Application of a primer combination and kit for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis

Abstract

The present invention discloses the application of a primer combination for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis in the preparation of a kit for detecting leukemia fusion genes. The primer combination consists of four major primer groups: the first major primer group includes the sequences shown in SEQ ID NO.1-48; the second major primer group includes the sequences shown in SEQ ID NO.47-84; the third major primer group includes the sequences shown in SEQ ID NO.85-134; the fourth major primer group includes the sequences shown in SEQ ID NO.47-48 and SEQ ID NO.135-189, and can detect 291 fusion subtypes of 121 fusion genes of leukemia. The kit prepared by using the primer combination of the present invention utilizes 4 multiplex PCR amplification systems, with comprehensive site coverage, can detect multiple fusion subtypes in one tube, and has high detection sensitivity, high resolution, fast detection speed and strong specificity.

Description

[0001] Cross - reference to related applications

[0002] This application is a divisional application based on the application with application number 202410013547.9, application date January 3, 2024, and invention title: Primer combination, kit and method for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis. Technical field

[0003] The present invention belongs to the field of molecular biology, and specifically relates to a primer combination, a kit and a method for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis. Background technique

[0004] Leukemia is a disease of malignant clonal proliferation of bone marrow hematopoietic stem cells, which is particularly common in children and adolescents. Research shows that chromosomal structural aberrations exist in most leukemia patients, including chromosomal deletions, insertions, inversions, duplications, translocations, etc., which cause chromosomal rearrangements and form new fusion genes. Fusion genes are important driving factors in tumorigenesis and development. The formed fusion genes can activate the expression of proto - oncogenes, cause structural variations of tumor suppressor genes, lose their activity, or form new fusion proteins, resulting in abnormal transcriptional regulatory factors, thereby activating or enhancing downstream signaling pathways, ultimately promoting cell growth and proliferation, inhibiting cell differentiation, and thus leading to tumorigenesis. So far, leukemia involves at least hundreds of fusion genes. Leukemia fusion genes can be used as molecular biological specific markers and diagnostic bases for diagnosing different types of leukemia. For example, in chronic myeloid leukemia (CML) or some acute lymphoblastic leukemia (ALL): BCR - ABL1; in acute T - lymphoblastic leukemia (T - ALL): SIL - TAL1; in acute promyelocytic leukemia (APL): PML - RARA, etc.

[0005] There are more than a hundred fusion genes mentioned in the WHO 2016 edition of the classification criteria for hematological malignancies. The identification of fusion genes provides important molecular indicators for the diagnosis, classification, selection of clinical treatment plans, efficacy monitoring, and research and development of targeted drugs for hematological malignancies. The development of fusion gene detection technology in hematological malignancies has great clinical significance and market value.

[0006] Currently, the detection methods for fusion genes mainly include chromosomal karyotype analysis, fluorescence in situ hybridization (FISH), multiplex nested RT-PCR, real-time fluorescence quantitative PCR, digital PCR quantitative detection, and high-throughput sequencing detection. However, each of these methods has its own defects or drawbacks. Chromosomal karyotype analysis has a low resolution, with a minimum resolution limit of 5MB, and cannot detect fusion genes with chromosomal "microdeletions" or "microduplications". The FISH technique is characterized by complex operations, high labor costs, low throughput, and low resolution. Compared with chromosomal karyotype analysis and fluorescence in situ hybridization techniques, PCR detection methods have the advantages of being fast, simple to operate, and highly sensitive. However, although real-time fluorescence quantitative RT-PCR and digital RT-PCR have high detection sensitivity, their throughput is very low, and only 1-2 fusion forms can be detected in one tube, which is not suitable for use as a screening reagent for fusion genes in hematological diseases and is only suitable for minimal residual monitoring, etc. Although high-throughput sequencing technology has relatively high sensitivity and can also detect unknown fusions, its high cost, the need for very professional bioinformatics analysts, and long detection cycles limit its application in the detection of fusion genes.

[0007] Therefore, currently on the market, the method used for qualitative screening of multiple fusion genes in hematological diseases is multiplex nested RT-PCR technology, but this technology also has the following defects:

[0008] (1) For nested RT-PCR, at least two rounds of PCR amplification are required. After the products generated by the first round of amplification are opened and diluted, they are used as amplification templates for the second round of amplification to obtain specific target fragments. The addition of this experimental process of diluting the PCR products and returning them to the template processing room for re-amplification not only increases the complexity of the experiment but also greatly increases the probability of contamination and does not conform to the smooth progress of the routine operation procedures in a PCR laboratory.

