Primer set for detecting leukemia BCR-ABL fusion gene and its application

Abstract

The present invention relates to a primer set for detecting the leukemia BCR-ABL fusion gene and its application, belonging to the technical field of medical detection. The primer set includes amplification primer pairs, and each amplification primer pair includes an upstream primer and a downstream primer. The upstream primer is selected from a sequence complementary to the exon region e1 to b3 of the BCR gene of the BCR-ABL fusion gene, and the downstream primer is selected from a sequence complementary to the exon region a2 to a3 of the ABL gene of the BCR-ABL fusion gene. The primer set for detecting the leukemia BCR-ABL fusion gene of the present invention can be used in the CRISPR / Cas detection system, can achieve the detection of the BCR-ABL fusion gene, and has the advantages of simple and rapid detection process, high detection sensitivity, and does not depend on complex instrument equipment and professional operators, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and particularly relates to a primer set for detecting the BCR-ABL fusion gene in leukemia and its application. Background Art

[0002] In recent years, the global incidence of leukemia has shown an increasing trend year by year. The incidence of leukemia in Chinese men is at a high level, especially in the adolescent group, where the incidence and mortality of leukemia are the highest. At present, according to the degree of differentiation of leukemia cells, it is medically divided into acute lymphoblastic leukemia (ALL) and chronic myelogenous leukemia (CML).

[0003] In 1960, Haugerforor and Nowell found that chromosomes 9 and 22 translocated in granulocytes of chronic myelogenous leukemia, causing the ABL gene on the long arm of chromosome 9 and the BCR gene on chromosome 22 to recombine, forming the Philadelphia chromosome (Ph chromosome) and generating the BCR-ABL fusion gene. Among them, ABL is a proto-oncogene that is prone to fuse with various genes in cancer cells, and the most common is to fuse with the BCR gene. The BCR-ABL fusion gene encodes a class of proteins with strong tyrosine kinase activity, which regulates the degree of tyrosine phosphorylation of cell-related proteins, and then controls the proliferation and self-renewal of hematopoietic stem cells, enhances cell division, inhibits the cell's response to apoptosis signals, prolongs the survival time of cells, and is closely related to the onset of leukemia.

[0004] Currently, the diagnostic techniques for detecting the BCR-ABL fusion gene in clinical practice mainly include chromosome analysis, FISH, real-time PCR, nested real-time PCR, etc. However, these techniques all rely on complex instrument equipment and complex operation processes, and it is difficult to achieve rapid and convenient identification of the BCR-ABL fusion gene. Summary of the Invention

[0005] Based on this, in view of the above problems, it is necessary to provide a primer set for detecting the BCR-ABL fusion gene in leukemia. Using this primer set, rapid and convenient detection of common BCR-ABL fusion genes in chronic myelogenous leukemia can be achieved.

[0006] A primer set for detecting the leukemia BCR-ABL fusion gene, including an amplification primer pair, where the amplification primer pair includes an upstream primer and a downstream primer. The upstream primer is selected from sequences complementary to the exon regions e1 to b3 of the BCR gene of the BCR-ABL fusion gene, and the downstream primer is selected from sequences complementary to the exon regions a2 to a3 of the ABL gene of the BCR-ABL fusion gene.

[0007] In previous studies, the present inventors found that during the formation of the BCR-ABL fusion gene, the break site of the ABL gene is relatively fixed, usually fusing with the BCR gene at the 5' end of the second exon of the ABL gene; while the break site of the BCR gene is relatively diverse. According to different break sites, the fusion gene can be divided into 3 different transcript types: ela2 type (p190 type), b3a2 or b2a2 type (M or p210 type), and el9a2 type (u or p230 type). 90%-95% of CML patients are detected with the appearance of the Ph chromosome at the initial diagnosis. All CML patients have the BCR-ABL fusion gene, and 98% of the patients have the BCR-ABL fusion gene type of p210 type.

[0008] Therefore, designing the amplification primer pair as described above can detect the vast majority of leukemia patients with the BCR-ABL fusion gene type, meeting the requirements of clinical detection.

[0009] In one embodiment, the amplification primer pair includes a PCR primer pair and / or an RPA primer pair. The upstream primer of the PCR primer pair is selected from the sequences shown in SEQ ID NO.1 - SEQ ID NO.2, and the downstream primer of the PCR primer pair is selected from the sequence shown in SEQ ID NO.3; the upstream primer of the RPA primer pair is selected from the sequences shown in SEQ ID NO.4 - SEQ ID NO.7, and the downstream primer of the RPA primer pair is selected from the sequences shown in SEQ ID NO.8 - SEQ ID NO.10. Selecting the above primers to form a primer pair can perform reverse transcription PCR amplification well and has a good amplification effect when used for isothermal nucleic acid amplification (RT-RPA or RT-RAA).

