Novel BCR / ABL fusion gene fluorescence in-situ hybridization probe and preparation method thereof

Abstract

The invention relates to the field of medical detection, provides a preparation method of a novel BCR / ABL fusion gene fluorescence in-situ hybridization probe, solves the problems that an existing common fluorescence in-situ hybridization probe is complex in preparation process and is limited by BAC cloning, and overcomes the problems that an existing probe is non-uniform in cell permeation, high in background and weak in signal intensity and is combined with non-specific fragments. The preparation method of the fluorescent probe provided by the invention is suitable for practical application, the fluorescent probe which is uniform in size, high in cell transmittance, uniform in penetration, high in targeting specificity, low in background, clear and obvious in signal, simple and efficient in production mode and capable of being synthesized at one time to meet permanent application requirements is prepared, and through process exploration, the fluorescent probe has a good application prospect. The dye, the dye concentration, the primer and the high-fidelity polymerase which are most suitable for the fluorescence labeling process of the BCR / ABL fusion gene fluorescence in-situ hybridization probe are screened out.

Description

[0001] This application claims priority to the prior Chinese application, application number: 202311045510.6, filing date August 18, 2021; all contents thereof are regarded as part of the present invention. Technical Field

[0002] The present invention relates to the field of medical detection, and in particular to a novel BCR / ABL fusion gene fluorescent in situ hybridization probe and a preparation method thereof. Background Art

[0003] According to the Chinese Diagnosis and Treatment Guidelines for Myeloid Leukemia (2020 Edition): Chronic myeloid leukemia (CML) is a malignant tumor formed by clonal proliferation of bone marrow hematopoietic stem cells, accounting for 15% of adult leukemia, with an annual global incidence of 1.6 / 100,000 to 2 / 100,000. A national leukemia incidence survey conducted in 46 survey points in 22 provinces (municipalities, autonomous regions) across the country from 1986 to 1988 showed that the annual incidence of CML was 0.36 / 100,000. Subsequently, epidemiological surveys in several regions of the country showed that the annual incidence of CML was 0.39 / 100,000 to 0.55 / 100,000. Chinese CML patients are younger than those in the West. Epidemiological surveys in several regions of the country showed that the median age of onset of CML was 45 to 50 years old, while the median age of onset of CML in Western countries was 67 years old.

[0004] As a first-line treatment drug, imatinib, a first-generation tyrosine kinase inhibitor, has increased the 10-year survival rate of CML patients to 85% to 90%. Nilotinib, dasatinib and other second-generation TKIs can achieve faster and deeper molecular responses in the first-line treatment of CML, and have gradually become one of the first-line treatment options for CML patients. Currently, more and more clinical research data show that some patients who have achieved a sustained deep molecular response (DMR) for more than 2 years after TKI treatment can achieve long-term treatment free remission (TFR), that is, functional cure. Obtaining a complete cytogenetic response (CCyR) and a deeper molecular response as soon as possible is the short-term goal of CML treatment, and improving the quality of life and functional cure are the long-term goals of CML treatment. Functional cure has become the treatment goal pursued by more and more CML patients. It should be noted that drug withdrawal has strict requirements on DMR levels, post-discontinuation monitoring and follow-up. The long-term safety of drug withdrawal is still unclear, and it is recommended to be conducted in clinical studies. Centers with conditions should carefully select patients in clinical practice, stop taking medication with full knowledge, and conduct regular follow-up and monitoring. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was once the first-line treatment for CML, but its application is limited by factors such as the availability of donors, patient age, and transplant-related risks. Currently, TKIs represented by imatinib have replaced hematopoietic stem cell transplantation as the first-line treatment for CML patients. In the treatment of CML, the patient's overall condition should be evaluated in detail, and the dominant treatment option should be recommended to the patient, and the next step of treatment should be carried out based on the patient's treatment willingness.

[0005] The fluorescence in situ hybridization (FISH) probe of BCR / ABL1 can be used to diagnose chronic myeloid leukemia (CML) and observe the course of the disease. It is one of the most commonly used FISH diagnostic reagents in hematological pathology. The BCR gene at 22q11 and the ABL1 gene at 9q34 undergo recombination to form a fusion gene, which can be detected in 95% of CML patients. The BCR / ABL1 fusion gene expresses an abnormally activated tyrosine kinase, which causes uncontrolled cell growth through anti-apoptotic effects. Animal experiments have also shown that the fusion gene can cause lesions similar to CML, suggesting that it plays a very important role in the onset of leukemias such as CML. Leukemia patients with positive BCR / ABL1 translocation can be effectively targeted with the tyrosine kinase inhibitor imatinib mesylate. Therefore, the detection of BCR-ABL1 translocation is of great significance for clinical diagnosis and treatment. Quantification of the number of BCR-ABL1-positive cells in bone marrow or peripheral blood specimens using FISH can also help monitor the clinical course and detect minimal residual disease.

[0006] Significance of detection: 1) Auxiliary diagnosis: BCR / ABL fusion gene formed by t(9;22)(q34;q11) translocation is a marker of CML. Typical t(9;22)(q34;q11) translocation can be detected in 95% of CML patients. Ph chromosome is difficult to detect in about 5% of CML patients through karyotype analysis, but the fusion gene can be detected and they are still classified as Ph+CML. 2) Guidance of medication: BCR / ABL detection can be performed on newly diagnosed patients, and the beneficiary population of tyrosine kinase inhibitors (including Imatinib / Dasatinib / Nilotibib, etc.) can be screened. In May 2001, the US Food and Drug Administration approved imatinib for marketing under the trade name Glivec (imatinib mesylate tablets). Glivec can be used to treat chronic phase, accelerated phase or blast phase of Philadelphia chromosome-positive chronic myeloid leukemia (Ph+CML). 3) Treatment monitoring: Ph chromosomes exist throughout the course of CML and continue to exist after treatment remission. Only by eliminating Ph-positive clones can a final cure be achieved. The "Expert Consensus on Treatment of Chronic Myeloid Leukemia (2010 Edition)" emphasizes that CML treatment must achieve cytogenetic and even molecular responses. The FISH method is an important method for monitoring cytogenetic responses. During treatment, it can reflect the dynamic changes in the amount of BCR / ABL fusion gene cells in the patient's body in real time, which can be used for subsequent treatment plan selection and drug use effect evaluation.

[0007] At the same time, according to the Chinese Guidelines for the Diagnosis and Treatment of Adult Acute Lymphoblastic Leukemia (Full Version):

[0008] Acute leukemia accounts for 20% to 30%. At present, there are relatively unified diagnostic criteria and systematic treatment plans reported by different research groups internationally. The complete remission (CR) rate can reach 70% to 90%, and the 3-5-year disease-free survival (DFS) rate can reach 30% to 60%. The National Comprehensive Cancer Network (NCCN) first published the diagnosis and treatment guidelines for acute lymphoblastic leukemia (ALL) in 2012. my country published the first edition of the expert consensus on the diagnosis and treatment of adult ALL in my country in 2012, which was recognized by domestic peers. Recently, the 2016 edition of the WHO Classification of Hematopoietic and Lymphoid Tumors was published, which updated the classification of ALL and proposed some new concepts. The NCCN clinical guidelines for adult ALL have also been revised several times. Based on this, the expert consensus on the diagnosis and treatment of adult ALL in my country has been updated. BCR-ABL1-like ALL is also one of the main types of acute leukemia.

[0009] Fluorescence in situ hybridization (FISH) is a method that uses fluorescently labeled target-specific nucleotide fragments - probes to display sequence locations on cell nucleus chromosomes based on the principles of denaturation, renaturation, and double-strand complementation. This technology is fast, safe, highly sensitive, and the probes can be stored for a long time. It is one of the technical methods for clinical molecular pathology diagnosis and has been widely used in cytogenetics, tumor biology, prenatal diagnosis and other fields.

