Primer combination for analyzing and detecting leukemia fusion gene based on capillary electrophoresis fragment and application of kit

Abstract

The invention discloses application of a primer combination for analyzing and detecting a leukemia fusion gene based on capillary electrophoresis fragments in preparation of a kit for detecting the leukemia fusion gene. The primer combination consists of four primer groups, wherein the first primer group comprises sequences as shown in SEQ ID NO.1-48; the second primer group comprises a sequence as shown in SEQ ID NO. 47-84; the third primer large group comprises a sequence as shown in SEQ ID NO. 85-134; the fourth primer group comprises sequences as shown in SEQ ID NO.47-48 and SEQ ID NO.135-189, and the fourth primer group can be used for detecting 291 fusion subtypes of 121 fusion genes of leukemia. According to the kit prepared by applying the primer combination, four multiple composite PCR amplification systems are utilized, site coverage is comprehensive, multiple fusion subtypes can be detected in one tube, and the kit is high in detection sensitivity, high in resolution ratio, high in detection speed and high in specificity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application based on application number 202410013547.9, application date January 3, 2024, and invention name: Primer combination, kit and method for detecting leukemia fusion gene based on capillary electrophoresis fragment analysis. Technical Field

[0002] The invention belongs to the field of molecular biology, and in particular relates to a primer combination, a kit and a method for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis. Background Art

[0003] Leukemia is a disease of malignant clonal proliferation of bone marrow hematopoietic stem cells, especially common in children and adolescents. Studies have shown that most leukemia patients have some kind of chromosomal structural aberration, including chromosome deletion, insertion, inversion, duplication, translocation, etc., which cause chromosome rearrangement and form new fusion genes. Fusion genes are important driving factors for the occurrence and development of tumors. The formed fusion genes can activate the expression of proto-oncogenes, cause the structure of tumor suppressor genes to mutate, lose activity, or form new fusion proteins, leading to abnormal transcriptional regulatory factors, thereby causing the activation or enhancement of downstream signaling pathways, ultimately promoting cell growth and proliferation, inhibiting cell differentiation, and thus leading to the occurrence of tumors. To date, leukemia involves at least hundreds of fusion genes. Leukemia fusion genes can be used as molecular biological specific markers and diagnostic evidence for different types of leukemia. For example, in chronic myeloid leukemia CML or some acute lymphocytic leukemia ALL: BCR-ABL1; in acute T lymphocytic leukemia (T-ALL): SIL-TAL1; in acute promyelocytic leukemia APL: PML-RARA, etc.

[0004] There are more than 100 fusion genes mentioned in the WHO 2016 classification standard for blood tumors. The identification of fusion genes provides important molecular indicators for the diagnosis, classification, selection of clinical treatment plans, efficacy monitoring and development of targeted drugs for blood tumors. The development of fusion gene detection technology in blood tumors has great clinical significance and market value.

[0005] At present, the detection methods of fusion genes mainly include chromosome karyotype analysis, fluorescence in situ hybridization (FISH), multiple nested RT-PCR, real-time fluorescence quantitative PCR and digital PCR quantitative detection, and high-throughput sequencing detection. However, these methods have their own defects or disadvantages. The resolution of chromosome karyotype analysis is low, and the minimum resolution is limited to 5MB, and it cannot detect fusion genes of chromosome "microdeletion" or "microduplication". FISH technology has the characteristics of complex operation, high labor cost, low throughput and low resolution. Compared with chromosome karyotype analysis and fluorescence in situ hybridization technology, PCR detection method has the advantages of rapidity, simple operation and high sensitivity. However, although real-time fluorescence quantitative RT-PCR and digital RT-PCR have high detection sensitivity, their throughput is very low. One tube can only detect 1-2 fusion forms, which is not suitable for blood disease fusion gene screening reagents, but only suitable for micro-residue monitoring. Although high-throughput sequencing technology has high sensitivity and can also detect unknown fusions, its high cost, the need for very professional bioinformatics analysts, and the long detection cycle limit its application in fusion gene detection.

[0006] Therefore, currently on the market, the method used for qualitative screening of multiple fusion genes in blood diseases is multiple nested RT-PCR technology, but this technology also has the following defects: (1) Nested RT-PCR requires at least two rounds of PCR amplification. The product produced by the first amplification is opened and diluted, and then used as an amplification template for the second round of amplification to obtain the specific target fragment. The addition of the experimental process of diluting the PCR product and returning it to the template processing room for re-amplification not only increases the complexity of the experiment, but also greatly increases the probability of contamination, which is not in line with the smooth implementation of the routine operation process of the PCR laboratory.

[0007] (2) The final product obtained by nested PCR is currently analyzed by agarose gel electrophoresis. Whether a corresponding fusion gene is produced is determined based on whether there is a corresponding target fragment and the length of the target fragment. Since the resolution of agarose gel electrophoresis for amplified fragments is 50-100bp, this greatly reduces the number of fragments that can be amplified in each tube, and each tube can amplify at most 5-10 amplified fragments.

[0008] (3) Also due to the resolution limitation of agarose gel electrophoresis, this technology is difficult to distinguish different fusion forms of the same fusion gene. For example, the PML-RARA fusion forms include S, V, and L types. The breakpoints between the S and V types are close, and the fragments of some fusion forms differ by less than 100 bp. It is difficult to determine the specific fusion form through agarose gel electrophoresis.

[0009] (4) It is also difficult to detect gene expression increases due to gene fusion, such as the enhanced expression of Hox11, HOX11L2, and EVI genes using this type of method.

[0010] With the continuous deepening of leukemia research, more and more leukemia fusion genes have been discovered. The current method of detecting leukemia fusion genes is far from meeting the market demand for one-time screening of most fusion genes at the same time. It is urgent to develop a fast, convenient, effective, accurate and easy-to-standardize blood disease fusion gene detection method and reagent. Summary of the invention

[0011] In order to solve one of the above problems, the present invention provides a primer combination, a kit and a method for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis. The detection method of the present invention is simple to operate, has strong specificity, is rapid and efficient, has high throughput and low cost, and can achieve qualitative detection of 291 fusion subtypes of 121 leukemia fusion genes at one time.