[0009] (2) Currently, the final products obtained by nested PCR are all analyzed by agarose gel electrophoresis. Whether there are corresponding target fragments and the lengths of the target fragments are used to determine whether there are corresponding fusion genes. Since the resolution of agarose gel electrophoresis for amplified fragments is 50-100bp, this greatly reduces the number of fragments that can be amplified multiplexly in each tube, and at most 5-10 amplified fragments can be performed in each tube of amplification.

[0010] (3) Also due to the resolution limitation of agarose gel electrophoresis, it is very difficult for this technology to specifically distinguish different fusion forms of the same fusion gene. For example, the PML-RARA fusion form has S type, V type, and L type. The breakpoints between the S type and the V type are relatively close, and the fragments between some fusion forms differ by less than 100bp. It is very difficult to determine the specific fusion form through agarose gel electrophoresis.

[0011] (4)For gene fusions that result in increased gene expression levels, such as enhanced expression of Hox11, HOX11L2, and EVI genes, it is also very difficult to achieve detection by such methods.

[0012] With the continuous in-depth study of leukemia, more and more leukemia fusion genes have been discovered. Currently, the methods for detecting leukemia fusion genes far cannot meet the market demand for a one-time screening of most fusion genes. There is an urgent need to develop a rapid, convenient, effective, accurate, and easily standardized detection method and reagent for hematological fusion genes. Summary of the Invention

[0013] To solve one of the above problems, the present invention provides a primer combination, kit, and method for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis. The detection method of the present invention is simple to operate, highly specific, rapid and efficient, has a high throughput, and low cost, and can qualitatively detect 291 fusion subtypes of 121 fusion genes in leukemia at one time.

[0014] To achieve the above object, the present invention adopts the following technical means:

[0015] 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 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 - SEQ ID 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 in leukemia, and the fusion genes also include the fusion gene types for monitoring the expression level.

[0016] In some embodiments of the present invention, each primer group in the primer combination is respectively composed of two primer subgroups: the first primer group includes a first primer subgroup composed of primer sequences shown in SEQ ID NO.1 - SEQ ID NO.24 and a second primer subgroup composed of primer sequences shown in SEQ ID NO.25 - SEQ ID NO.48; the second primer group includes a first primer subgroup composed of primer sequences shown in SEQ ID NO.47 - SEQ ID NO.64 and a second primer subgroup composed of primer sequences shown in SEQ ID NO.65 - SEQ ID NO.84; the third primer group includes a first primer subgroup composed of primer sequences shown in SEQ ID NO.85 - SEQ ID NO.116 and a second primer subgroup composed of primer sequences shown in SEQ ID NO.117 - SEQ ID NO.134; the fourth primer group includes a first primer subgroup composed of primer sequences shown in SEQ ID NO.135 - SEQ ID NO.164 and a second primer subgroup composed of primer sequences shown in SEQ ID NO.165 - SEQ ID NO.189 and SEQ ID NO.47 - SEQ ID NO.48, wherein SEQ ID NO.47 - SEQ ID NO.48 are the quality control primers for the first primer group, the second primer group and the fourth primer group, and SEQ ID NO.133 - SEQ ID NO.134 are the quality control primers for the third primer group.

[0017] In some embodiments of the present invention, the primer sequences in different primer subgroups of the first primer group, the second primer group, the third primer group and the fourth primer group are respectively labeled with different fluorescent labels; the fluorescent group of the fluorescent label is selected from any one of FAM, HEX, VIC, TAMRA, ROX, TEXRED. Preferably, the fluorescent group is labeled on the primers with the most common amplification of various fusion genes as much as possible, reducing the primer synthesis cost and the fluorescence interference caused by too many fluorescent primers in the amplification.

[0018] In some embodiments of the present invention, 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, and SEQ ID NO.133 in the third primer group 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 fluorescent labels of the primer sequences in the same subgroup are the same.

[0019] 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, SEQ ID NO.147, SEQ ID NO.154, SEQ ID NO.156, SEQ ID NO.163, SEQ ID NO.165, SEQ ID NO.168 are universal primers.

[0020] The second aspect of the present invention provides a kit for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis, including the primer combination described in the first aspect of the present invention.