[0010] In one embodiment, the upstream primer of the PCR primer pair is selected from the sequence shown in SEQ ID NO.2, and the downstream primer of the PCR primer pair is selected from the sequence shown in SEQ ID NO.3; the upstream primer of the RPA primer pair is selected from the sequence shown in SEQ ID NO.7, and the downstream primer of the RPA primer pair is selected from the sequence shown in SEQ ID NO.10. It has the best amplification effect.

[0011] In one embodiment, it further includes crRNA. The crRNA includes a backbone sequence and a targeting sequence. The backbone sequence binds to the Cas protein, and the targeting sequence is complementary to the BCR-ABL fusion gene fragment. It can be understood that the backbone sequence can be designed conventionally according to the type and requirements of the used Cas protein, and the targeting sequence can also be designed according to the general design in the art, with any fragment site within the BCR-ABL fusion gene region as the target site for design.

[0012] In one embodiment, the crRNA is selected from the sequences shown in SEQ ID NO:11 - SEQ ID NO.14. Designing the crRNA as the above sequences has good targeting specificity and can achieve better detection effects.

[0013] In one embodiment, the crRNA is selected from the sequence shown in SEQ ID NO.14, which has the best detection effect.

[0014] The present invention also discloses the application of the above primer set in the preparation of a reagent for detecting the leukemia BCR-ABL fusion gene.

[0015] It can be understood that after amplifying the target fragment with the amplification primer pair in the above primer set, it can be used in conventional gene detection or in the CRISPR / Cas detection system, and has the advantages of rapidity and high sensitivity when used in the CRISPR / Cas detection system.

[0016] The present invention also discloses a kit for detecting the leukemia BCR-ABL fusion gene, including the above primer set.

[0017] In one embodiment, the kit further includes crRNA, Cas protein, and a signal reporting probe. The crRNA includes a backbone sequence and a targeting sequence. The backbone sequence binds to the Cas protein, and the targeting sequence is complementary to the BCR-ABL fusion gene fragment.

[0018] In one embodiment, the Cas protein is Cas12a, and the crRNA is selected from the sequences shown in SEQ ID NO:11 - SEQ ID NO.14. Detecting with the above crRNA in combination with the Cas12a protein has good detection effects. In particular, LbacrRNA-a2 shown in SEQ ID NO:10 has the best detection effect.

[0019] In one embodiment, the kit further includes a lateral flow test strip, which is composed of a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane, and a blotting paper that are sequentially laminated and attached to the bottom plate; the conjugate pad is embedded with gold nanoparticles conjugated with anti-FAM antibody, the nitrocellulose membrane is provided with a control line and a test line, and the control line is embedded with streptavidin, and the test line is embedded with a secondary antibody. It can be understood that for the specific assembly and manufacture of this lateral flow test strip, reference can be made to the test strips used for the detection of other targets in the conventional technology. The key of this technical solution lies in the reagents used for the CRISPR reaction.

[0020] The present invention also discloses a method for detecting the leukemia BCR-ABL fusion gene for non-diagnostic and therapeutic purposes, including the following steps:

[0021] Sample extraction: Take the sample to be tested and extract the RNA therein;

[0022] RPA amplification: Using the above amplification primer pair, amplify the BCR-ABL fusion gene fragment of the above-mentioned extracted sample to be tested to obtain an amplification product;

[0023] CRISPR reaction detection: Take the above amplification product, add a signal reporting probe, a Cas protein, and the above-mentioned crRNA, perform a CRISPR reaction, drop the reaction solution onto the sample pad area of the above-mentioned lateral flow test strip, and obtain a test result through the color development of the test line of the test strip.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] A primer set for detecting the leukemia BCR-ABL fusion gene of the present invention can be used in a CRISPR / Cas detection system, can realize the detection of the BCR-ABL fusion gene, and has the advantages of simple and rapid detection process, high detection sensitivity, and does not rely on complex instrument equipment and professional operators. Description of the Drawings

[0026] Figure 1 Schematic diagram of primer design in the embodiment;

[0027] Figure 2 Electrophoresis result diagram of RT-PCR products in Example 1;

[0028] Figure 3 Electrophoresis result diagram of RPA products in Example 1;