[0012] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a primer combination for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis, the primer combination consisting of four primer groups: the first primer group includes primer sequences shown in SEQ ID NO.1-SEQ ID NO.48; the second primer group includes primer sequences shown in SEQ ID NO.47-SEQ ID NO.84; the third primer group includes primer sequences shown in SEQ ID NO.85-SEQ ID NO.134; the fourth primer group includes primer sequences shown in SEQID NO.47-SEQ ID NO.48 and SEQ ID NO.135-SEQ ID NO.189. The primer combination can detect 291 fusion subtypes of 121 fusion genes of leukemia, and the fusion genes also include fusion gene types for monitoring expression levels.

[0013] In some embodiments of the present invention, each primer group in the primer combination is composed of two primer groups: the first primer group includes a first primer group composed of primer sequences shown in SEQ ID NO.1-SEQ ID NO.24 and a second primer group composed of primer sequences shown in SEQ ID NO.25-SEQ ID NO.48; the second primer group includes a first primer group composed of primer sequences shown in SEQ ID NO.47-SEQ ID NO.64 and a second primer group composed of primer sequences shown in SEQ ID NO.65-SEQ ID NO.84; the third primer group includes a first primer group composed of primer sequences shown in SEQ ID NO.85-SEQ ID NO.116 and a second primer group composed of primer sequences shown in SEQ ID NO.117-SEQ ID NO.134; the fourth primer group includes a first primer group composed of primer sequences shown in SEQ ID NO.135-SEQ ID NO.164 and a second primer group composed of primer sequences shown in SEQ ID NO.165-SEQ ID NO.189 and SEQ ID NO.47-SEQ ID NO. The second primer group is composed of the primer sequences shown in NO.48, wherein SEQ ID NO.47-SEQ IDNO.48 are the quality control primers of the first primer group, the second primer group and the fourth primer group, and SEQ ID NO.133-SEQ IDNO.134 are the quality control primers of the third primer group.

[0014] In some embodiments of the present invention, the primer sequences in different primer groups of the first primer group, the second primer group, the third primer group, and the fourth primer group are respectively labeled with different fluorescent markers; the fluorescent group of the fluorescent marker is selected from any one of FAM, HEX, VIC, TAMRA, ROX, and TEXRED. Preferably, the fluorescent group is labeled on the most common primers for amplification of multiple fusion genes as much as possible to reduce the cost of primer synthesis and reduce the fluorescence interference caused by too many fluorescent primers in amplification.

[0015] In some embodiments of the present invention, the 5' end of the primer sequences shown in SEQ ID NO.5, SEQ ID NO.15, SEQ ID NO.22, SEQ ID NO.27, SEQ ID NO.43, SEQ ID NO.46, and SEQ ID NO.47 in the first primer group is fluorescently labeled; the 5' end of the primer sequences shown in SEQ ID NO.49, SEQ ID NO.59, SEQ ID NO.62, SEQ ID NO.64, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.70, SEQ ID NO.72, SEQ ID NO.74, SEQ ID NO.77, SEQ ID NO.79, SEQ ID NO.80, and SEQ ID NO.83 in the second primer group is fluorescently labeled; the 5' end of the primer sequences shown in SEQ ID NO.85, SEQ ID NO.115, SEQ ID NO.116, SEQ ID NO.117, SEQ ID NO.124, SEQ ID NO.125, SEQ ID NO.128, SEQ ID The 5' ends of the primer sequences shown in SEQ ID NO.132 and SEQ ID NO.133 are fluorescently labeled; the 5' ends of the primer sequences shown in SEQ ID NO.135, SEQ ID NO.137, SEQ ID NO.147, SEQ ID NO.154, SEQ ID NO.156, SEQ ID NO.163, SEQ ID NO.165, SEQ ID NO.168, SEQ ID NO.175, SEQ ID NO.181, SEQ ID NO.182, SEQ ID NO.183, SEQ ID NO.184, SEQ ID NO.47, SEQ ID NO.186, and SEQ ID NO.188 in the fourth primer group are fluorescently labeled, and the primer sequences in the same group have the same fluorescent label.

[0016] Among them, the primers shown in SEQ ID NO.5, SEQ ID NO.15, SEQ ID NO.22, SEQ ID NO.27, SEQ ID NO.49, SEQ ID NO.64, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.70, SEQ ID NO.72, SEQ ID NO.74, SEQ ID NO.80, SEQ ID NO.83, SEQ ID NO.132, SEQ ID NO.135, SEQ ID NO.137, SEQID NO.147, SEQ ID NO.154, SEQ ID NO.156, SEQ ID NO.163, SEQ ID NO.165, and SEQ ID NO.168 are universal primers.

[0017] The second aspect of the present invention provides a kit for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis, comprising the primer combination described in the first aspect of the present invention.

[0018] In some embodiments of the present invention, the kit further comprises an amplification enzyme mixture, a positive typing reference standard substance, a negative control, and a capillary electrophoresis internal standard reagent.

[0019] In some embodiments of the present invention, the positive typing reference standard material is prepared by using a primer combination for positive plasmids of 121 fusion genes and 291 fusion subtypes of leukemia: primers from SEQ ID NO.1 to SEQ ID NO.189 are used to perform PCR amplification to obtain positive fragments, and the four primer groups are mixed separately.

[0020] In some embodiments of the present invention, the amplification enzyme mixture is a PCR amplification buffer, Mg 2+ , a mixture of hot start enzymes, and the negative control is cDNA obtained by reverse transcription of normal human blood RNA.