[0010] The traditional probe preparation method uses bacterial plasmid or phage recombinant artificial genome (BAC or PAC) cloning, and extracts BAC or PAC clone DNA containing human genomic DNA fragments containing target sequences through bacterial culture amplification and separation methods. Then, using this as a template, the polymerase gap translation or chain reaction PCR (random primer method) two methods are used for fluorescent labeling to prepare probes. Using BAC clones as templates and using gap translation technology for probe production is the mainstream of probe products at home and abroad, and is the first generation of probes. The main disadvantage of this type of probe is that it contains repetitive sequences and non-specific sequences. Repetitive and non-specific sequences are the source of hybridization background and non-specific signals. Cot 1DNA rich in human genome repetitive sequences is required to suppress background and non-specific signals, which increases the cost of probe production. At the same time, the inhibition of Cot 1DNA can greatly reduce the hybridization background, but the background is still high and the non-specific signal cannot be completely eliminated. In addition, the size of this type of probe varies, and the probe fragments range from 50bp to more than 1000bp, and the cell or tissue penetration is uneven, resulting in imbalances such as hybridization efficiency. Probes without repetitive sequences also use BAC clones as templates. After amplification by PCR technology, they are mixed with Cot 1 DNA, denatured and renatured, and treated with double-stranded specific DNA hydrolases to remove repetitive sequences, and then fluorescently labeled. Probes without repetitive sequences are an improvement on traditional probe preparation technology, which reduces hybridization background, eliminates nonspecific signals, increases cell penetration of probes, and improves hybridization efficiency. They are second-generation probes. The common point of these two generations of probes is that they both need to use BAC clones as templates, so the design and preparation of probes are limited by BAC clones. Because the number and size of BAC clones are accessible, fixed and limited, the size is generally around 100-200kb, and the extraction of BAC clone DNA is time-consuming and labor-intensive and requires large equipment, resulting in high preparation costs. The completion of human whole genome sequencing and the development of nucleic acid synthesis technology have made it possible to artificially synthesize fluorescent in situ hybridization probes. The new generation of probes are designed using modern bioinformatics and produce oligonucleotide probes through chemical synthesis, completely getting rid of the use of BAC clones.

[0011] Therefore, in the field of CML diagnosis, treatment, and drug efficacy judgment of BCR / ABL gene testing, which requires extremely high precision, a new BCR / ABL fluorescent in situ hybridization probe with good cell penetration, clear signal, low background, and efficient and convenient production process is needed. Summary of the invention

[0012] To solve the above problems, the present invention provides a novel method for preparing a BCR / ABL fusion gene fluorescent in situ hybridization probe, which is suitable for practical application to prepare a fluorescent probe with uniform size, high cell permeability and uniform penetration, high targeting specificity, low background, clear and obvious signal, simple and efficient production method, and can be synthesized once to meet permanent application requirements. The method comprises the following steps:

[0013] (1) The nucleic acid sequences of the BCR gene and ABL gene target region specific sequences are annotated and the repeated sequences are removed;

[0014] (2) converting the nucleic acid sequence obtained in step (1) into an oligonucleotide sequence according to the length of the oligonucleotide to be synthesized, wherein the oligonucleotide sequences obtained for the BCR and ABL genes have the same length for each oligonucleotide;

[0015] (3) designing and synthesizing primers for amplifying oligonucleotide fragments, adding fluorescent dyes to the 5' end during primer synthesis to obtain fluorescently labeled forward and reverse amplification primers, synthesizing the oligonucleotide sequence obtained in step (2) into a specific oligonucleotide pool with consistent length using oligonucleotide chemical synthesis technology, and introducing forward primers and reverse primers at both ends of the sequence during synthesis, so that the oligonucleotides in the constructed BCR and ABL oligonucleotide pools have consistent lengths;

[0016] (4) using the fluorescently labeled primers of the BCR and ABL genes obtained in step (3) and the oligonucleotide pool obtained in step (2) as templates to perform PCR to obtain BCR and ABL gene oligonucleotide fluorescent probe sets;

[0017] (5) After purifying the probe set, dissolve it in hybridization solution and store it.

[0018] In some optional methods, the oligonucleotide pool of BCR gene and ABL gene of uniform size can be obtained by the following steps: the relevant sequences of BCR gene and ABL gene are found through (http: / / genome.ucsc.edu), wherein the BCR gene probe region starts from SHGC-10404 (STS site) at the 5' end to the 3' end of the BCR gene, covering an area of ​​about 600 kb, with a 320 kb region left in the middle, and the BCR gene 3' end probe starts from RH8078 (STS site) to SHGC-17034 (STS site), covering an area of ​​about 640 kb; the ABL gene probe region starts from SHGC-147754 (STS site) at the 5' end to SHGC-8190 (STS site) at the 3' end of the ABL gene, covering an area of ​​about 700 kb. Use the searcher's repetitive sequence identification function to mark the repetitive sequences, and then copy the target region-specific sequence that does not contain repetitive sequences to file processing software such as Microsoft "Word" or "Excel". According to the length of the oligonucleotide to be synthesized, 60-100bp can be selected, and the online FASTA mode converter http: / / avermitilis.ls.kitasato-u.ac.jp / readseq.cgi is used for mode conversion. Then, specific amplification forward and reverse primers are added to both ends of each oligonucleotide for chemical synthesis.

[0019] In some embodiments, the primers used to amplify the probe template library in step (3) are the forward primer shown in SEQ ID: 1 and the reverse primer shown in SEQ ID: 2, the forward primer shown in SEQ ID: 3 and the reverse primer shown in SEQ ID: 4, the forward primer shown in SEQ ID: 5 and the reverse primer shown in SEQ ID: 6, the forward primer shown in SEQ ID: 7 and the reverse primer shown in SEQ ID: 8, the forward primer shown in SEQ ID: 9 and the reverse primer shown in SEQ ID: 10, the forward primer shown in SEQ ID: 11 and the reverse primer shown in SEQ ID: 12, the forward primer shown in SEQ ID: 13 and the reverse primer shown in SEQ ID: 14, the forward primer shown in SEQ ID: 15 and the reverse primer shown in SEQ ID: 16, the forward primer shown in SEQ ID: 17 and the reverse primer shown in SEQ ID: 18, the forward primer shown in SEQ ID: 19 and the reverse primer shown in SEQ ID: 20, the forward primer shown in SEQ ID: 21 and the reverse primer shown in SEQ ID: 22, The forward primer shown in SEQ ID: 23 and the reverse primer shown in SEQ ID: 24, the forward primer shown in SEQ ID: 25 and the reverse primer shown in SEQ ID: 26, the forward primer shown in SEQ ID: 27 and the reverse primer shown in SEQ ID: 28, the forward primer shown in SEQ ID: 29 and the reverse primer shown in SEQ ID: 30, at least one of the above primer pairs, experiments have shown that the above primers can be used to amplify the oligonucleotide fragments of ABL and BCR genes. In some preferred embodiments, the primers used to amplify the probe template library in step (3) are the forward primer shown in SEQ ID: 21 and the reverse primer shown in SEQ ID: 22. Experiments have shown that the fluorescent probe target gene obtained after amplification using the above primer sequences has better fluorescence effect under a fluorescence microscope. The oligonucleotide pool described in step (3) can be diluted and packaged for repeated use.

[0020] Furthermore, the PCR amplification step in step (4) can be amplified in two steps. The first step is to obtain a probe template library of multiple copies of BCR and ABL genes, which is obtained by amplifying using forward and reverse primers without fluorescent markers, and the target concentration can be achieved by adjusting the number of amplifications; the second step is to obtain a single-use fluorescent probe, which is amplified using forward and reverse primers containing fluorescent markers to obtain in situ hybridization fluorescent probes of ABL and BCR genes using the probe template library as a template; the probe template library can be stored and reused. The advantage of performing two-step amplification over one-step amplification is that a probe template library of multiple copies for storage can be obtained, and a purification step is added in the two amplifications, and the second step amplification PCR system is adjusted to obtain a large number of desired oligonucleotide probes.

[0021] Furthermore, the high-fidelity enzyme used in the PCR amplification system in step (4) is Phantaax Super-Fidelity DNA Polymerase. Experiments have shown that in the application of the present invention, the use of high-fidelity enzyme K can best meet the need for one-time multiple PCR. The fluorescent probe amplified by high-fidelity enzyme K has the most obvious reflection under a fluorescent microscope.

[0022] Furthermore, the oligonucleotide sequence obtained in step (2) can be screened once before being used in step (3) to remove oligonucleotide sequences containing TTTT and AAAA, and to remove fragments with a GC content of more than 50% or less than 10%.

[0023] Furthermore, in step (2), the length of the oligonucleotide fragment sequence screened is 60-100 bp.

[0024] Furthermore, in step (4), the fluorescent dye for marking the BCR gene is Fluorescein (fluorescein, green), the fluorescent dye for marking the ABL gene is ATTO (orange), and the amount of 20uM primer is 7.5μL per 50μL system. Experiments have shown that the fluorescent probe obtained by this staining combination has a clearer boundary and lower background in the fluorescence microscopy observation image compared with the control ROX, TAMRA, Alexa, and FAM dyes, and the size of the fluorescent spots presented is uniform, which is very convenient for distinguishing the fusion situation.

[0025] Furthermore, it is characterized in that the method provided by the present invention also includes a hybridization solution and a staining solution used to dissolve the probe, wherein the hybridization solution contains 30-60% by mass of formamide and 1g-3g / 10ml of dextran sulfate, and the staining solution contains components of DAPI and 1,4-diazobicyclo (2,2,2) octane (DABCO) and anti-quenching agents such as p-phenylenediamine and propyl gallate.