[0029] Figure 4 Schematic diagram of the design principle of crRNA in Example 2;

[0030] Figure 5 Electrophoresis identification diagram of in vitro transcribed crRNA in Example 2;

[0031] Figure 6 The activity evaluation results of typical crRNAs targeting and recognizing the BCR-ABL fusion gene in Example 3;

[0032] Figure 7 Schematic diagram of the detection principle of the lateral flow test strip in Example 4;

[0033] Wherein: A is the detection principle of negative results; B is the detection principle of positive results;

[0034] Figure 8 The evaluation results of detection sensitivity in Example 4;

[0035] Figure 9 The detection results of the lateral flow test strip in Example 5;

[0036] Wherein: A is the color development result of the lateral flow test strip; B is the gray scale statistical result of the color development band; C is the ROC curve. Detailed implementation manners

[0037] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0039] Unless otherwise specified, the reagents used in the following examples are all commercially available; unless otherwise specified, the methods used in the following examples can be realized by conventional methods.

[0040] Example 1

[0041] Design and verification of specific primers for amplifying the BCR-ABL fusion gene.

[0042] Considering that the BCR-ABL fusion genotypes include ela2 type, b3a2 type, b2a2 type, etc., the design and verification are carried out according to the following method steps.

[0043] 1. Design of amplification primers

[0044] Retrieve the gene sequences of exons e1 and b2 of the BCR gene and exon a2 of the ABL gene from the NCBI database, and design specific amplification primers with the BCR gene and ABL gene sequence regions as the upstream and downstream targets respectively (as shown in Figure 1 ), and some of the primers are shown in the following table.

[0045] Table 1. Amplification primers for BCR-ABL fusion gene

[0046]

[0047] 2. Verification of amplification primers

[0048] In this method, the amplification of the target gene can be achieved by two amplification methods: RT-PCR and RT-RPA.

[0049] (1) RT-PCR method

[0050] When using the RT-PCR amplification method, taking the RT-PCR primers (b2-F1 and a2-R1) screened through experiments as an example, one-step RT-PCR is used to amplify the BCR-ABL fusion gene in clinical samples (collected from samples in this hospital), and the reaction reagent is PrimeScript TM One Step RT-PCR Kit.

[0051] Taking a 25 μL amplification reaction as an example, add 12.5 μL of 2×1Step Buffer and 1 μL of PrimeScript 1Step Enzyme Mix to each reaction, the final concentrations of the upstream and downstream primers are both 400 nM, 9 μL of RNase-free water, and 1 μL of RNA sample. After mixing well, place it in a PCR instrument for reaction. The reaction program is 50 °C, 30 min; 94 °C, 2 min; (94 °C, 30 s; 58 °C, 30 s; 72 °C, 1 min; 35 cycles); 72 °C, 3 min. After the reaction, take 6 μL of the amplification product for electrophoresis detection.

[0052] The electrophoresis results are as shown in Figure 2 , Figure 2 which is the electrophoresis result of the amplification product of the BCR-ABL fusion gene in the RNA clinical sample amplified with the primer pair b2-F1 and a2-R1. Among them, samples 1-8 are the amplification results of BCR-ABL fusion gene positive samples, and samples 9-20 are the amplification results of BCR-ABL fusion gene negative samples. From the above results, it can be seen that this primer pair can specifically amplify the BCR-ABL fusion gene fragment without interference from negatives.

[0053] The above samples 1-8 are different case samples collected clinically respectively.

[0054] (2) Isothermal amplification method (RT-RPA)

[0055] To screen out RPA primers with better amplification ability, the amplification sensitivity of the primers to the target was first evaluated. Since there is no commercially available BCR-ABL mRNA reference material on the market, the synthesized b2a2 BCR-ABL fusion gene DNA ordered was used as the template (the template sequence is shown in SEQ ID NO.19), and the indicators of different RPA primer pairs were evaluated and compared. The RPA reaction reagent was TwistAmp Basic Kit.

[0056] Taking the 50 μL RPA amplification reaction as an example, 29.5 μL of Primer free Rehydration buffer, 2.4 μL of upstream primer (10 μM), 2.4 μL of downstream primer (10 μM), 9.2 μL of H2O, and 2 μL of target gene were added to each tube of the RPA TwistAmp Basic Kit lyophilized powder. After thorough mixing, 2.5 μL of MgOAc (280 mM) was added, and the mixture was incubated at 39 °C for 20 min. After the reaction, 4 μL of the amplification product was taken for electrophoresis detection.