[0021] The third aspect of the present invention provides a method for detecting leukemia fusion gene based on capillary electrophoresis fragment analysis, comprising the following steps: (1) Obtaining the cDNA template of the sample to be tested; (2) The primer combination described in the first aspect of the present invention is mixed according to four primer groups to obtain four detection primer groups, and multiplex PCR amplification is performed in four groups to obtain amplification products; (3) Detect the amplified product by capillary electrophoresis; In data analysis, positive typing reference standard substances were used to accurately adjust the position of the target bin; when a target peak higher than 100RFU appeared in a target bin other than the fusion type position for quality control and expression monitoring, the fusion gene in the corresponding bin of the sample was determined to be positive; for the fusion type for expression monitoring, if a target peak appeared in the bin position, a peak height ratio was required, and if the peak at the target gene position was greater than 1 / 4 of the peak at the quality control position, it indicated that the corresponding gene expression was positive.

[0022] In some embodiments of the present invention, the amplification system of four groups of PCR amplification is: 5×PCR master MIX 2 μL, detection primer set (Primer mix) 1 μL, cDNA to be tested 2 μL, deionized pure water 2 μL, and the total volume of the system is 10 μL. The primers shown in SEQ ID NO.1-SEQ ID NO.48 constitute the first detection primer set; the primers shown in SEQ ID NO.47-SEQ ID NO.84 constitute the second detection primer set; the primers shown in SEQ ID NO.85-SEQ ID NO.134 constitute the third detection primer set; the primers shown in SEQ ID NO.135-SEQ ID NO.189 and SEQ ID NO.47, SEQ ID NO.48 constitute the fourth detection primer set.

[0023] In some specific embodiments of the present invention, in order to minimize the generation of non-specific fragments during multiple PCR amplification, a touchdown PCR amplification program is used for PCR amplification, and the amplification program is as follows: pre-denaturation at 95°C for 10 min for 1 cycle; denaturation to extension for 5 cycles: denaturation at 95°C for 10s, annealing at 66°C for 30s, decreasing by 1°C in each cycle, and extension at 70°C for 40s; denaturation to extension for 25 cycles: denaturation at 95°C for 10s, annealing at 60°C for 30s, and extension at 70°C for 40s; post-amplification extension at 60°C for 30min for 1 cycle; the amplified products are stored at 4°C or 16°C.

[0024] In some embodiments of the present invention, in the amplification system, the primer concentrations of the primer combinations of SEQ ID NO.1-SEQ ID NO.24 are all 1 pmol / μL, and the primer concentrations of SEQ ID NO.25-SEQ ID NO.48 are all 1.5 pmol / μL; the primer concentrations of SEQ ID NO.47-SEQ ID NO.64 are all 1.5 pmol / μL, and the primer concentrations of SEQ ID NO.64-SEQ ID NO.84 are all 1 pmol / μL; the primer concentrations of SEQ ID NO.85-SEQ ID NO.116 are 1 pmol / μL, and the primer concentrations of SEQ ID NO.117-SEQ ID NO.134 are 1.5 pmol / μL; the primer concentrations of SEQ ID NO.135-SEQ ID NO.164 are 1 pmol / μL, and the primer concentrations of SEQ ID NO.165-SEQ ID NO.189 are 1.5 pmol / μL.

[0025] In some specific embodiments of the present invention, the PCR amplification product capillary electrophoresis detection method is as follows: Prepare a sample loading mixture containing molecular weight internal standard and formamide (the ratio of molecular weight internal standard and formamide is 1:500), and then calculate the number of tubes according to the number of PCR amplification products of the test sample (each test sample - containing negative control × 4 tubes of PCR product) + 4 positive typing reference standard substances. Use a pipette to dispense 9μL of the molecular weight internal standard and formamide mixture into each test well, take 1μL of PCR amplification product or 1μL of positive typing reference standard substance and add it to the mixture, cover with a sealing film, centrifuge instantly, and remove bubbles in the sample loading tube. Denature the loading plate at 95℃ for 5 minutes, and quickly ice bath for 2 minutes to completely denature the PCR product into single-stranded nucleic acid. Perform capillary electrophoresis detection on the machine according to the steps in the user manual of the genetic analyzer. The parameters are set as injection time set to 15s, injection voltage set to 3kV, and running time set to 2000s.

[0026] In some specific embodiments of the present invention, the result interpretation scheme is as follows: (1) Interpretation of experimental validity: In the negative control test results, the target peak appeared only at the quality control position in the first, second, and third group amplification results, and the peak height was greater than 300 RFU, and there were no obvious non-specific peaks (peak height > 100 RUF) in other bins. In the fourth group amplification results, the target peak appeared at the quality control position, and the peak height was greater than 300 RFU. The target fragment positions for measuring gene expression, Hox11, HOX11L2, and EVI, may also have target fragment peaks, but the ratio of their peak heights to the quality control peak height is no more than 1 / 4.

[0027] (2) Interpretation of sample validity: For the test results of the sample to be tested, the target peak must appear at the quality control bin position in the amplification of groups 1 to 4, and the peak height must be no less than 300 RFU, indicating that the sample cDNA template is normal.

[0028] (3) Result interpretation: Under the premise that the experimental operation and samples are valid, the amplification results of the four groups of samples are analyzed. If a target peak higher than 100 RFU appears in the target bin other than the quality control and expression monitoring fusion types: Hox11, HOX11L2, and EVI positions, the fusion gene in the corresponding bin of the sample is judged to be positive; for the fusion types monitored for expression: Hox11, HOX11L2, and EVI bin positions, if a target peak appears, a peak height ratio is required. If the peak at the target gene position is greater than 1 / 4 of the peak at the quality control position, it indicates that the corresponding gene expression is positive.