[0026] Further, the primers used to amplify the probe template library are the forward primer shown in SEQ ID: 1 and the reverse primer shown in SEQ ID: 2, the forward primer shown in SEQ ID: 3 and the reverse primer shown in SEQ ID: 4, the forward primer shown in SEQ ID: 5 and the reverse primer shown in SEQ ID: 6, the forward primer shown in SEQ ID: 7 and the reverse primer shown in SEQ ID: 8, the forward primer shown in SEQ ID: 9 and the reverse primer shown in SEQ ID: 10, the forward primer shown in SEQ ID: 11 and the reverse primer shown in SEQ ID: 12, the forward primer shown in SEQ ID: 13 and the reverse primer shown in SEQ ID: 14, the forward primer shown in SEQ ID: 15 and the reverse primer shown in SEQ ID: 16, the forward primer shown in SEQ ID: 17 and the reverse primer shown in SEQ ID: 18, the forward primer shown in SEQ ID: 19 and the reverse primer shown in SEQ ID: 20, the forward primer shown in SEQ ID: 21 and the reverse primer shown in SEQ ID: 22, At least one of the forward primers shown in SEQ ID: 23 and the reverse primer shown in SEQ ID: 24, the forward primer shown in SEQ ID: 25 and the reverse primer shown in SEQ ID: 26, the forward primer shown in SEQ ID: 27 and the reverse primer shown in SEQ ID: 28, and the forward primer shown in SEQ ID: 29 and the reverse primer shown in SEQ ID: 30 is used for synthesis. Amplification using at least one of the above sequences can successfully prepare a large number of fluorescent probes.

[0027] In actual operation, all sequences of the probe template library are in one tube and all sequences need to be amplified. Therefore, the same forward primer and backward primer need to be loaded on all sequences and all sequences need to be amplified by special enzymes.

[0028] Further, the primers have a forward primer as shown in SEQ ID: 21 and a reverse primer as shown in SEQ ID: 22 as preferred primer sequences. The amplification effect of the preferred primer sequence is more stable, and the amplified probe has a large signal intensity in the fluorescence microscope image, which proves that the preferred primer sequence can be applied to the amplification of more specific oligonucleotide sequences without repetitive sequences in BCR and ABL genes, and has better repetitive stability than non-preferred primers.

[0029] On the other hand, the specific amplification primers provided by the present invention have the function of improving the fluorescence effect after the probe target obtained in the preparation of the BCR / ABL fusion gene fluorescence in situ hybridization probe, and the specific amplification primer pair sequence is the forward primer shown in SEQ ID: 21 and the reverse primer shown in SEQ ID: 22. The above primers can be used for amplification of a large number of BCR and ABL gene-specific oligonucleotide sequences without repetitive sequences, do not contain matching fragments with any nucleic acid sequence in the BCR and ABL oligonucleotide pools, and are suitable for stable amplification of BCR and ABL oligonucleotide sequence pools of consistent size.

[0030] In summary, the beneficial effects of the present invention are as follows:

[0031] 1. The production of the BCR / ABL probe of the present invention is not limited by BAC cloning and only needs to be synthesized once, thereby reducing the batch-to-batch difference caused by repeated synthesis. The constructed oligonucleotide probe template library is easy to store and ensures stable probe performance. The preparation process is simple, the constructed probe template library can be stored for a long time, the labeling is simple, and a large number of probes can be prepared in a short time, which is suitable for large-scale production.

[0032] 2. The BCR / ABL probe of the present invention has uniform and controllable size and strong penetration. The probe sizes prepared by the commonly used bacterial artificial chromosome BAC cloning and polymerase chain reaction PCR (random primer method) are all random and uncontrollable, and ultimately vary between 100-1500 bp. The size of all BCR / ABL probes of the present invention is 60-100 bp, which can easily penetrate the cell nuclear membrane, improve hybridization efficiency, and the probe has better signal intensity under a fluorescence microscope.

[0033] 3. BCR / ABL probe of the present invention: high specificity, bright and clear signal points, low background and clear without noise. The BCR / ABL dual-color probe of the present invention has eliminated repetitive sequences and non-specific sequences when designing the probe, and the detection accuracy is higher than that of the original BAC clone probe, and no Cot 1 DNA needs to be added for blocking.

[0034] 4. Based on the designed primers, the present invention selects the most suitable probe scheme for preparing BCR / ABL fusion gene detection through screening and testing of oligonucleotide amplification enzymes, dyes, and dye concentrations. The obtained fluorescent hybridization probe has a clearer signal under a fluorescent microscope, is easy to observe, and has a stable effect in repeated tests, which is suitable for large-scale preparation of probes in long-term practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 :Schematic diagram of the preparation process of BCR / ABL fusion gene fluorescence in situ hybridization probe;

[0037] Figure 2 :Schematic diagram of the sequence of BCR gene in the human genome;

[0038] Figure 3 :Schematic diagram of the sequence of ABL gene in the human genome;

[0039] Figure 4 :Schematic diagram of BCR probe sites;

[0040] Figure 5 :Schematic diagram of ABL probe site;

[0041] Figure 6 : Agarose gel electrophoresis detection of BCR and ABL genes after oligonucleotide pool amplification;

[0042] Figure 7 :BCR / ABL fusion gene fluorescence in situ hybridization results of IM-9 samples;

[0043] Figure 8 :The results of fluorescence in situ hybridization of BCR / ABL fusion gene in K-562 sample;

[0044] Fig. 9 :BCR / ABL fusion gene fluorescence in situ hybridization results of HL-60 samples;

[0045] Fig.10 : Preparation method control experiment results: oligo probe set;

[0046] Fig.11 : Preparation method control experiment results: BAC cloning method probe set;

[0047] Fig.12 : Primer control experiment results: Primer 1;

[0048] Fig.13 : Primer control experiment results: Primer 2;

[0049] Fig.14 : Primer control experiment results: Primer 3;

[0050] Fig.15: Primer control experiment results: Primer 4;

[0051] Fig.16 : Dye screening experiment results: Group 1;

[0052] Fig.17 : Dye screening experiment results: Group 2;

[0053] Fig.18 : Dye screening experiment results: Group 3;

[0054] Fig.19 : Dye screening experiment results: Group 4;

[0055] Fig. 20 : Dye screening experiment results: Group 5;

[0056] Fig.21 : Dye screening experiment results: Group 6;

[0057] Fig. 22 : Dye screening experiment results: Group 7;

[0058] Fig.23 : Dye screening experiment results: Group 8;

[0059] Fig.24 : Experimental results of primer dosage: 2.5 μL primer;

[0060] Fig.25 : Primer dosage experimental results: 3.75 μL primer;

[0061] Fig.26 : Experimental results of primer dosage: 5 μL primer;

[0062] Fig. 27 : Experimental results of primer dosage: 6.25 μL primer;

[0063] Fig.28 : Experimental results of primer dosage: 7.5 μL primer;

[0064] Fig.29 : Enzyme screening experiment results: Enzyme K repeat 1;

[0065] Fig.30 : Enzyme screening experiment results: Enzyme K was repeated 2 times;

[0066] Fig.31 : Enzyme screening experiment results: Enzyme K was repeated 3 times;

[0067] Fig.32 : Enzyme screening experiment results: Enzyme T repeat 1;

[0068] Fig.33 : Enzyme screening experiment results: Enzyme T repeat 2;

[0069] Fig.34 : Enzyme screening experiment results: Enzyme T was repeated 3 times;

[0070] Fig.35 : Enzyme screening experiment results: Enzyme N repeat 1;

[0071] Fig.36 : Enzyme screening experiment results: Enzyme N was repeated 2 times;

[0072] Fig.37 : Enzyme screening experiment results: Enzyme N was repeated 3 times.

[0073] Specific implementation cases

[0074] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0075] Example 1: Complete preparation process of BCR / ABL fusion gene fluorescence in situ hybridization probe

[0076] The complete preparation process diagram is as follows Figure 1 shown.

[0077] 1. Obtain oligonucleotide pools with specific sequences of BCR gene and ABL gene target regions and remove repeated sequences

[0078] 1.1 Use the UCSC Genome Browser to find the BCR gene sequence (BCR----NC_000022.11) and the ABL gene sequence (ABL---NC 000009.12), respectively. Figure 2 and Figure 3 shown.

[0079] 1.2 Use the browser's built-in bioinformatics analysis function to annotate the BCR / ABL gene sequence after removing non-repetitive sequences, and copy the target-specific gene sequence to a document processor such as Microsoft Word or Excel. The BCR gene probe region starts from SHGC-10404 (STS site) at the 5 end to the 3 end of the BCR gene, covering an area of ​​about 600kb, leaving a space of about 320kb in the middle. The BCR gene 3 end probe starts from RH8078 (STS site) to SHGC-17034 (STS site), covering an area of ​​about 640kb (chromosome 22) ( Figure 4 ); The 5 end of the ABL gene probe region starts at SHGC-147754 (STS site) to the 3 end of the ABL gene SHGC-8190 (STS site), covering an area of ​​approximately 700kb (chromosome 9) ( Figure 5 ).