[0021] In some embodiments of the present invention, the kit further includes an amplification enzyme mixture, a positive genotyping reference standard substance, a negative control, and a capillary electrophoresis internal standard reagent.

[0022] In some embodiments of the present invention, the positive genotyping reference standard substance is prepared by performing PCR amplification on positive plasmids of 291 fusion subtypes of 121 leukemia fusion genes with primer combinations: primers of SEQ ID NO.1 to SEQ ID NO.189 to obtain positive fragments, and then mixing them according to four major primer groups.

[0023] In some embodiments of the present invention, the amplification enzyme mixture is a mixed solution composed of a PCR amplification buffer, Mg 2+ , and a hot start enzyme mixture, and the negative control is cDNA reverse transcribed from normal human blood RNA.

[0024] The third aspect of the present invention provides a method for detecting leukemia fusion genes based on capillary electrophoresis fragment analysis, including the following steps:

[0025] (1) Obtaining a cDNA template of the sample to be tested;

[0026] (2) Mixing the primer combinations described in the first aspect of the present invention according to four major primer groups to obtain four detection primer groups, and performing multiplex PCR amplification in four groups to obtain amplification products;

[0027] (3) Detecting the amplification products by capillary electrophoresis;

[0028] In data analysis, a positive genotyping reference standard substance is used to accurately adjust the position of the target bin; when a target peak higher than 100 RFU appears on the target bin other than the positions of the quality control and the fusion types for monitoring the expression level, it is determined that the fusion gene on the corresponding bin of the sample is positive; for the fusion types for monitoring the expression level, when a target peak appears at the bin position, the peak height ratio needs to be calculated. 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] In some embodiments of the present invention, the amplification systems for the four groups of PCR amplifications are as follows: 2 μL of 5×PCR master MIX, 1 μL of the detection primer set (Primer mix), 2 μL of the cDNA to be tested, 2 μL of deionized pure water, and the total volume of the system is 10 μL. Among them, the primer sets shown in SEQ ID NO.1 - SEQ ID NO.48 form the first group of detection primer sets; the primer sets shown in SEQ ID NO.47 - SEQ ID NO.84 form the second group of detection primer sets; the primer sets shown in SEQ ID NO.85 - SEQ ID NO.134 form the third group of detection primer sets; the primer sets shown in SEQ ID NO.135 - SEQ ID NO.189 and SEQ ID NO.47, SEQ ID NO.48 form the fourth group of detection primer sets.

[0030] In some specific embodiments of the present invention, in order to minimize the generation of non-specific fragments during multiplex PCR amplification, a touchdown PCR amplification program is used for PCR amplification. 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 10 s, annealing at 66°C for 30 s, decreasing 1°C for each cycle, and extension at 70°C for 40 s; denaturation to extension for 25 cycles: denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 70°C for 40 s; post-amplification extension at 60°C for 30 min for 1 cycle; the amplified product is stored at 4°C or 16°C.

[0031] In some embodiments of the present invention, in the amplification system, the primer concentrations of SEQ ID NO.1 - SEQ ID NO.24 are all 1 pmol / μL, 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, 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, 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, the primer concentrations of SEQ ID NO.165 - SEQ ID NO.189 are 1.5 pmol / μL.

[0032] In some specific embodiments of the present invention, the method for capillary electrophoresis detection of PCR amplification products is as follows:

[0033] Prepare a loading mixture mixed with a molecular weight internal standard and formamide (the mixing ratio of the molecular weight internal standard and formamide is 1:500). Then, calculate the number of tubes according to the number of PCR amplification products of the test sample (each test sample - including the negative control × 4 tubes of PCR products) + 4 positive genotyping reference standards. Use a pipette to dispense 9 μL of the mixture of the molecular weight internal standard and formamide into each detection well. Take 1 μL of the PCR amplification product or 1 μL of the positive genotyping reference standard and add it to the mixture. Cover with a sealing film and centrifuge instantaneously to remove the bubbles in the sample loading tube. Denature the loading plate at 95°C for 5 minutes and then quickly ice-bath for 2 minutes to completely denature the PCR products into single-stranded nucleic acids. Perform capillary electrophoresis detection on the genetic analyzer according to the steps in the user manual. The parameter settings are as follows: the injection time is set to 15 s, the injection voltage is 3 kV, and the running time is 2000 s.