[0029] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: (1) A super-multiplex PCR amplification system can simultaneously amplify multiple fusion gene fusion types: The patent of this invention uses 4 multiplex composite PCR amplification systems to detect 121 fusion genes and 291 fusion subtypes of leukemia. Its advantages are: ① Comprehensive coverage of sites: The fusion genes detected by this kit basically cover the fusion types that can be detected in transcribed cDNA in the WHO 2016 version of the blood tumor classification standard, including chronic / acute myeloid leukemia (CML / AML), acute B-lymphocyte and T-lymphocyte leukemia (B- / T-ALL), myeloid / lymphoid leukemia with eosinophilia, acute promyelocytic leukemia (APL) and other common fusion types in various types of leukemia. It can perform qualitative screening for the presence of fusion genes in leukemia patients at one time, providing more information for clinicians.

[0030] ② Greatly reduce the operation intensity and detection cost. One tube of PCR multiplex amplification can detect an average of more than 70 fusion subtypes. Compared with traditional agarose electrophoresis, one tube can only detect 5-10 fusion subtypes at most. The number of fusion subtypes that can be detected in one tube is 10-15 times more than the conventional one.

[0031] (2) Detect the amplified product by capillary electrophoresis using a genetic analyzer: The detection platform adopted by the present invention is a widely used detection platform, which detects the amplification product by capillary electrophoresis, and its main advantages are: ①High detection sensitivity The use of QF-PCR combined with capillary electrophoresis greatly improves the detection sensitivity, which is more than 100 times higher than that of agarose electrophoresis. It can sensitively detect the amplified product and clearly distinguish the specific fusion subtypes. On the other hand, due to the high detection sensitivity, the requirements for the amount of PCR amplification product are reduced, and the detection limit can be reached without nested PCR, which can further reduce the detection cost, simplify the operation process, and avoid contamination.

[0032] ②High detection resolution Usually, it can be clearly detected within the detection range of 70-600bp. It can effectively distinguish the difference of 1bp. Such a high fragment length resolution can make the product size separated by 2-3bp clearly distinguishable to prevent the occurrence of misjudgment of different fusion types. At the same time, high resolution also makes it possible to detect more fusion subtypes in the same fluorescence channel.

[0033] ③The test results can be quantified The test results have specific peak heights with specific values, and the peak height and the template starting amount have a linear correspondence within a certain range. It can be compared with the internal reference (quality control gene) to a certain extent to detect fusion types such as gene overexpression caused by fusion genes.

[0034] ④Six fluorescent signals can be detected simultaneously The genetic analysis instrument can simultaneously receive six fluorescent signals, and there will not be much mutual interference between different fluorescent signals. One channel is needed to detect the molecular internal standard, and the other five channels can be used to detect different fusion subtypes of the sample. This patent only shows the use of two channels to detect the sample. In fact, this method can be used to detect more gene fusions in one tube.

[0035] ⑤Fast detection speed Automatic large-scale testing is possible: the capillary electrophoresis process completes the test in 40 minutes. Depending on the amount of samples tested at one time, 16 wells, 32 wells, or 96 wells can be tested at one time.

[0036] ⑥The test results can be automatically analyzed and interpreted using analysis software. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The detection profile of TCF3-HLF gene fusion in the HAL-01 cell line in Example 4 is shown; Figure 2 The detection profile of the MV-4-11 cell line containing KMT2A-AF4 fusion in Example 4 is shown; Figure 3 The detection profile of the HL60 cell line without fusion gene in Example 4 is shown; Figure 4 The BCR-ABL1 P210 positive detection profile in Example 5 is shown; Figure 5 The positive detection profile of PML-RARA L-type in Example 5 is shown; Figure 6 The detection spectrum of the positive typing reference standard substance is shown. DETAILED DESCRIPTION

[0038] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0040] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0041] Example 1 Primer Design Based on the latest leukemia fusion gene database and the typical fusion gene types mentioned in the WHO 2016 version of the blood tumor classification standard, detection primers for detecting leukemia fusion genes were designed to achieve qualitative detection of 121 fusion genes and 291 fusion subtypes of leukemia. The specific information of the detected fusion genes and fusion subtypes is shown in Table 1.

[0042] Table 1: Comparison of fusion genes and fusion subtypes

[0043] Primers are designed specifically based on the upstream and downstream sequences of the relevant fusion gene breakpoints. The following principles should be followed in primer design: (1) Target sequences with the same fusion gene should be grouped together for design. For example, genes and fusion forms involved in ABL1 gene fusion should be designed simultaneously in the first group of primer combinations to observe the possible breakpoint characteristics of related genes. If the breakpoints are close, a primer can be designed near the breakpoint sequence of the gene as a universal primer.

[0044] For example, ABL1-related fusion genes involve exon 2, exon 3, and exon 4 as 3' sequences and fusion with other paired genes. In this case, a universal primer can be designed on exon 4, which can take into account all ABL1 fusions as the 3' end.

[0045] The specific location of primer design also needs to take into account the criteria listed in (2) and (3).

[0046] (2) The length of the target sequence amplified by all primer pairs needs to be controlled between 75 and 600 bp. Within this length range, capillary electrophoresis detection can clearly and effectively distinguish a difference of 1 bp.

[0047] (3) The corresponding products generated by each group of primers with the same fluorescent label are at least 3 bp apart to facilitate the identification of the specific target fragments amplified.

[0048] (4) The Tm value of the primers designed in the primer combination needs to be around 60°C so that when combined with other paired primers, they can be efficiently amplified in an amplification system, avoid the generation of primer dimers, and improve the efficiency and specificity of multiplex amplification.

[0049] In each set of primer combinations, for the forward or reverse primers for amplification of each form of each specific fusion, it is necessary to label the fluorescent group described in the above group. The forward or reverse primers of the primers in the first group of the four major groups are labeled with FAM fluorescent group modification at the 5' end, and the forward or reverse primers of the primers in the second group of the four major groups are labeled with HEX fluorescent group modification at the 5' end.