[0080] 1.3 The obtained BCR / ABL gene specific sequence without repetitive sequence was processed using nucleic acid sequence FASTA format software to generate oligonucleotide fragments with a size between 60-100 bp, and forward and reverse PCR primers were designed by extending both ends of all specific oligonucleotide fragments of uniform size (the primer set is shown in Table 1), and a specific oligonucleotide pool of uniform size was obtained through chemical synthesis (the specific oligonucleotide pool is shown in Tables 2 and 3 at the end of the specification, wherein Table 2 is the BCR specific oligonucleotide pool and Table 3 is the ABL specific oligonucleotide pool).

[0081] Table 1. Specific amplification primers

[0082] Primer pairs Forward Primer Serial Number Reverse primer Serial Number 1 5'-CGCACCAGCGTATTCGTGTC-3' SEQ ID: 1 5'-AGTATGGTGCTCCGTGTTCG-3' SEQ ID:2 2 5'-AACCAGGCGGGCTCGCCACG-3' SEQ ID:3 5'-AAGCGCGTTGCTACCTAGAC-3' SEQ ID:4 3 5'-TTCGATTGTCGTGGCCACCC-3' SEQ ID:5 5'-AGTGAGCTGCGAGAGCTTTG-3' SEQ ID:6 4 5'-AGTTAACATGTAGCCCGGCC-3' SEQ ID:7 5'-CGTCGGTACCACGTATCGGG-3' SEQ ID:8 5 5'-ATCTAGGAGGGGCGCGTAGG-3' SEQ ID:9 5'-GGGCATCCTGTCACAAGGAG-3' SEQ ID:10 6 5'-ACCCCAATCGTTTTCCGTCG-3' SEQ ID:11 5'-CTGTCAAGGGATTCCCGTGG-3' SEQ ID:12 7 5'-GGGCAATACGAGCTCAAGCC-3' SEQ ID:13 5'-ACTCAGCGTCGCTCCCATTG-3' SEQ ID:14 8 5'-AAGCGGACAGCGGTTCTACG-3' SEQ ID:15 5'-ACCCCGGTCCGTTCTGAGTC-3' SEQ ID:16 9 5'-GGCCACGCGTCGACTAGTAC-3' SEQ ID:17 5'-AACTTGTATCCGACCCCCGC-3' SEQ ID:18 10 5'-CCCGGCGCAGAGAGGAAGCC-3' SEQ ID:19 5'-CGAGTGTCACGTCTGACCTC-3' SEQ ID:20 11 5'-GCTGTAGCAGCAGGCTGTGC-3' SEQ ID:21 5'-AGCCAAAAAAGCACGTGGTG-3' SEQ ID:22 12 5'-CACCCGGGGGATGTCTAAAC-3' SEQ ID:23 5'-TATGGCACTCATCCCAGCTG-3' SEQ ID:24 13 5'-GGTGCCTGGTTTCACTACCC-3' SEQ ID:25 5′-TAACGGAGAGACCCTGCGGC-3′ SEQ ID:26 14 5'-GAGCCTAGGTCCACTGGAGG-3' SEQ ID:27 5'-CAGGGTTTTCCCAGTCACGAC-3' SEQ ID:28 15 5'-GGCTCTGCGACCCGTTCCGG-3' SEQ ID:29 5'-ATGGTCCTGCTGGAGTTCGTG-3' SEQ ID:30

[0083] The above 15 primer pairs can all be used. In this embodiment, the forward primer shown in SEQ ID: 21 and the reverse primer sequence shown in SEQ ID: 22 are added to both ends of the oligonucleotide sequence.

[0084] 2. Obtain a specific BCR and ABL probe template library of uniform size

[0085] 2.1 The BCR gene oligonucleotide pool and ABL gene oligonucleotide pool designed as above have been synthesized.

[0086] 2.2 Dilute and aliquot the above oligonucleotide pool.

[0087] 2.3 Preparation of BCR / ABL probe template library

[0088] The amplification system shown in Table 4 was used for preparation:

[0089] Table 4. Amplification reaction system

[0090] Components Final concentration BUFFER(GC) / <![CDATA[MgCl2]]> 2.5mM dNTP 0.3mM Primer F 2uM Primer R 2uM Enzymes 0.02U Template DNA 2ng / ul

[0091] In this embodiment, the oligonucleotide pool is amplified twice to increase the concentration of the obtained template library. After the first amplification, the concentration of the purified BCR oligonucleotide pool is 6.1 ng / μL, and the concentration of the ABL oligonucleotide pool is 4.4 ng / μL. After the second amplification, the concentration of the purified BCR oligonucleotide pool is 17.8 ng / μL, and the concentration of the ABL oligonucleotide pool is 47.4 ng / μL.

[0092] 2.4 Purification of BCR / ABL probe template library

[0093] The gel microcolumn centrifugation method is used: the molecular sieving effect of the gel and the centrifugation method are used to separate the macromolecular DNA and small molecular dNTP and other substances. The gel microcolumn medium used is Sephadex G-50. After purification, the BCR / ABL probe oligonucleotide library is obtained.

[0094] 2.4.1 Preparation of purification column: 800 μl of Sephadex G50 was added to the sterilized column and centrifuged at 1100 rcf for 4 minutes; 500 μl of TES was added to the column and centrifuged at 1100 rcf for 4 minutes;

[0095] 2.4.2 Sample purification by column: Put the column into a clean 50ml centrifuge tube; take out all the reaction solution, do not touch the tube wall when sucking, and then add the sample to the mattress in the column; Note: Be sure to add the sample to the mattress, do not add the sample to the gap between the mattress and the tube wall; Centrifuge for 6 minutes at a speed of 2200rpm, and discard the purification column;

[0096] 2.4.3 Result detection: Prepare 1.5% agarose gel and run electrophoresis for about 30 minutes. The electrophoresis results are as follows: Figure 6 As shown, the markers from left to right are: BCR first amplification result, BCR second amplification result, ABL first amplification result, ABL second amplification result.

[0097] 1. The sequencing results of BCR and ABL oligonucleotide libraries are shown in Table 5 to confirm the correctness of their sites.

[0098] Table 5. Sequencing results of BCR and ABL oligonucleotide libraries

[0099] Sample Probe sequence number Number of consistency probes Proportion of probes measured oligo sequence length Number of bases not covered by oligo oligo coverage ratio ABL1-1 11951 10892 91.14％ 1434120 4531 99.68％ ABL1-2 11951 11730 98.15％ 1434120 1 99.99％ BCR-1 11974 8776 73.29％ 1436880 18301 98.73％ BCR-2 11974 9740 81.34％ 1436880 1965 99.86％

[0100] Based on the above experimental data, it can be concluded that the BCR / ABL fluorescent probe prepared by this method can cover a longer area and is therefore feasible for marking target genes.

[0101] 2. BCR / ABL probe labeling

[0102] 2.1 Labeling reaction system

[0103]

[0104] Table 7. Labeling reaction template

[0105] Template Name Date of expansion concentration Corresponding primer dye BCR-G 2022.11.29 17.8 Fluorescein (green) ABL-O 2022.06.10 47.4 ATTO (orange)

[0106] Table 8. Actual dosage of labeling reaction

[0107] component V(ul) BCR-G ABL-O Labeling mixture (2×) 25 25 25 Fluorescent primer F (20uM) 5 5 5 Fluorescent primer R (20uM) 5 5 5 DNA template (100ng) 50ng / ul 8.5 3.2 Purified water (up to 50ul) 1.6 6.8 total 50 50 50

[0108] Table 9. Labeling reaction procedure

[0109]

[0110] Add the labeling mixture (the ingredients are shown in Table 6, prepared in advance if no primers are added), primer mixture, DNA template, and purified water to a sterilized clean PCR tube in sequence, flick the tube to mix, cover the tube, and centrifuge briefly to remove liquid from the tube wall.

[0111] 2.2 Labeling reaction procedure:

[0112] Place the above reaction system reagents into the thermal cycler, cover the thermal cycler, set the thermal cycler to the above reaction program (Table 9), set the top cover temperature to 104°C, and run the program. After the program is finished, take out the reaction solution and place it in an ice box.

[0113] 2.3 Purification

[0114] The purification steps for each probe labeling reaction are as follows (one labeling reaction corresponds to one spin column):

[0115] 2.3.1 Preparation of spin columns

[0116] 2.3.1.1 Take 0.8 ml of Sephadex G50 soaking solution and add it to a sterilized clean centrifuge column. Centrifuge at 1100 rcf for 4 min at low temperature and discard the waste liquid at the bottom of the column.