[0034] In some specific embodiments of the present invention, the result interpretation scheme is as follows:

[0035] (1) Interpretation of experimental validity: Detection results of negative controls. Among the amplification results of the first group, the second group, and the third group, only the target peak appears at the quality control position, and the peak height value is greater than 300 RFU, and there are no obvious non-specific peaks (peak height > 100 RUF) on other bins; for the amplification result of the fourth group, the target peak appears at the quality control position, and the peak height is greater than 300 RFU. There may also be target fragment peaks at the positions of the target fragments Hox11, HOX11L2, and EVI for measuring gene expression, but the ratio of their peak heights to the quality control peak height is not higher than 1 / 4.

[0036] (2)Interpretation of sample validity: For the test results of the sample to be tested, in the amplification of the first to fourth groups, the target peak must appear at the quality control bin position, and the peak height must not be lower than 300 RFU, indicating that the cDNA template of the sample is normal.

[0037] (3)Result interpretation: On the premise of proper experimental operation and valid samples, analyze the amplification results of the four groups of samples. If a target peak higher than 100 RFU appears at the target bin other than the quality control and the fusion types for monitoring expression levels: Hox11, HOX11L2, and EVI positions, it is determined that the fusion gene at the corresponding bin of the sample is positive; for the bin positions of the fusion types for monitoring expression levels: Hox11, HOX11L2, and EVI, if a target peak appears, the peak height ratio needs to be calculated. 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.

[0038] Advantages of the present invention

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] (1)Through one ultra-multiplex PCR amplification system, it is possible to simultaneously amplify the fusion types of multiple fusion genes:

[0041] This invention patent utilizes four multiplex PCR amplification systems to achieve the detection of 291 fusion subtypes of 121 fusion genes in leukemia. Its advantages are as follows:

[0042] ①Comprehensive site coverage: The fusion genes detected by this kit basically cover the fusion types that can be detected in the transcribed cDNA included in the WHO 2016 edition of the classification criteria for hematological malignancies, including common fusion types in various types of leukemia such as chronic / acute myeloid leukemia (CML / AML), acute B-lymphocyte and T-lymphocyte leukemia (B- / T-ALL), myeloid / lymphoid leukemia with eosinophilia, and acute promyelocytic leukemia (APL). It can qualitatively screen whether leukemia patients have fusion genes at one time, providing more information for clinicians.

[0043] ②Greatly reduce the operation intensity and detection cost. On average, one tube of PCR multiplex amplification can detect more than 70 fusion subtypes. Compared with traditional agarose gel electrophoresis, one tube can detect at most 5 - 10 fusion subtypes, and the number of fusion subtypes that can be detected in one tube is 10 - 15 times more than that of the conventional method.

[0044] (2)Use a genetic analyzer to detect the amplification products by capillary electrophoresis:

[0045] The detection platform adopted by the present invention is a widely used detection platform, which detects the amplification products by capillary electrophoresis. The main advantages are as follows:

[0046] ① High detection sensitivity

[0047] The combination of QF-PCR and capillary electrophoresis greatly improves the detection sensitivity, which is more than 100 times higher than that of agarose electrophoresis. It can sensitively detect the amplification products and clearly distinguish specific fusion subtypes. On the other hand, due to the high detection sensitivity, the requirement for the amount of PCR amplification products is reduced. It is possible to reach the detection limit without nested PCR, which can further reduce the detection cost, simplify the operation process and avoid contamination, etc.

[0048] ② High detection resolution

[0049] Usually, within the detection range of 70 - 600bp, the difference of 1bp can be clearly and effectively distinguished. Such a high fragment length resolution enables the product sizes separated by 2 - 3bp to be clearly resolved, preventing misjudgment of different positive fusion types. At the same time, the high resolution also makes it possible to detect more fusion subtypes in the same fluorescence channel.

[0050] ③ The detection results can be quantified

[0051] The detection results have specific peak heights with specific numerical displays, and there is a linear corresponding relationship between the peak height and the initial amount of the template within a certain range. It can be compared with the internal reference (quality control gene) to a certain extent to achieve the detection of the fusion type of gene overexpression caused by the fusion gene.

[0052] ④ Six fluorescence signals can be detected simultaneously

[0053] The genetic analysis instrument can simultaneously receive six fluorescence signals, and there is no great mutual interference between different fluorescence signals. One channel is used to detect the molecular internal standard, and the other five channels can detect different fusion subtypes of the sample. In this patent, only two channels are shown to detect the sample, and actually more gene fusions can be detected in one tube using this method.