[0050] Furthermore, the fluorescent group is labeled on the most common primers for amplification of multiple fusion genes as much as possible to reduce the cost of primer synthesis and reduce the fluorescence interference caused by too many fluorescent primers in amplification.

[0051] Based on the above primer design principle, for the one-time detection of 291 fusion subtypes formed by 121 fusion genes of leukemia, the specific primers and sequences designed by the present invention are shown in Table 2 below.

[0052] Table 2 Primer combination grouping table

[0053]

[0054]

[0055]

[0056]

[0057] Note: In the above table, the primer names marked with "—" indicate that the primers are fluorescently labeled at the 5' end. The fluorescent labeling group can be FAM, HEX, VIC, TAMRA, ROX or TEXRED. In the present invention, FAM and HEX are selected as fluorescent labels for different groups.

[0058] Example 2 Leukemia Fusion Gene Detection Kit A kit for detecting 121 fusion genes and 291 fusion subtypes of leukemia at one time, comprising the primer combination consisting of four primer groups described in Example 1, and also comprising: amplification enzyme mixture (PCR master MIX), positive typing reference standard material, negative control, and capillary electrophoresis internal standard reagent. The kit can be used to complete the detection of corresponding fusion genes through four groups of RT-PCR amplification systems.

[0059] The amplification enzyme mixture in the kit is a mixture of PCR amplification buffer, Mg2+, and hot start enzyme.

[0060] The positive typing reference standard substance is prepared by mixing the positive fragments obtained by PCR amplification of the positive plasmids of the 121 fusion genes and 291 fusion subtypes using the above primer combinations, and then forming four detection primer groups according to four primer groups. The positive typing reference standard substance is mainly a standard control substance used for the final detection result interpretation of the fusion gene.

[0061] The negative control was cDNA obtained by reverse transcription of normal human blood RNA.

[0062] Example 3 Leukemia fusion gene super multiplex RT-PCR detection method The method for qualitative detection of leukemia fusion gene super multiplex RT-PCR based on capillary electrophoresis fragment analysis comprises the following steps: 1. Obtain the cDNA template of the sample to be tested; 2. Amplification system configuration: using the 4 major primer mixtures in Example 1, the primers shown in SEQ ID NO.1-SEQ ID NO.48 constitute the first group of detection primers; the primers shown in SEQ ID NO.47-SEQ ID NO.84 constitute the second group of detection primers; the primers shown in SEQ ID NO.85-SEQ ID NO.134 constitute the third group of detection primers; the primers shown in SEQ ID NO.135-SEQ ID NO.189 and SEQ ID NO.47, SEQ ID NO.48 constitute the fourth group of detection primers, and the amplification enzyme mixture in the kit of Example 2, using the cDNA obtained in step 1 as a template, and performing PCR amplification in 4 tubes.

[0063] The total PCR amplification system in each tube (tubes 1-4) is 10 μL, and the amplification system is shown in Table 3.

[0064] Table 3 Amplification system

[0065] In the amplification system, the primer combination in Example 1 has a concentration of 1 pmol / μL for the primers (FAM group) shown in SEQ ID NO.1-SEQ ID NO.24 in the first group of the first large group, and a concentration of 1.5 pmol / μL for the primers (HEX group) shown in SEQ ID NO.25-SEQ ID NO.48 in the second group of the first large group; a concentration of 1.5 pmol / μL for the primers (HEX group) shown in SEQ ID NO.47-SEQ ID NO.64 in the first group of the second large group, and a concentration of 1 pmol / μL for the primers (FAM group) shown in SEQ ID NO.64-SEQ ID NO.84 in the second group of the second large group; a concentration of 1 pmol / μL for the primers (FAM group) shown in SEQ ID NO.85-SEQ ID NO.116 in the first group of the third large group, and a concentration of 1.5 pmol / μL for the primers (HEX group) shown in SEQ ID NO.117-SEQ ID NO.134 in the second group of the third large group; The concentration of the primers shown by ID NO.164 (FAM group) in the first subgroup of the fourth group was 1 pmol / μL, and the concentration of the primers shown by SEQ ID NO.165 to SEQ ID NO.189 (HEX group) in the second subgroup of the fourth group was 1.5 pmol / μL.

[0066] 3. PCR amplification: In order to minimize the generation of non-specific fragments during the multiplex PCR amplification process, the present invention adopts a touch down PCR amplification program for PCR amplification. The amplification program is shown in Table 4.

[0067] Table 4 Amplification procedures

[0068] 4. Capillary electrophoresis detection of PCR amplification products: Prepare a sample loading mixture containing molecular weight internal standard and formamide (the ratio of molecular weight internal standard and formamide is 1:500), and then calculate the number of tubes according to the number of PCR amplification products of the test sample (each test sample - containing negative control × 4 tubes of PCR product) + 4 positive typing reference standard substances. Use a pipette to dispense 9μL of the molecular weight internal standard and formamide mixture into each test well, take 1μL of PCR amplification product or 1μL of positive typing reference standard substance and add it to the mixture, cover with a sealing film, centrifuge instantly, and remove bubbles in the sample loading tube. Denature the loading plate at 95℃ for 5 minutes, and quickly ice bath for 2 minutes to completely denature the PCR product into single-stranded nucleic acid. Perform capillary electrophoresis detection on the machine according to the steps in the user manual of the genetic analyzer. The parameters are set as injection time set to 15s, injection voltage set to 3kV, and running time set to 2000s.

[0069] 5. Data Analysis Import relevant files into GeneMapper software, including Panel, Bin, corresponding Analysis Method, and Orange600 internal standard. Input sample source data (.fsa file), select the previously imported file in the relevant parameter selection column, and analyze the data. The establishment of the Bin file requires accurate position adjustment using positive typing reference standard substances.