[0117] 2.3.1.2 Add 0.5 ml of 1×TES to the centrifuge column, centrifuge at 1100 rcf, 4°C for 4 min, and discard the waste liquid at the bottom of the column;

[0118] 2.3.1.3 Place the prepared centrifuge column in a sterilized, clean 1.5 ml centrifuge tube.

[0119] 2.3.2 Probe purification: Add the labeled reaction solution to the center of the mattress in the centrifugal column; centrifuge at 2200rcf, 4℃ for 10min, collect all the same probe liquids that have passed through the column, mix them in a centrifuge tube, and the obtained probes are BCR probe intermediate and ABL probe intermediate; and detect their concentrations: BCR probe intermediate: 120.65ng / ul; ABL probe intermediate: 141.90ng / ul.

[0120] 2.4 Probe enrichment

[0121] 2.4.1 Mix the purified probes according to the following proportions and add them to the centrifuge tube for concentration:

[0122] Table 10. BCR / ABL fusion probe ratio table

[0123]

[0124] 2.5 Add the probe to the hybridization solution

[0125] 2.5.1 Add an appropriate amount of hybridization solution (10 μl / person) to the above-mentioned centrifuge tube for concentration, centrifuge briefly, and place in a 37°C water bath to dissolve for 60 minutes, mixing every 5 minutes.

[0126] Table 11. BCR / ABL fusion probe dissolution table

[0127] Probe Name Hybridization solution BCR / ABL fusion probe Universal Hybridization Solution

[0128] Hybridization solution formula: a general hybridization solution with a final concentration of 2×SSC buffer, containing 30-60% by mass formamide and 1g-3g / 10ml of dextran sulfate.

[0129] 2.6 Probe Packaging

[0130] Use a pipette to dispense the BCR / ABL dual-color probe into a natural color cryotube and cover it with a red cryotube cap. The BCR / ABL dual-color probe is obtained after dispensing.

[0131] Table 12. BCR / ABL fusion probe loading table

[0132]

[0133] 2.7 Staining solution packaging

[0134] Use a pipette to dispense the staining solution into amber cryotubes and cover the amber cryotube caps. The aliquoted solution is the staining solution.

[0135] Table 13, Dyeing liquid filling table

[0136]

[0137] Example 2: FISH method using BCR / ABL probe

[0138] 1. Sample processing

[0139] 1.1 Take cultured K-562, HL-60, and IM-9 cells;

[0140] 1.2 Add 10 ml of hypotonic solution preheated at 37°C into the centrifuge tube and mix gently by pipetting;

[0141] 1.3 Place in a 37℃ water bath for 30 min;

[0142] 1.4 Add 1 ml of fresh fixative, mix gently by pipetting, and pre-fix at room temperature for 8 minutes;

[0143] 1.5 Mix by pipetting and centrifuge at 1500 rpm for 8 minutes;

[0144] 1.6 Remove the supernatant, add 10 ml of fresh fixative to the precipitate, mix gently by pipetting, and let stand at room temperature for 8 minutes

[0145] 1.7 Centrifuge at 1500 rpm for 8 minutes;

[0146] 1.8 The above washing steps can be repeated until the cell pellet is washed clean.

[0147] 2. Production

[0148] 2.1 Take a clean slide;

[0149] 2.2 After resuspending the cells, take 3 μl of the suspension and drop it on different positions on the slide, making sure not to overlap;

[0150] 2.3 Dry at room temperature;

[0151] 2.4 Observe the cell density of the three areas under a phase contrast microscope with a 20× objective lens. The cells should not overlap significantly and the number should be between 100 and 200. ① If the cells overlap, add an appropriate amount of fresh fixative to dilute the cell suspension, mix well and take another 3μl of the cell suspension for slide preparation. ② If the cell density is low, centrifuge, carefully remove an appropriate amount of supernatant, mix well and take another 3μl of the suspension for slide preparation, dry and observe.

[0152] 2.5 Observe under a phase contrast microscope. If there are too many cell fragments, pretreatment is required and appropriate hybridization areas should be selected;

[0153] Note: At least one more slide should be prepared for each sample. The remaining cell suspension can be stored at 2-8°C for one month so that it can be re-slided when necessary.

[0154] 3. Instrument preparation

[0155] 3.1 Turn on the thermostatic water bath and set the temperature to 37°C. Pour 50ml of enzyme buffer into the Coplin jar, put it into the water bath, and preheat it to 37°C; before elution, set the thermostatic water bath temperature to 68°C, pour the wash solution into the Coplin jar, and put it into the water bath for at least 30 minutes, ensuring that the temperature of the wash solution reaches 68°C ± 1°C before use.

[0156] 4. Inspection process

[0157] 4.1 Sample preprocessing

[0158] 4.1.1 Soak the glass slide in 2×SSC solution at room temperature for 10 minutes;

[0159] 4.1.2 Digest in pepsin solution at 37±1℃ for 10 minutes;

[0160] 4.1.3 Then soak the slide in 2×SSC solution at room temperature for 2 minutes;

[0161] 4.1.4 Dehydrate the slides in 70%, 85%, and 100% alcohol solutions for 1 minute each;

[0162] 4.1.5 Air-dry the slides at room temperature.

[0163] 4.2 Sample denaturation and hybridization

[0164] 4.2.1 Soak the moisturizing strip in pure water and place it in the corresponding slot of the hybridizer, making sure the surface of the hybridizer is clean;

[0165] 4.2.2 Set the denaturation hybridization program as follows: denaturation: 85°C, 4 minutes, hybridization: 43°C, 16-18 hours;

[0166] 4.2.3 Take out the probe, put it to room temperature, vortex centrifuge, take 10 μl of the probe, and drop it on the target area of ​​the slide;

[0167] 4.2.4 Cover with a cover glass, be careful to avoid bubbles, and seal the edge of the cover glass with sealing glue;

[0168] 4.2.5 Place the sealed slide on the hybridizer and start the denaturation hybridization procedure.

[0169] 4.3 Post-hybridization processing

[0170] 4.3.1 Preheat Lotion 1 to 68±1℃ and keep Lotion 2 at room temperature;

[0171] 4.3.2 After hybridization is completed, take out the cell slice and remove the sealing glue and cover glass from the cell slice;

[0172] 4.3.3 Place the slide in 68±1℃ washing solution 1 and soak for 2 minutes;

[0173] 4.3.4 Place the slide in the room temperature washing solution 2 and soak for 1 minute;

[0174] 4.3.5 Place the slide in 70% alcohol solution and wash for 1 minute;

[0175] 4.3.6 Take out the slides and dry them naturally in a dark place.

[0176] 4.4 Counterstaining

[0177] 4.4.1 Add 10 μL of staining solution to the hybridization area, avoid bubbles, cover with a coverslip, carefully remove excess staining solution with absorbent paper, and place in a dark place at -20℃±5℃ for more than 20 minutes;

[0178] 4.4.2 Select appropriate filter sets to observe the results under a fluorescence microscope.

[0179] 4.5 Results Analysis

[0180] 4.5.1 Select appropriate filter sets to observe the results under fluorescence microscope ( Figures 7 to 9 BCR / ABL fluorescence in situ hybridization results of IM-9, K-562, and HL-60, respectively);

[0181] 4.5.2 Find the area under the 10× objective lens and count under the 100× objective lens;

[0182] 4.5.3 Adjust the focus and find the appropriate area, which requires the cell nucleus border to be intact, the staining solution to be evenly stained, the nuclei to have no overlap, and the signal points to be clear;

[0183] In the selected area, all signal points were found at different levels of the nucleus, and the fusion signals, separate orange signals and green signals were counted. When the orange-red and green signal points overlapped or the distance between them was less than the diameter of one signal point, it was counted as one fusion signal (yellow, F). Cell nuclei with no signal or weak signal were not counted.

[0184] Table 14. FISH results

[0185] name Sample Type Signal background Dual Channel BCR / ABL IM-9 Orange green medium strong / Observable BCR / ABL K-562 Orange green medium strong / Observable BCR / ABL HL-60 Orange green medium strong / Observable

[0186] It can be seen from the above table and the result image that the fluorescence intensity in the three test results all reached medium intensity, and the fluorescence signal was clear with extremely low background, which was convenient for counting.

[0187] Example 3: Fluorescence labeling effect of BCR / ABL probe prepared using BAC clones

[0188] The fluorescent labeling effect of the BCR / ABL probe prepared by BAC cloning was compared with that by synthetic oligonucleotide amplification. The preparation method of the fluorescent probe amplified by synthetic oligonucleotide was the same as in Example 1. The use methods of the two probes were the same as in Example 2. The fluorescent probe prepared in Example 1 and the BCR / ABL probe prepared by BAC cloning were used for BCR / ABL fusion gene detection in K-562 cells, respectively.