[0057] Partial electrophoresis results are as Figure 3 shown. The amplification sensitivity of the primer b2-F4 / a2-R4 to the target reached 100 aM, and the primer dimer generated in the negative control was the least among the three pairs of primers. Therefore, b2-F4 / a2-R4 was selected as the optimal amplification primer.

[0058] Example 2

[0059] Design, preparation and verification of crRNA targeting the BCR-ABL fusion gene.

[0060] 1. Design

[0061] There are two ways to prepare crRNA, including chemical synthesis or in vitro transcription. Since the cost of RNA chemical synthesis is too high, the in vitro transcription method is usually used for crRNA preparation.

[0062] In vitro transcription of crRNA can use synthetic double-stranded DNA or partially double-stranded DNA as the template. The DNA template consists of a T7 RNA polymerase promoter region and a crRNA sequence coding region. The design principle of crRNA is as Figure 4 shown, where A is the design principle of the backbone sequence and targeting sequence of crRNA, and B is the design principle of the transcription template of crRNA.

[0063] According to the sequences of the BCR and ABL genes retrieved, different regions of the BCR-ABL fusion gene were selected as the targets for crRNA to design crRNA. Taking the p190 and p210 types of BCR-ABL fusion genotypes as examples, there are three design strategies for crRNA:

[0064] 1) Target the BCR region of the BCR-ABL fusion gene;

[0065] 2) Target the fusion region of the BCR-ABL fusion gene, including the fusion regions of exon e1 and a2, exon b2 and a2, and exon b3 and a2;

[0066] 3) Target the ABL region of the BCR-ABL fusion gene.

[0067] In terms of the design principle, targeting the fusion region of the BCR-ABL fusion gene as the target for crRNA can achieve the specific detection of different fusion genotypes by crRNA. However, due to the requirement of the Cas12a system for the PAM site at the targeting site, which is generally the TTTN (or TTN) sequence, it is limited by the composition of the base sequence in the fusion site region. In this example, considering both the detection target region and the PAM site requirement, crRNA targeting the a2 exon region was designed, and some of them are shown in Table 2.

[0068] Table 2. crRNA

[0069]

[0070]

[0071] Note: The above sequences are RNA sequences, where T is the letter specification in the WIPO Sequence Listing and represents uracil U.

[0072] Since the primers for amplifying the fusion gene are primer pairs specific to the BCR-ABL fusion gene and cannot amplify the BCR and ABL genes without gene fusion, the specificity of the amplification product is ensured. Combined with the crRNA targeting the a2 gene, the specificity of detecting the BCR-ABL fusion gene can be guaranteed. Moreover, by combining the use of amplification primers and crRNA targeting the a2 gene, it is possible to judge different fusion genes with the same crRNA, which can simplify the detection system.

[0073] 2. Preparation

[0074] The DNA template sequences for transcribing crRNA are shown in Table 3 below.

[0075] Table 3. DNA template sequences for transcribing crRNA

[0076]

[0077] Note: The underlined part is the complementary region of the T7 promoter sequence.

[0078] Taking a 25 μL in vitro transcription system for RNA as an example, the T7 RNA polymerase was purchased from NEB. Each transcription reaction system consisted of 13 μL of RNase-free water, 2.5 μL of 10×RNA polymerase buffer, 5 μL of 10 mM NTP mixture, 2.5 μL of T7 RNA polymerase, 1 μL of recombinant RNase inhibitor (TaKaRa, Dalian), and 1 μL of template DNA (final concentration up to 200 - 1000 nM). After thorough mixing, it was placed in a PCR instrument and incubated at 37°C for 4 - 16 hours for transcription.

[0079] 3. Verification

[0080] Take 1 μL of the transcription product for PAGE electrophoresis identification. Add 1 μL of DNase I to the RNA transcription reaction solution and mix well. After incubating at 37°C for 30 minutes, purify the transcribed crRNA using an RNA purification kit (purchased from Tiangen Biotech Co., Ltd.), and take the RNA product for PAGE electrophoresis verification.

[0081] The verification results are as Figure 5 shown, Figure 5 is the electrophoretic identification diagram of in vitro transcribed crRNA. The electrophoresis results show that the crRNA was successfully prepared and can be used for subsequent experiments.

[0082] Example 3

[0083] Screening of crRNA.

[0084] Based on the base sequence of the BCR-ABL fusion gene obtained from NCBI, a double-stranded DNA target fragment was ordered and synthesized to simulate the BCR-ABL fusion gene for crRNA activity evaluation and screening.

