of ABL1 probe is 0.30 μM.

[0107] (4) System size test: the volume of each component in Table 6 of Example 2 was changed and reduced in proportion, and 20 μL and 25 μL systems were prepared respectively, using BCR / ABL1P210 standard products 1-4 and ABL1 standard products 1-4. The results are shown in Table 14 below:

[0108] Table 14 Effect of reaction system size on Q-PCR

[0109]

[0110] From Table 14, we know that the best reaction system is 25 μL.

[0111] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composition for detecting BCR-ABL1 P210 type fusion gene, characterized in that: It comprises a pair of BCR-ABL1P210 fusion gene detection primers and a BCR-ABL1P210 fusion gene detection probe, wherein the upstream primer sequence of the BCR-ABL1P210 fusion gene detection primer is 5'-GCTGACCAACTCGTGTGTGAA-3', the downstream primer sequence of the BCR-ABL1P210 fusion gene detection primer is 5'-CACTCAGACCCTGAGGCTCA-3', the sequence of the BCR-ABL1P210 fusion gene detection probe is: 5'-ACAGCATTCCGCTGAC CAT-3', and the 6th to 13th bases from the 5' end of the BCR-ABL1P210 fusion gene detection probe are LNA modification sites; It also includes a pair of internal reference gene ABL1 detection primers and an internal reference gene ABL1 detection probe, wherein the upstream primer sequence of the internal reference gene ABL1 detection primer is 5'-GGGTCCCAAGCAACT ACATCA-3', the downstream primer sequence of the internal reference gene ABL1 detection primer is 5'-GGCCAG GACTGCTCTCACTCT-3', the sequence of the internal reference gene ABL1 detection probe is 5'-TGGC AGCTTCTTGGTG-3', and the 5th to 12th bases from the 5' end of the ABL1 detection probe are LNA modification sites; Both ends of the BCR-ABL1 P210 fusion gene detection probe and the ABL1 detection probe are respectively labeled with a fluorescent group and a quenching group.

2. The composition for detecting BCR-ABL1 P210 type fusion gene according to claim 1, characterized in that: The fluorescent groups include but are not limited to FAM, VIC, and Cy5.

3. The composition for detecting BCR-ABL1 P210 type fusion gene according to claim 1, characterized in that: The quenching groups include but are not limited to MGB, Cy3, and BHQ.

4. The composition for detecting BCR-ABL1 P210 type fusion gene according to any one of claims 1 to 3, characterized in that: The fluorescent label is located at the 5' end of the probe and the quencher label is located at the 3' end of the probe.

5. A kit for detecting BCR-ABL1 P210 fusion gene, characterized in that: A composition comprising any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that It also includes Buffer, dNTP, Taq enzyme, strong positive quality control, weak positive quality control, negative quality control, BCR-ABL1 P210 gradient standard, ABL1 gradient standard. When the kit is used for Q-PCR, the reaction system is 25 μL, the amount of Buffer added in the system is not less than 5 μL, the final concentration of dNTP is not less than 0.6×, the amount of Taq enzyme added is not less than 5 μL, the final concentration of BCR / ABL1 P210 probe is 0.25 μM or more, the final concentration of ABL1 probe is 0.30 μM or more, and the concentration of primers and probes is (1-3):1; Among them, the contents of dATP, dCTP, and dGTP in 12.5×dNTP are all 2.5mM, and the content of dUTP is 7.5mM.

7. A method for detecting BCR-ABL1 P210 fusion gene for non-disease diagnosis purposes, characterized in that: The steps include: S1, extract sample RNA; S2, using the sample RNA, BCR-ABL1 P210 gradient standard, ABL1 gradient standard, strong positive quality control, weak positive quality control, and negative quality control in step S1 as templates, and using the fusion gene detection primers and detection probes and the internal reference gene detection primers and detection probes in the composition of any one of claims 1 to 4 to perform Q-PCR amplification reactions respectively; S3, result determination: If the negative control product has no amplification curve, the MR of the weak positive control product is ≥3.0, and the MR of the positive control product is ≤1.5, then the experimental result is considered reliable; otherwise, the experiment is considered invalid. If the BCR-ABL1 P210 copy number is ≥7, the ABL1 copy number is ≥32000, and (BCR-ABL1 P210 copy number / ABL1 copy number) × 100% is ≥ 0.002%, it is considered positive; If the BCR-ABL1 P210 copy number is <7 or the Ct value is no amplification, and the ABL1 copy number is ≥32,000, it is judged as negative.

8. The method according to claim 7, characterized in that The Q-PCR reaction system is 25 μL, the amount of buffer added in the system is not less than 5 μL, the final concentration of dNTP is not less than 0.6×, the amount of Taq enzyme added is not less than 5 μL, the final concentration of BCR / ABL1 P210 probe is above 0.25 μM, the final concentration of ABL1 probe is above 0.30 μM, and the concentration of primers and probes is (1-3):1; Among them, the contents of dATP, dCTP, and dGTP in 12.5×dNTP are all 2.5mM, and the content of dUTP is 7.5mM.

9. The method according to claim 8, characterized in that The Q-PCR reaction system is 25 μL, wherein the amount of buffer added is 5 μL, the amount of 12.5×d NT P added is 1.2 μL, the amount of Taq enzyme added is 0.5 μL, the final concentration of BCR / ABL1 P210 primer is 0.5 μM, the final concentration of BCR / ABL1 P210 probe is 0.25 μM, the amount of ROX dye added is 0.5 μL, and the amount of template added is 10 μL; or the amount of buffer added is 5 μL, the amount of 12.5×d NT P added is 1.2 μL, the amount of Taq enzyme added is 0.5 μL, the final concentration of ABL1 primer is 0.5 μM, the final concentration of ABL1 probe is 0.3 μM, the amount of ROX dye added is 0.5 μL, and the amount of template added is 10 μL.

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