[0189] The specific steps of the BAC method are as follows:

[0190] 1. Clone extraction

[0191] 1.1 Experimental Reagents

[0192] REPLI-g Ultrafast Mini Kit(25), QIAprep Spin Miniprep Kit(250)

[0193] 1.2 Experimental equipment

[0194] Biochemical incubator, air bath oscillator, desktop centrifuge, high-speed refrigerated centrifuge, constant temperature water bath, fluorescence quantifier Qubit 3.0

[0195] 1.3 Experimental process

[0196] 1.3.1 Culture medium preparation

[0197] LB agar: Weigh 17.5 g of LB agar, dissolve in 500 ml of purified water, autoclave at 121°C for 15 minutes, and set aside;

[0198] LB broth: Weigh 12.5 g of LB broth, dissolve in 500 ml of purified water, sterilize at 121°C for 15 min, and set aside.

[0199] 1.3.2 Bacterial recovery

[0200] (1) In a clean bench, add 200 μl of chloramphenicol to LB agar, shake well, pour about 10 ml of solid culture medium into a sterile plate, place horizontally until cooled, turn the plate upside down and set aside;

[0201] (2) Inoculation: Dissolve the glycerol stock, shake the bacteria, dip an inoculation loop into the plate, and then streak the plate onto the plate. Incubate the plate upside down at 37°C overnight until a single colony grows.

[0202] 1.3.3 Culture

[0203] (1) In a clean bench, add 200 μl of chloramphenicol to LB broth, shake well, and pour about 15 ml of liquid culture medium into a sterile 50 ml centrifuge tube for later use;

[0204] (2) Inoculation: Pick a single colony with an inoculation loop, inoculate it into liquid culture medium, and culture it overnight in an air bath constant temperature oscillator until the culture medium becomes turbid (temperature 37°C, rotation speed: 220 rpm).

[0205] 1.3.4 Plasmid extraction

[0206] (1) Centrifuge the overnight culture at 8000g for 5 minutes, remove the supernatant, remove the liquid on the tube wall with dust-free paper, and then invert to dry;

[0207] (2) Add 426 μl of Buffer P1 to resuspend the bacterial solution, remove it completely, and place it in a 2 ml centrifuge tube;

[0208] (3) Add 426 μl of Buffer P2, mix by inverting 4-6 times, and let stand at room temperature for 5 minutes;

[0209] (4) Add 426 μl of pre-cooled Buffer P3 and immediately invert several times to mix thoroughly until a white flocculent precipitate is formed. Incubate on ice for 10 min.

[0210] (5) Centrifuge at 20,000 g, 4°C for 15 min, and carefully remove the supernatant and place it in a new 2 ml centrifuge tube;

[0211] (6) Add 0.65 times the volume of isopropanol to the supernatant, mix well, centrifuge at 20,000 g, 4°C for 10 min, and carefully discard the supernatant;

[0212] (7) Add 500 μl of room temperature 70% ethanol, centrifuge at 20,000 g for 5 min, and carefully remove the supernatant without disturbing the precipitate at the bottom;

[0213] (8) Empty the centrifuge tube so that the liquid on the tube wall collects at the bottom of the tube, and carefully remove the liquid at the bottom of the tube with a pipette.

[0214] (9) Open the tube cap and dry at room temperature for 10 minutes to evaporate the excess liquid;

[0215] (10) Add 190ul of buffer EX and gently tap the bottom of the tube to resuspend the DNA. Add 4ul of exonuclease solution and 6ul of 100mM ATP solution, mix well, and incubate in a 37°C water bath for 60 minutes.

[0216] (11) Add 200 μl of Buffer QS, flick to mix thoroughly, and centrifuge briefly;

[0217] (12) Add all samples to the purification column, centrifuge at 12000 rpm, 4°C for 30 seconds, and discard the filtrate;

[0218] (13) Add 800 μl of Buffer PE and centrifuge for 30 seconds;

[0219] (14) Centrifuge the empty tube at 12000 rpm at 4°C for 2 min and transfer the column to a new 1.5 ml centrifuge tube;

[0220] (15) Carefully add 36 μl of 65°C preheated Buffer EB to the center of the purification column, centrifuge at 12,000 rpm, 4°C for 2 min, and discard the purification column.

[0221] (16) Take 2 μl of ECoRI enzyme, 1 μl of ECoRI buffer, and 6 μl of purified water and incubate in a 37°C water bath for 60 min;

[0222] (17) The concentration was detected using Qubit3. The results are shown in Table 15.

[0223] 2. Template amplification

[0224] 2.1 Experimental Purpose

[0225] Amplify the template required for BCR / ABL probe labeling.

[0226] 2.2 Experimental reagents

[0227] GE Amplification Kit

[0228] 2.3 Experimental equipment

[0229] PCR instrument, high-speed refrigerated centrifuge, fluorescence quantitative instrument Qubit 3.0

[0230] 2.4 Experimental Procedure

[0231] 1) Add samples according to the reaction system shown in Table 15:

[0232] Table 15. Reaction system

[0233] Template Name Concentration (ng / ul) 10ng DNA dosage (ul) H2O is added to 10ul(ul) BCR-1 2.88 3.47 6.53 BCR-2 7.05 1.42 8.58 BCR-3 13.4 0.75 9.25 BCR-4 4.61 2.17 7.83 BCR-5 4.33 2.31 7.69 BCR-6 10.1 0.99 9.01 BCR-7 15.1 0.66 9.34 ABL1-1 5.2 1.92 8.08 ABL1-2 6.5 1.54 8.46 ABL1-3 17.6 0.57 9.43 ABL1-4 765 131 869 ABL1-5 7.2 1.39 8.61 ABL1-6 3.04 3.29 6.71

[0234] Mix by flicking the tube gently and centrifuge briefly.

[0235] 2. Sample denaturation: Denature at 95°C for 3 minutes in a PCR instrument, quickly insert into ice, flick the tube wall after cooling, and centrifuge briefly;

[0236] 3. Amplification reaction: Add the sample to the Ready-To-Go Genomiphi V3 cake, a total of 13 tubes, cover the lid and incubate in a PCR instrument at 30°C for 120 minutes, then inactivate at 65°C for 10 minutes;

[0237] 4. DNA concentration determination: Take the above reaction solution, add 180ul of purified water to dilute to 200ul, tap the tube wall lightly, centrifuge briefly, and detect its concentration using Qubit3.0. The results are shown in Table 16.

[0238] Table 16. Template amplification results

[0239] Template Name Concentration after amplification (ng / ul) BCR-1 77.2 BCR-2 67.8 BCR-3 94.2 BCR-4 56.2 BCR-5 77.0 BCR-6 72.2 BCR-7 92.6 ABL1-1 73.8 ABL1-2 120 ABL1-3 120 ABL1-4 120 ABL1-5 120 ABL1-6 120

[0240] 5. Template DNA purification: Add 1 / 10 volume of 3.0M sodium acetate and 2.5 times volume of anhydrous ethanol according to the volume, mix well and centrifuge briefly, and precipitate on ice for 30 minutes. Centrifuge at 15000rpm and 4℃ in a high-speed refrigerated centrifuge for 15 minutes, discard the supernatant, add 500ul pre-cooled 70% anhydrous ethanol, mix well, centrifuge at 15000rpm and 4℃ for 5 minutes, discard the supernatant and centrifuge briefly, then aspirate the remaining supernatant. Leave at room temperature for a few minutes until the alcohol is evaporated, add appropriate amount of purified water, and dissolve at 4℃ overnight;

[0241] 6. Qubit3.0 was used to detect its concentration. The results are shown in Table 17.

[0242] Table 17. Purification results

[0243]

[0244]

[0245] 3. Probe labeling

[0246] The experimental materials and instruments are shown in Tables 18 and 19.

[0247] Table 18. Experimental raw materials

[0248] name Specification dNTP 100mM / tube dTTP 100mM / tube DNA Polymerase I 10U / ul dUTP*(orange) 50nmol / tube dUTP*(green) 50nmol / tube DNase I working solution / DNA Pol I /

[0249] Table 19. Experimental instruments

[0250] name Pipette Mini centrifuge Vortex Oscillator PCR instrument High-speed refrigerated centrifuge

[0251] 3.1 Add samples according to the system shown in Table 20:

[0252] Table 20, Sample addition method

[0253]

[0254] After adding the reaction solution, mix and centrifuge.

[0255] 3.2 Labeling reaction: In a metal bath, react at 15°C for 16 hours. (The temperature of the PCR instrument cover is set to 42°C)

[0256] 3.3 Stop the reaction:

[0257] After denaturation at 70°C for 10 minutes, take out the centrifuge tube, quickly insert it into ice, and then briefly centrifuge the reagent in a mini centrifuge until there is no liquid on the tube wall.