[0085] Taking a 20 μL reaction system as an example, add 12 μL of RNase-free water, 2 μL of NEB buffer 2.1, 1 μL of 10 uM DNA reporter (FAM-TTTTT-BHQ), 2 μL of crRNA (1 uM), 1 μL of LbaCas12a (1 uM), and 2 μL of target DNA (100 nM) to an EP tube. After thorough mixing, place it in a fluorescence quantitative PCR instrument and incubate at 37°C for 30 minutes, then read the fluorescence value once every 1 minute. Statistically analyze the reaction rates of different crRNAs recognizing the target DNA.

[0086] The results are asFigure 6 As shown Figure 6 This is the activity evaluation result of a typical crRNA targeting the BCR-ABL fusion gene. The activity results of crRNA-a2, crRNA-b2a2, crRNA-b3a2, and crRNA-e1a2 targeting the fusion regions of exons b2 and a2, b3 and a2, and e1 and a2 of the BCR-ABL fusion gene, respectively.

[0087] The above results show that crRNA-a2 exhibits the best detection activity for different fusion gene fragments (b2a2, b3a2, e1a2). Therefore, crRNA-a2 with the optimal reaction rate (LbacrRNA-a2, i.e., SEQ ID NO.14) was selected to conduct subsequent experiments.

[0088] Example 4

[0089] Evaluation of detection sensitivity.

[0090] Based on LbacrRNA-a2 with the optimal reaction rate, a Cas12a gene recognition system was constructed to evaluate the detection sensitivity of the Cas12a-lateral flow dipstick method for the BCR-ABL fusion gene in clinical samples.

[0091] The lateral flow dipstick is assembled by a bottom plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent paper that are sequentially laminated and attached to the bottom plate. Gold nanoparticles conjugated with anti-FAM antibody are embedded in the conjugate pad; streptavidin and a secondary antibody are scribed and embedded on the nitrocellulose membrane as the control line (C) and the test line (T), respectively. The detection principle of the lateral flow dipstick is as Figure 7 shown: In the detection of negative samples ( Figure 7 A), Cas12a / crRNA does not recognize the target gene sequence (i.e., the fusion gene fragment), and Cas12a has no nuclease activity. Therefore, the single-stranded DNA reporter probe is not cleaved and remains intact. When the Cas reaction solution is dropped onto the lateral flow dipstick, FAM at the end of the reporter probe binds to the gold nanoparticles conjugated with anti-FAM antibody (FAM-GNP). When further flowing through the C line, streptavidin binds to the biotin labeled at the other end of the reporter probe, and the colloidal gold is intercepted at the C line. Therefore, a red band can be observed at the C line and no band at the T line. In the detection of positive samples ( Figure 7B), The target gene is amplified into double-stranded DNA, so Cas12a / crRNA recognizes the target gene sequence, Cas12a is activated and exhibits nuclease activity, and the single-stranded DNA reporter probe is cleaved. Therefore, when the Cas reaction solution is dropped onto the lateral flow test strip, FAM-GNP will not be retained by streptavidin on the C line and will flow through the C line. When it flows through the T line, the secondary antibody against the FAM antibody embedded binds to the FAM antibody labeled on the surface of GNP, causing FAM-GNP to accumulate on the T line. Therefore, a red band can be observed on the T line.

[0092] Using the synthetic BCR-ABL DNA mimicking target gene ordered in Example 1, perform RPA amplification of different concentrations of the mimicking target gene with primers b2-F4 / a2-R4. Take 2 μL of the RPA amplification product, add 12 μL of RNase-free water, 2 μL of NEBbuffer2.1, 1 μL of 10 uM DNA reporter (Biotin-TTATTATT-FAM), 2 μL of crRNA (crRNA-a2, 1 uM), and 1 μL of LbaCas12a (1 uM). After thorough mixing, incubate at 37 °C in a fluorescence quantitative PCR instrument for 20 minutes. Then, add 60 μL of HybriDetect Assay buffer to the reaction solution, drop it onto the lateral flow test strip, and observe the results.

[0093] The results are as Figure 8 shown. The detection sensitivity of this method for the mimicking target gene reaches 100 aM, which is approximately equivalent to 60 copies / μL.

[0094] Example 5

[0095] Construct a Cas12a gene recognition system based on LbacrRNA-a2 with the optimal reaction rate, and evaluate the detection sensitivity of the Cas12a-lateral flow test strip method for the BCR-ABL fusion gene in clinical samples.