[0070] 6. Interpretation of results (1) Interpretation of experimental validity: In the negative control test results, the target peak appeared only at the quality control position in the first, second, and third group amplification results, and the peak height was greater than 300 RFU, and there were no obvious non-specific peaks (peak height > 100 RUF) in other bins. In the fourth group amplification results, the target peak appeared at the quality control position, and the peak height was greater than 300 RFU. The target fragment positions for measuring gene expression, Hox11, HOX11L2, and EVI, may also have target fragment peaks, but the ratio of their peak heights to the quality control peak height is no more than 1 / 4.

[0071] (2) Interpretation of sample validity: For the test results of the sample to be tested, the target peak must appear at the quality control bin position in the amplification of groups 1 to 4, and the peak height must be no less than 300 RFU, indicating that the sample cDNA template is normal.

[0072] (3) Result interpretation: Under the premise that the experimental operation and samples are valid, the amplification results of the four groups of samples are analyzed. If a target peak higher than 100 RFU appears in the target bin other than the quality control and expression monitoring fusion types: Hox11, HOX11L2, and EVI positions, the fusion gene in the corresponding bin of the sample is judged to be positive; for the fusion types monitored for expression: Hox11, HOX11L2, and EVI bin positions, if a target peak appears, a peak height ratio is required. If the peak at the target gene position is greater than 1 / 4 of the peak at the quality control position, it indicates that the corresponding gene expression is positive.

[0073] Example 4 Primer combination and kit specific detection The HAL-01 cell line with TCF3-HLF gene fusion, the MV-4-11 cell line of human acute B lymphoblastic leukemia cell line with KMT2A-AF4 gene fusion, and the HL60 negative cell line without fusion gene were selected to verify the specificity of the system. The HAL-01 cell line, MV-4-11 cell line, and HL60 cell line were purchased from Hangzhou Hawk Biotechnology Co., Ltd.

[0074] The detection method is as follows: 1. Cell culture The purchased cell lines were cultured according to the standard cell culture operation and cultured in a 37°C, 5% CO2 incubator. After the cells reached about 5×10^5 cells / ml culture medium, the cell RNA was extracted.

[0075] 2. Cell RNA Extraction Use Tiangen Bio's RNA Easy Fast Animal Tissue Cell Total RNA Extraction Kit to extract RNA from three cell lines: HAL-01 cell line, MV-4-11 cell line, and HL60 cell line according to the kit instructions. And measure the concentration of RNA. The extracted RNA concentration needs to reach more than 10ng / μL, and the OD260 / OD280 should be between 1.9-2.1.

[0076] 3. Reverse transcription Promega GoScript™ Reverse Transcriptase Kit was used to obtain cDNA according to the kit instructions.

[0077] 4. Multiplex RT-PCR Amplification The four major groups of primer mixtures described in Example 1 were used to prepare the reaction solution according to the amplification system of Example 3; and a total PCR amplification system of four tubes (1-4 tubes) was prepared for the reverse transcribed cDNA of each cell line.

[0078] After the system is configured, PCR amplification is performed, and the amplification procedure is the same as in Example 3 5. Capillary electrophoresis detection Prepare a sample loading mixture containing molecular weight internal standard and formamide (the ratio of molecular weight internal standard and formamide is 1:500), and then calculate the number of tubes according to the number of PCR amplification products of the test samples (3 cell samples × 4 tubes of PCR products) + 4 positive typing reference standard substances. Use a pipette to dispense 9μL of the molecular weight internal standard and formamide mixture into each test well, take 1μL of PCR amplification product or 1μL of positive typing reference standard substance and add it to the mixture, cover with a sealing film, centrifuge instantly, and remove bubbles in the sample loading tube. Denature the loading plate at 95℃ for 5 minutes, and quickly ice bath for 2 minutes to completely denature the PCR product into single-stranded nucleic acid. Perform capillary electrophoresis detection on the machine according to the steps in the user manual of the genetic analyzer. The parameters are set as injection time of 15s, injection voltage of 3kV, and running time of 2000s.

[0079] 6. Data Analysis Import relevant files into GeneMapper software, including Panel, Bin, corresponding Analysis Method, and Orange600 internal standard. Input sample source data (.fsa file), select the previously imported file in the relevant parameter selection column, and analyze the data.

[0080] The results of the TCF3-HLF gene fusion test in the HAL-01 cell line are as follows Figure 1 As shown; the detection results of MV-4-11 cell line containing KMT2A-AF4 fusion are shown Figure 2 As shown; the test results of HL60 cell line without fusion gene are as follows Figure 3 shown.

[0081] from Figure 1 It can be seen that the TCF3-HLF gene fusion detection in the HAL-01 cell line showed a specific peak in the bin position of the E16insE4 fusion form of TCF3-HLF in the second group of amplification maps. The positive internal reference quality control in the amplification maps of groups 1-4 was also normal, and there were normal peak graphs at the positions of the quality control peaks, and there were no non-specific peaks at other positions.

[0082] from Figure 2 It can be seen that the detection results of the MV-4-11 cell line containing KMT2A-AF4 fusion also showed a specific peak in the bin position of the E10A4 fusion form of the KMT2A-AFF1 fusion gene in the second group of amplification maps. The positive internal reference quality control in the amplification maps of groups 1-4 was also normal, and there were normal peak graphs at the positions of the quality control peaks, and there were no non-specific peaks at other positions.

[0083] from Figure 3 It can be seen that the HL60 cell line does not contain the test results of the fusion gene. In amplification groups 1-4, except for the positive internal reference quality control, there are normal peak graphs at the quality control peak position, and there are no non-specific peaks at other positions.

[0084] The results showed that the results of the three cell lines verified using the kit were 100% consistent with the actual fusion status of the samples, demonstrating high specificity.

[0085] Example 5 Application of the kit in detecting leukemia fusion genes Using the super multiplex primer combination and kit for 121 leukemia fusion genes in Example 1, the 5 bone marrow samples with confirmed typing were tested using a fluorescent quantitative PCR detection platform.