[0258] 3.4 Filling columns: Take 0.8 ml of Sephadex G50 for each column and add it to the sterilized column. Centrifuge at 1100 g in a high-speed centrifuge for 5 minutes at room temperature. Discard the waste liquid at the bottom of the column. Then take 0.5 ml of 1×TES and add it to the sterilized column. Centrifuge at 1100 g in a high-speed refrigerated centrifuge for 5 minutes at room temperature. Fill 2 columns in total.

[0259] 3.5 Sample purification: Place the purification column (filled column) in a clean 50ml centrifuge tube, add the BCR probe / ABL probe to the purification column facing the mattress in the column, centrifuge at 1100g in a high-speed refrigerated centrifuge at room temperature for 10 minutes (Note: add the sample facing the mattress, do not add the sample to the gap between the mattress and the tube wall), and detect its concentration. The results are shown in Table 21. The amount of template required for labeling and the amount of H2O added are shown in Table 22, the probe drying results are shown in Table 23, and the BAC cloning reagent labeling method is shown in Table 24.

[0260] Table 21. Template dosage and H2O quantity required for labeling

[0261]

[0262]

[0263] Table 22. Probe volume measurement and purity and concentration determination after labeling

[0264]

[0265] Table 23. Probe Drying Record

[0266]

[0267] Table 24. BAC cloning reagent labeling method

[0268] System composition Dosage (batch 1μg or 50μl) DNA template (Calculate the volume corresponding to 1μg according to the concentration) Xμl Sterilized water (40-X)μl 10mM dNTP (without dTTP) 0.5μl 1mM dTTP 1μl 1mM dUTP* (label) 0.5μl 10× Reaction Buffer (Polymerase) 5μl DNA polymerase I 1μl DNase I working solution 2μl Total volume 50μl

[0269] The above-mentioned labeling reaction mixture was dispensed into 0.5 ml centrifuge tubes, with each tube not exceeding 50 μl, and centrifuged until there was no liquid on the tube wall; the centrifuge tubes containing the labeling reaction mixture were transferred to a thermal cycler and reacted at 15°C for 16 hours; after denaturation at 70°C in the thermal cycler for 10 minutes, the temperature was immediately cooled, and then centrifuged until there was no liquid on the tube wall, and placed in an ice box.

[0270] The probe obtained by the BAC clone prepared by the above operation and the probe prepared in Example 1 were respectively used to detect the BCR / ABL fusion gene in K-562 cells using the method of Example 2. The obtained result is a fluorescence detection image: Fig.10 This is the detection image of the oligo probe prepared in Example 1. Fig.11 This is the detection image of the probe prepared by the BAC cloning method in this example.

[0271] The results show that the fluorescence detection image of oligo probe in situ hybridization has a clearer background than the detection image of the probe prepared by BAC cloning method, and shows less non-specific fragment signals on the image, so it is more conducive to judgment. The detection image of the probe prepared by BAC cloning method has a very obvious background, and non-specific fragments in the cell are marked, which has a more accurate result for judging gene fusion. It can be observed that Fig.11 There are multiple signals in the cells with unclear boundaries, which are difficult to identify in clinical applications.

[0272] Example 4: BCR / ABL gene oligo probe primer screening

[0273] In the initial screening, four pairs of primers, including the forward primer shown in SEQ ID: 21 and the reverse primer sequence shown in SEQ ID: 22, the forward primer shown in SEQ ID: 3 and the reverse primer sequence shown in SEQ ID: 4, the forward primer shown in SEQ ID: 9 and the reverse primer sequence shown in SEQ ID: 10, and the forward primer shown in SEQ ID: 27 and SEQ ID: 28, had good amplification effects.

[0274] In Example 1, the forward primer shown in SEQ ID: 21 and the reverse primer sequence shown in SEQ ID: 22 (primer 3) were added to both ends of the oligonucleotide sequence. In this example, the forward primer shown in SEQ ID: 3 and the reverse primer sequence shown in SEQ ID: 4 (primer 1), the forward primer shown in SEQ ID: 9 and the reverse primer sequence shown in SEQ ID: 10 (primer 2), the forward primer shown in SEQ ID: 27 and the reverse primer sequence shown in SEQ ID: 28 (primer 4) were respectively used to participate in the labeling effect of the prepared oligo probe. The preparation method refers to Example 1, and the probe labeling method refers to Example 2. The prepared probe detects the BCR / ABL fusion gene in K-562 cells in the manner of Example 2.

[0275] The experimental results are fluorescence detection images: Fig.12 is the fluorescence detection image corresponding to primer 1, Fig.13 is the fluorescence detection image corresponding to primer 2, Fig.14 is the fluorescence detection image corresponding to primer 3, Fig.15 This is the fluorescence detection image corresponding to primer 4.

[0276] As can be seen from the images, all four primer sets can obtain clear images labeled with fluorescent probes, among which the fluorescence signals in the images of primers 1 and 4 are relatively weak, and the fluorescence signal in the image corresponding to primer 3 is strong and clear. Primer 3 corresponds to the forward primer shown in SEQ ID: 21 and the reverse primer sequence shown in SEQ ID: 22, so this primer set has the effect of improving the fluorescence effect of the obtained probe target.

[0277] Table 25. Comparison of hybridization efficiency of 4 sets of primers for preparing probes for detection

[0278] Primer 1F / 1R Primer 2F / 2R Primer 3F / 3R Primer 4F / 4R 88％ 95％ 100％ 94％

[0279] According to the test results, the hybridization efficiency (the ability of the probe in the reagent to bind to the target sequence) after detection using primer groups 1 to 4 was calculated respectively. The calculation method of the hybridization efficiency was normal signal count cells / counted cells; the results are shown in Table 25. The hybridization efficiency of primer 4 was 94%, the hybridization efficiency of primer 3 was 100%, the hybridization efficiency of primer 2 was 95%, and the hybridization efficiency of primer 1 was 88%. It can be seen that the hybridization efficiency of primer 3 was the highest and the signal intensity was relatively excellent.

[0280] Example 5: Dye and dye gradient screening of BCR / ABL gene oligo probe fluorescent labeling process

[0281] In Example 1, the primers used in the probe fluorescent labeling step are BCR: Fluorescein (fluorescein, green, provided by Shanghai Jierui with dye primers) and ABL: TAMRA (tetramethylrhodamine, orange). In this example, ABL: ROX (X-rhodamine, red), ABL: ATTO (orange), ABL: TAMRA (tetramethylrhodamine, orange 1), ABL: Alexa (orange 2), BCR: Fluorescein (fluorescein, green 1), BCR: FAM (carboxyfluorescein, green 2) primers are used for experiments. The 20uM concentration primer used in Example 1 contains 7.5μL primer per 50μL, and the fluorescent probe effect is tested at 2.5μL, 3.75μL, 5μL, 6.25μL, and 7.5μL primer dosages. The prepared probe detects the BCR / ABL fusion gene in K-562 cells in the manner of Example 2.

[0282] The probe preparation method is referred to Example 1, and the probe usage is referred to Example 2. Different dye combinations are used in the fluorescent labeling step to conduct experiments: the first group: red / green 1 (ABL / BCR), the second group: orange / green 1 (ABL / BCR), the third group: orange 1 / green 1 (ABL / BCR), the fourth group: orange 2 / green 1 (ABL / BCR), the fifth group: red / green 2 (ABL / BCR), the sixth group: orange / green 2 (ABL / BCR), the seventh group: orange 1 / green 2 (ABL / BCR), and the eighth group: orange 2 / green 1 (ABL / BCR). The obtained probes are used to detect the BCR / ABL fusion gene in K-562 cells in the manner of Example 2.

[0283] The experimental results are fluorescence detection images: Figures 16 to 23 The result images are the first group: red / green 1, the second group: orange-red / green 1, the third group: orange 1 / green 1, the fourth group: orange 2 / green 1, the fifth group: red / green 2, the sixth group: orange-red / green 2, the seventh group: orange 1 / green 2, and the eighth group: orange 2 / green 1. Figures 24 to 28 These are the fluorescent labeling result images of the labeling system with primer (orange / green 1) contents of 2.5 μL, 3.75 μL, 5 μL, 6.25 μL, and 7.5 μL, respectively.

[0284] Comparing the fluorescence detection images of 8 different primers, it can be concluded that BCR: Green Primer 2 has a weak signal in the present invention, and the fluorescent probe of Green Primer 1 has a better fluorescence signal. From the screening of ABL gene probe primers, it can be found that the hybridization efficiency and signal intensity of various dye probes can meet the standards. Among them, the best effect is the orange-red dye, i.e. ATTO, with obvious and clear signals, high contrast with the background, and the cleanest background.