[0054] ⑤ Fast detection speed

[0055] Automated large - batch detection can be carried out: the capillary electrophoresis process can complete the detection in 40 minutes. According to the amount of samples detected at one time, 16 wells, or 32 wells, or 96 wells can be detected at one time.

[0056] ⑥ The detection results can be automatically analyzed and interpreted using analysis software. Description of the Drawings

[0057] Figure 1Shows the detection map of the TCF3-HLF gene fusion in the HAL-01 cell line in Example 4;

[0058] Figure 2 Shows the detection map of the KMT2A-AF4 fusion in the MV-4-11 cell line in Example 4;

[0059] Figure 3 Shows the detection map of the absence of fusion genes in the HL60 cell line in Example 4;

[0060] Figure 4 Shows the positive detection map of BCR-ABL1 P210 in Example 5;

[0061] Figure 5 Shows the positive detection map of PML-RARA L type in Example 5;

[0062] Figure 6 Shows the detection map of the positive typing reference standard substance. Detailed implementation mode

[0063] The following examples are used here to demonstrate the preferred implementation modes of the present invention. Those skilled in the art will understand that the technologies disclosed in the following examples represent the technologies discovered by the inventors that can be used to implement the present invention, and therefore can be regarded as the preferred solutions for implementing the present invention. However, those skilled in the art should understand according to this specification that many modifications can be made to the specific embodiments disclosed here, and still obtain the same or similar results, without departing from the spirit or scope of the present invention.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The materials cited herein and their citations will be incorporated by reference. Those skilled in the art will realize or through routine experimentation will understand many equivalent technologies of many specific embodiments of the invention described here. These equivalents will be included in the claims.

[0065] The technical solutions of this patent will be further described in detail below in conjunction with the specific implementation modes.

[0066] Example 1 Primer design

[0067] Based on the latest leukemia fusion gene database and the typical fusion gene types mentioned in the WHO 2016 edition of the classification criteria for hematological malignancies, detection primers for detecting leukemia fusion genes are designed to achieve qualitative detection of 291 fusion subtypes of 121 fusion genes in leukemia. The specific situations of the detected fusion genes and fusion subtypes are shown in Table 1.

[0068] Table 1: Comparison of fusion genes and fusion subtypes

[0069]

[0070] 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:

[0071] (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.

[0072] 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.

[0073] The specific location of primer design also needs to take into account the criteria listed in (2) and (3).

[0074] (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.

[0075] (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.

[0076] (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.

[0077] 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.

[0078] Furthermore, the fluorescent group is preferably labeled on the primers that are most commonly used for the amplification of various fusion genes, so as to reduce the primer synthesis cost and the fluorescence interference caused by too many fluorescent primers during amplification.

[0079] Based on the above primer design principle, for the 291 fusion subtypes formed by the one-time detection of 121 leukemia fusion genes, the specific primers and sequences designed by the present invention are shown in Table 2 below.

[0080] Table 2 Grouping of primer combinations

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Note: In the above table, the primers marked with "-" indicate that the primer is 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 the fluorescent labels for different groups.

[0087] Example 2 Leukemia fusion gene detection kit

[0088] A kit that can be used for the one-time detection of 291 fusion subtypes of 121 leukemia fusion genes, which contains the primer combination composed of four major primer groups described in Example 1, and also includes: an amplification enzyme mixture (PCR master MIX), a positive typing reference standard substance, a negative control, and a capillary electrophoresis internal standard reagent. Using this kit, the detection of the corresponding fusion genes can be completed through four groups of RT-PCR amplification systems.

[0089] The amplification enzyme mixture in the kit is composed of a PCR amplification buffer, Mg2+, and a hot start enzyme.

[0090] The positive typing reference standard substance is prepared by mixing the positive fragments obtained by PCR amplification of the positive plasmids of the 291 fusion subtypes of the 121 fusion genes with the above primer combination, and then forming four detection primer groups according to the four major primer groups and mixing them respectively. The positive typing reference standard substance is mainly a standard control substance for the final determination of the fusion gene detection results.

[0091] The negative control is cDNA reverse transcribed from normal human blood RNA.