[0096] The steps included in this detection are: extraction of cellular RNA from blood, amplification of the fusion gene, CRISPR recognition reaction, analysis of the detection results by lateral flow test strip, etc.

[0097] This lateral flow test strip detection method includes the following steps:

[0098] 1. Sample extraction:

[0099] Take the sample to be tested and extract the RNA therein using a commercially available RNA extraction kit.

[0100] 2. Nucleic acid amplification (RPA):

[0101] The BCR-ABL fusion gene fragment of the sample to be tested extracted above was amplified using primers b2-F1 and a2-R1 according to the conditions in Example 1 to obtain an amplified product.

[0102] 3. CRISPR reaction:

[0103] Take the above amplification product, add 12 μL RNase-free water, 2 μL NEB buffer 2.1, 1 μL 10uM DNA reporter (Biotin-TTATTATT-FAM), 2 μL crRNA (crRNA-a2, 1uM), 1 μL LbaCas12a (1uM), and 2 μL RT-PCR amplification results of the target BCR-ABL fusion gene. After fully mixing, place it in a fluorescent quantitative PCR instrument and incubate it at 37°C for 20 minutes, and then add 60 μL HybriDetect Assay buffer to the reaction solution.

[0104] 4. Lateral flow test paper:

[0105] The above reaction solution was dripped into the lateral flow test strip and the result was observed.

[0106] The results are as follows Figure 9 As shown, Figure 9 It is the application result of Cas12a / crRNA- lateral flow test paper system in clinical sample detection in the present embodiment. Wherein, A is the visual detection result of clinical sample BCR-ABL fusion gene based on Cas12a / crRNA system and lateral flow test paper; B is the result of quantitative statistics of gray value of control line (C) and test line (T) of lateral flow test paper in A figure result; C is ROC analysis of visual detection result of clinical sample BCR-ABL fusion gene.

[0107] The above results show that for samples No. 1-8 described in Example 1, the amplification primer pair of this example can successfully amplify the BCR-ABL fusion gene fragment, and the selected crRNA-a2 can be quickly and accurately detected to obtain accurate results, and the presence of the BCR-ABL fusion gene can be determined by directly observing the color of the detection line of the lateral flow test paper with the naked eye ( Figure 9 A), grayscale statistical analysis of the color bands of the lateral flow test paper further confirmed the test results ( Figure 9 B), by further comparing the grayscale statistical results with the clinical test results and performing ROC curve analysis ( Figure 9C), the detection accuracy of this method was 93.75%. According to the cut-off value obtained from the ROC curve analysis, the detection sensitivity of this method was calculated to be 88.9% (true positive / (true positive + false negative)), and the specificity was 100% (true negative / (true negative + false positive)).

[0108] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0109] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A primer set for detecting leukemia BCR-ABL fusion gene, characterized in that, Comprising amplification primer pairs and crRNA, the amplification primer pairs include PCR primer pairs and / or RPA primer pairs. The upstream primer of the PCR primer pair is selected from the sequence shown in SEQ ID NO.2, and the downstream primer of the PCR primer pair is selected from the sequence shown in SEQ ID NO.3; the upstream primer of the RPA primer pair is selected from the sequence shown in SEQ ID NO.7, and the downstream primer of the RPA primer pair is selected from the sequence shown in SEQ ID NO.10; The crRNA is selected from the sequence shown in SEQ ID NO.

14.

2. Use of the primer set according to claim 1 in the preparation of a reagent for detecting the leukemia BCR-ABL fusion gene.

3. A kit for detecting the leukemia BCR-ABL fusion gene, characterized in that, Comprising the primer set according to claim 1.

4. The kit for detecting the leukemia BCR-ABL fusion gene according to claim 3, wherein It further includes a Cas protein and a signal reporter probe, with one end of the signal reporter probe labeled with FAM and the other end labeled with biotin.

5. The kit for detecting the leukemia BCR-ABL fusion gene according to claim 4, wherein The Cas protein is Cas12a.

6. The kit for detecting the leukemia BCR-ABL fusion gene according to claim 4, wherein It further includes a lateral flow test strip, which is composed of a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane and a blotting paper that are sequentially laminated and attached to the bottom plate; the conjugate pad is embedded with gold nanoparticles conjugated with an anti-FAM antibody, and the gold nanoparticles of the anti-FAM antibody are used to bind to the FAM; the nitrocellulose membrane is provided with a control line and a detection line, and the control line is embedded with streptavidin, and the streptavidin is used to bind to the biotin; the detection line is embedded with a secondary antibody.

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