[0285] Comparing the fluorescence labeling with different primer concentrations, when the primer dosage is lower than 7.5 μL per 50 μL, the fluorescence signal will be too weak. Therefore, in the application of the present invention, the primer dosage should reach 7.5 μL per 50 μL to meet the demand of large-scale preparation of oligonucleotide probes.

[0286] When the primer dosage was 7.5 μL per 50 μL, the hybridization efficiency after the probes were prepared using 8 sets of dye combinations for detection was calculated respectively. The detection results are shown in Table 26.

[0287] Table 26. Comparison of hybridization efficiency of 8 dye combinations for preparing probes for detection

[0288] Group Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Hybridization efficiency 98％ 100％ 98％ 95％ 96％ 96％ 99％ 95％

[0289] It can be seen from Table 26 that there are obvious differences in the hybridization efficiency of the probes prepared by the eight groups of dyes. Among them, the hybridization efficiency and signal intensity of the dye probes in the second group are relatively the best, and the hybridization efficiency reaches 100%. Therefore, the dye combination of the second group (orange / green 1) is preferred to prepare probes.

[0290] Example 6: High-fidelity enzyme screening during fluorescent labeling of BCR / ABL gene oligo probe

[0291] In the probe fluorescent labeling step in Example 1, the high-fidelity enzyme contained in the labeling mixture used is enzyme K. This example uses different high-fidelity enzymes for probe labeling research, and finally performs FISH testing to observe the final labeling effects of different enzymes. Enzyme K: Phantaax Super-Fidelity DNA Polymerase (purchased from Norwegian), enzyme T: TIANSeq HiFi Amplification Mi (purchased from TIANGEN), enzyme N: High-Fidelity DNA Polymerase (purchased from Yisheng Bio) were used to conduct experiments respectively, and each experiment was repeated 3 groups to observe its stability. The prepared probe detects the BCR / ABL fusion gene in K-562 cells in the manner of Example 2.

[0292] The probe preparation method is referred to Example 1, and the probe usage is referred to Example 2. Different high-fidelity enzyme combinations are used in the fluorescent labeling step to conduct experiments. The resulting images are as follows: Figures 29 to 31 This is the fluorescence detection result image of enzyme K. Figures 32-34 This is the fluorescence detection result image of enzyme T. Figures 35-37 This is the fluorescence detection result image of enzyme N.

[0293] From the results, it can be seen that the image fluorescence intensity of the fluorescently labeled probe involving enzyme K is the highest and has good repeatability, that is, Phantaax Super-Fidelity DNA Polymerase is most suitable for the fluorescent labeling process in the present invention, while the fluorescence signal intensity in the image corresponding to enzyme T is inconsistent and has poor repeatability. The fluorescence signals in the three result images corresponding to enzyme N are all weak, which is not suitable for the present invention.

[0294] According to the detection results, the average hybridization efficiency of the three enzymes after probe labeling was calculated respectively, among which the hybridization efficiency of enzyme K was 100%, the hybridization efficiency of enzyme T was 94%, and the hybridization efficiency of enzyme N was 95%.

[0295] It can be seen that enzyme K has the highest hybridization efficiency detection rate, while enzyme T and enzyme N both have low hybridization efficiency detection rates due to insufficient signal intensity or clarity. Therefore, enzyme K is preferred for probe labeling.

[0296] Table 2. BCR-specific oligonucleotide pool

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] Table 3. ABL specific oligonucleotide pool

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] The embodiments described in the present invention are all preferred embodiments and features. Any person skilled in the art can make some changes and modifications based on the essence of the description of the present invention. These changes and modifications are also considered to be within the scope of the present invention and the scope limited by the independent claims and the appended claims.

Claims

1. A novel method for preparing a BCR / ABL fusion gene in situ hybridization probe, characterized in that: The following steps are involved: (1) The nucleic acid sequences of the BCR gene and ABL gene target region specific sequences are annotated and the repeated sequences are removed; (2) converting the nucleic acid sequence obtained in step (1) into an oligonucleotide sequence according to the length of the oligonucleotide to be synthesized, wherein the length of each oligonucleotide in the oligonucleotide sequences obtained for the BCR and ABL genes is the same; (3) designing and synthesizing primers for amplifying oligonucleotide fragments, adding fluorescent dyes to the 5' end during primer synthesis to obtain fluorescently labeled forward and reverse amplification primers, synthesizing the oligonucleotide sequence obtained in step (2) into a specific oligonucleotide pool with consistent length using oligonucleotide chemical synthesis technology, and introducing forward primers and reverse primers at both ends of the sequence during synthesis, so that the length of the oligonucleotide fragments in the constructed BCR and ABL oligonucleotide pools is consistent; (4) using the fluorescently labeled primers of the BCR and ABL genes obtained in step (3) and the oligonucleotide pool obtained in step (2) as templates to perform PCR to obtain BCR and ABL gene oligonucleotide fluorescent probe sets; (5) After purifying the probe set, dissolve it in hybridization solution and store it.

2. The method according to claim 1, characterized in that Step (4) PCR amplification can be divided into two steps. The first step is to obtain a probe template library of multiple copies of BCR and ABL genes, which is obtained by amplification using forward and reverse primers without fluorescent labels. The target concentration can be achieved by adjusting the number of amplifications. The purpose of the second step amplification is to obtain a single-use fluorescent probe. Using the probe template library as a template, forward and reverse primers containing fluorescent markers are used to amplify the in situ hybridization fluorescent probes of the ABL and BCR genes; the probe template library can be stored and reused.

3. The method according to claim 2, characterized in that The primers used for amplifying the probe template library in step (4) are the forward primer shown in SEQ ID: 1 and the reverse primer shown in SEQ ID: 2, the forward primer shown in SEQ ID: 3 and the reverse primer shown in SEQ ID: 4, the forward primer shown in SEQ ID: 5 and the reverse primer shown in SEQ ID: 6, the forward primer shown in SEQ ID: 7 and the reverse primer shown in SEQ ID: 8, the forward primer shown in SEQ ID: 9 and the reverse primer shown in SEQ ID: 10, the forward primer shown in SEQ ID: 11 and the reverse primer shown in SEQ ID: 12, the forward primer shown in SEQ ID: 13 and the reverse primer shown in SEQ ID: 14, the forward primer shown in SEQ ID: 15 and the reverse primer shown in SEQ ID: 16, the forward primer shown in SEQ ID: 17 and the reverse primer shown in SEQ ID: 18, the forward primer shown in SEQ ID: 19 and the reverse primer shown in SEQ ID: 20, the forward primer shown in SEQ ID: 21 and the reverse primer shown in SEQ ID: 22, and the reverse primer shown in SEQ ID:

23. The forward primer shown in SEQ ID: 23 and the reverse primer shown in SEQ ID: 24, the forward primer shown in SEQ ID: 25 and the reverse primer shown in SEQ ID: 26, the forward primer shown in SEQ ID: 27 and the reverse primer shown in SEQ ID: 28, the forward primer shown in SEQ ID: 29 and the reverse primer shown in SEQ ID: 30, at least one pair among the above 15 primer pairs.

4. The method according to claim 3, characterized in that The primers used to amplify the probe template library in step (4) are the forward primer shown in SEQ ID: 21 and the reverse primer shown in SEQ ID:

22.

5. The method according to claim 1, characterized in that The high-fidelity enzyme used in the PCR amplification system in step (4) is Phantaax Super-Fidelity DNA Polymerase.

6. The method according to claim 1, characterized in that: The oligonucleotide sequence obtained in step (2) may be screened once before being used in step (3) to remove oligonucleotide sequences containing TTTT and AAAA.

7. The method according to claim 1, characterized in that The length of the oligonucleotide sequence obtained in step (2) is 60-100 bp.

8. The method according to claim 1, characterized in that In step (4), the fluorescent dye used to label the BCR gene is Fluorescein, and the fluorescent dye used to label the ABL gene is Alexa. In the labeling system, the amount of 20uM concentration primer is 7.5μL per 50μL system.

9. A novel BCR / ABL fusion gene fluorescence in situ hybridization probe, characterized in that: The method is prepared by the method according to any one of claims 1 to 8, and further comprises a hybridization solution and a staining solution used to dissolve the probe, wherein the hybridization solution contains 30-60% formamide by mass and 1g-3g / 10ml of dextran sulfate, and the staining solution contains the components DAPI and 1,4-diazobicyclo (2,2,2) octane (DABCO) and p-phenylenediamine and propyl gallate as anti-fading agents.

10. The specific amplification primers are used to improve the fluorescence effect of the obtained probe target in the preparation of BCR / ABL fusion gene fluorescence in situ hybridization probes. The specific amplification primer pair sequence is the forward primer shown in SEQ ID: 21 and the reverse primer shown in SEQ ID: 22.

Images