[0086] The specific detection methods are as follows: 1. RNA extraction from bone marrow samples RNAprep Pure High-Efficiency Blood Total RNA Extraction Kit (DP443) was used to extract RNA from 5 clinical samples according to the kit instructions. The concentration of RNA was determined. The extracted RNA concentration needed to reach more than 50 ng / μL, and the OD260 / OD280 was between 1.9-2.1.

[0087] 2. Reverse transcription The present invention uses Promega GoScript™ Reverse Transcriptase kit and operates according to the kit instructions to obtain cDNA.

[0088] 3. Multiplex RT-PCR Amplification The four major primer mixtures described in Example 1 were used to prepare the reaction solution according to the same amplification system as in Example 3. The cDNAs of the reverse transcribed samples of the five confirmed typing samples and one negative control sample were subjected to a 4-tube (1-4) PCR amplification system.

[0089] After the system is configured, PCR amplification is performed, and the amplification procedure is the same as in Example 3.

[0090] 4. Capillary electrophoresis detection Prepare a sample loading mixture mixed with molecular weight internal standard and formamide (the mixing ratio of molecular weight internal standard and formamide is 1:500), and then calculate the number of tubes according to the number of PCR amplification products of the test samples (5 clinical samples + negative control) × 4 tubes of PCR products + 4 positive typing reference standard substances. Use a pipette to dispense 9μL of the molecular weight internal standard and formamide mixture into each test well, take 1μL of PCR amplification product or 1μL of positive typing reference standard substance and add it to the mixture, cover with a sealing film, centrifuge instantly, and remove bubbles in the sample loading tube. Denature the loading plate at 95℃ for 5 minutes, and quickly ice bath for 2 minutes to completely denature the PCR product into single-stranded nucleic acid. Perform capillary electrophoresis detection on the machine according to the steps in the user manual of the genetic analyzer. The parameters are set as injection time set to 15s, injection voltage set to 3kV, and running time set to 2000s.

[0091] 5. Data Analysis Import relevant files into GeneMapper software, including Panel, Bin, corresponding Analysis Method, and Orange600 internal standard. Input sample source data (.fsa file), select the previously imported file in the relevant parameter selection column, and analyze the data.

[0092] 6. Interpretation of results According to the data analysis spectrum interpretation criteria, 5 clinical samples were analyzed.

[0093] The results were as follows: 3 BCR-ABL1 P210 positive, 1 PML-RARA L type positive and 1 negative sample.

[0094] Figure 4 This is a BCR-ABL1 P210 positive pattern in one of the cases. Figure 4 It can be seen that a specific peak appears at the corresponding position of the first group of amplification maps, and a specific peak appears at the position of the sample quality control peak.

[0095] Figure 5 It is a PML-RARA L-type positive spectrum. Figure 5 It can be seen that specific peaks appear at the corresponding positions of the first group of amplification maps, which are specific peaks for sample quality control.

[0096] For interpretation criteria, please refer to the spectrum of positive typing reference standard material, such as Figure 6 As shown, a specific peak graph with a peak height RFU value ≥ 300 appears in the corresponding bin for the established bin, indicating that the fusion form of the fusion gene corresponding to the bin is positive.

[0097] The clinical test was performed using a fluorescent quantitative test kit that is currently recognized to have high sensitivity. The test results of this embodiment were consistent with the clinical results, with a compliance rate of 100%.

[0098] It can be seen that the leukemia fusion genes detected by the super-multiple primer combination and kit for detecting 121 fusion genes of leukemia of the present invention are consistent with the clinical diagnosis, indicating that the super-multiple primer combination, kit and detection method for detecting leukemia fusion genes based on capillary electrophoresis analysis of the present invention have high specificity and can be fully applied to the clinical comprehensive and qualitative screening and detection of 121 fusions and 291 fusion subtypes of leukemia. The detection method of the present invention is simple to operate, has strong specificity, is fast and efficient, has high throughput, is low cost, and has extremely high clinical application value.

[0099] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. Use of a primer combination for detecting leukemia fusion gene based on capillary electrophoresis fragment analysis in the preparation of a kit for detecting leukemia fusion gene, characterized in that: The primer combination consists of four primer groups: the first primer group includes the primer sequences shown in SEQ ID NO.1-SEQ ID NO.48; the second primer group includes the primer sequences shown in SEQ ID NO.47-SEQID NO.84; the third primer group includes the primer sequences shown in SEQ ID NO.85-SEQ ID NO.134; the fourth primer group includes the primer sequences shown in SEQ ID NO.47-SEQ ID NO.48 and SEQ ID NO.135-SEQ ID NO.

189. The primer combination can detect 291 fusion subtypes of 121 fusion genes of leukemia, and the fusion genes also include fusion gene types for monitoring expression levels.

2. The use according to claim 1, characterized in that: Each primer group in the primer combination is composed of two primer groups: the first primer group includes a first primer group composed of primer sequences shown in SEQ ID NO.1-SEQ ID NO.24 and a second primer group composed of primer sequences shown in SEQ ID NO.25-SEQ ID NO.48; the second primer group includes a first primer group composed of primer sequences shown in SEQ ID NO.47-SEQ ID NO.64 and a second primer group composed of primer sequences shown in SEQ ID NO.65-SEQ ID NO.84; the third primer group includes a first primer group composed of primer sequences shown in SEQ ID NO.85-SEQ ID NO.116 and a second primer group composed of primer sequences shown in SEQ ID NO.117-SEQ ID NO.134; the fourth primer group includes a first primer group composed of primer sequences shown in SEQ ID NO.135-SEQ ID NO.164 and a second primer group composed of primer sequences shown in SEQ ID NO.165-SEQ ID NO.189 and SEQ ID NO.47-SEQ ID NO. The second primer group is composed of the primer sequences shown in NO.48, wherein SEQ ID NO.47-SEQ IDNO.48 are the quality control primers of the first primer group, the second primer group and the fourth primer group, and SEQ ID NO.133-SEQ IDNO.134 are the quality control primers of the third primer group.

3. The use according to claim 2, characterized in that: The primer sequences in different primer groups of the first primer group, the second primer group, the third primer group and the fourth primer group are respectively labeled with different fluorescent markers; the fluorescent group of the fluorescent marker is selected from any one of FAM, HEX, VIC, TAMRA, ROX and TEXRED.

4. The use according to claim 3, characterized in that: The 5' ends of the primer sequences shown in SEQ ID NO.5, SEQ ID NO.15, SEQ ID NO.22, SEQ ID NO.27, SEQ ID NO.43, SEQ ID NO.46, and SEQ ID NO.47 in the first primer group are fluorescently labeled; the 5' ends of the primer sequences shown in SEQ ID NO.49, SEQ ID NO.59, SEQ ID NO.62, SEQ ID NO.64, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.70, SEQ ID NO.72, SEQ ID NO.74, SEQ ID NO.77, SEQ ID NO.79, SEQ ID NO.80, and SEQ ID NO.83 in the second primer group are fluorescently labeled; the 5' ends of the primer sequences shown in SEQ ID NO.85, SEQ ID NO.115, SEQ ID NO.116, SEQ ID NO.117, SEQ ID NO.124, SEQ ID NO.125, SEQ ID NO.128, SEQ ID NO.132, SEQ ID The 5' end of the primer sequence shown in NO.133 is fluorescently labeled; the 5' end of the primer sequences shown in SEQ ID NO.135, SEQ ID NO.137, SEQ ID NO.147, SEQ ID NO.154, SEQ ID NO.156, SEQ ID NO.163, SEQ ID NO.165, SEQ IDNO.168, SEQ ID NO.175, SEQ ID NO.181, SEQ ID NO.182, SEQ ID NO.183, SEQ ID NO.184, SEQ ID NO.47, SEQ ID NO.186, and SEQ ID NO.188 in the fourth primer group are fluorescently labeled, and the primer sequences in the same group have the same fluorescent label.

5. The use according to any one of claims 1 to 4, characterized in that: The kit also includes an amplification enzyme mixture, a positive typing reference standard substance, a negative control, and a capillary electrophoresis internal standard reagent.

6. The use according to claim 5, characterized in that: The positive typing reference standard material is prepared by using a primer combination for positive plasmids of 121 fusion genes and 291 fusion subtypes of leukemia: primers from SEQ ID NO.1 to SEQ ID NO.189 are used to perform PCR amplification to obtain positive fragments, and four primer groups are mixed to obtain the positive fragments.

7. The use according to claim 6, characterized in that: The amplification enzyme mixture is a PCR amplification buffer, Mg 2+ , a mixture of hot start enzymes, and the negative control is cDNA obtained by reverse transcription of normal human blood RNA.

8. Use of the kit containing the primer combination for detecting leukemia fusion gene according to any one of claims 1 to 7 in leukemia fusion gene typing detection, characterized in that: The application is to perform typing detection on leukemia fusion gene by capillary electrophoresis, comprising the following steps: (1) Obtain RNA from the sample to be tested and reverse transcribe it to obtain a cDNA template; (2) Configuration of amplification system: Four sets of primer mixtures were used: the primers shown in SEQ ID NO.1 to SEQ ID NO.48 constituted the first set of detection primers; The primers shown in SEQ ID NO.47-SEQ ID NO.84 constitute the second set of detection primers; The third group of detection primers is composed of the primers shown in SEQ ID NO.85-SEQ ID NO.134; the fourth group of detection primers is composed of the primers shown in SEQ ID NO.135-SEQ ID NO.189 and SEQ ID NO.47 and SEQ ID NO.48, and the PCR amplification buffer, Mg 2 + , an amplification mixture prepared by mixing the hot start enzyme, using the cDNA obtained in step (1) as a template, and performing PCR amplification in 4 tubes; (3) Use capillary electrophoresis to detect the amplified product: calculate the number of tubes according to the number of PCR amplified products of the test sample: test sample and negative control × 4 tubes of PCR product + 4 positive typing reference standard substances, use a pipette to dispense the capillary electrophoresis internal standard reagent into each detection well, then take the PCR amplified product or the positive typing reference standard substance and add it to the mixed solution, cover with a sealing film, and centrifuge instantly; denature the sample plate at 95℃ for 5 minutes, and quickly ice bath for 2 minutes; capillary electrophoresis detection machine: set the parameters as injection time 15s, injection voltage 3kV, and running time 2000s; During data analysis, positive typing reference standard substances were used to accurately adjust the position of the target bin; when a target peak higher than 100RFU appeared in a target bin other than the fusion type position for quality control and expression monitoring, the fusion gene in the corresponding bin of the sample was determined to be positive; for the fusion type for expression monitoring, if a target peak appeared in the bin position, a peak height ratio was required, and if the peak at the target gene position was greater than 1 / 4 of the peak at the quality control position, it indicated that the corresponding gene expression was positive.

9. The use according to claim 8, characterized in that: In the amplification system, the primer concentration of SEQ ID NO.1-24 is 1 pmol / μL, and the primer concentration of SEQ ID NO.25-48 is 1.5 pmol / μL; the primer concentration of SEQ ID NO.47-64 is 1.5 pmol / μL, and the primer concentration of SEQ ID NO.64-84 is 1 pmol / μL; the primer concentration of SEQ ID NO.85-116 is 1 pmol / μL, and the primer concentration of SEQ ID NO.117-134 is 1.5 pmol / μL; the primer concentration of SEQ ID NO.135-164 is 1 pmol / μL, and the primer concentration of SEQ ID NO.165-189 is 1.5 pmol / μL.

10. The use according to claim 8, characterized in that: The capillary electrophoresis internal standard reagent is prepared by mixing a molecular weight internal standard and formamide in a volume ratio of 1:500.

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