[0092] Example 3 Detection Method for Super-Multiplex RT-PCR of Leukemia Fusion Genes

[0093] The method for qualitatively detecting super-multiplex RT-PCR of leukemia fusion genes based on capillary electrophoresis fragment analysis comprises the following steps:

[0094] 1. Obtain the cDNA template of the sample to be tested;

[0095] 2. Configure the amplification system: Use the four major groups of primer mixtures in Example 1. The primers shown in SEQ ID NO.1 - SEQ ID NO.48 form the first group of detection primers; the primers shown in SEQ ID NO.47 - SEQ ID NO.84 form the second group of detection primers; the primers shown in SEQ ID NO.85 - SEQ ID NO.134 form 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 form the fourth group of detection primers, and mix with the amplification enzyme mixture in the kit of Example 2. Using the cDNA obtained in step 1 as the template, perform PCR amplification in 4 tubes.

[0096] The total volume of the PCR amplification system for each tube (tubes 1 - 4) is 10 μL, and the amplification system is shown in Table 3.

[0097] Table 3 Amplification System

[0098]

[0099] In the primer combination of Example 1 in the amplification system, the concentration of the primers shown in SEQ ID NO.1 - SEQ ID NO.24 (FAM group) in the first subgroup of the first major group is 1 pmol / μL, and the concentration of the primers shown in SEQ ID NO.25 - SEQ ID NO.48 (HEX group) in the second subgroup of the first major group is 1.5 pmol / μL; the concentration of the primers shown in SEQ ID NO.47 - SEQ ID NO.64 (HEX group) in the first subgroup of the second major group is 1.5 pmol / μL, and the concentration of the primers shown in SEQ ID NO.64 - SEQ ID NO.84 (FAM group) in the second subgroup of the second major group is 1 pmol / μL; the concentration of the primers shown in SEQ ID NO.85 - SEQ ID NO.116 (FAM group) in the first subgroup of the third major group is 1 pmol / μL, and the concentration of the primers shown in SEQ ID NO.117 - SEQ ID NO.134 (HEX group) in the second subgroup of the third major group is 1.5 pmol / μL; the concentration of the primers shown in SEQ ID NO.135 - SEQ ID NO.164 (FAM group) in the first subgroup of the fourth major group is 1 pmol / μL, and the concentration of the primers shown in SEQ ID NO.165 - SEQ ID NO.189 (HEX group) in the second subgroup of the fourth major group is 1.5 pmol / μL.

[0100] 3. PCR amplification: In order to minimize the generation of non-specific fragments during the multiplex PCR amplification process, the present invention uses the Touch down PCR amplification program for PCR amplification, and the amplification program is shown in Table 4.

[0101] Table 4 Amplification program

[0102]

[0103] 4. Capillary electrophoresis detection of PCR amplification products:

[0104] Prepare a loading mixture mixed with a molecular weight internal standard and formamide (the mixing ratio of the molecular weight internal standard and formamide is 1:500). Then, calculate the number of tubes according to the number of PCR amplification products of the test sample (for each test sample - including the negative control × 4 tubes of PCR products) + 4 positive genotyping reference standards. Use a pipette to dispense 9 μL of the mixture of the molecular weight internal standard and formamide into each test well. Take 1 μL of the PCR amplification product or 1 μL of the positive genotyping reference standard and add it to the mixture. Cover with a sealing film and centrifuge instantaneously to remove the bubbles in the sample loading tube. Denature the sample loading plate at 95 °C for 5 minutes and then quickly ice-bath for 2 minutes to completely denature the PCR products into single-stranded nucleic acids. Perform capillary electrophoresis detection on the genetic analyzer according to the steps in the user manual. The parameter settings are: the injection time is set to 15 s, the injection voltage is 3 kV, and the running time is 2000 s.

[0105] 5. Data Analysis

[0106] Import relevant files into the GeneMapper software, including Panel, Bin, the corresponding Analysis Method, and the Orange600 internal standard. Input the sample source data (.fsa file), select the previously imported files in the relevant parameter selection column, and analyze the data. The establishment of the Bin file requires accurate position adjustment using the positive genotyping reference standard.

[0107] 6. Result Interpretation

[0108] (1) Interpretation of experimental validity: For the detection results of the negative control, in the amplification results of the first group, the second group, and the third group, only the target peak appears at the quality control position, and the peak height value is greater than 300 RFU, and there are no obvious non-specific peaks (peak height > 100 RUF) on other positions of the bin; for the amplification result of the fourth group, the target peak appears at the quality control position, and the peak height is greater than 300 RFU. There may also be target fragment peaks at the positions of the target fragments Hox11, HOX11L2, and EVI for measuring gene expression, but the ratio of their peak heights to the quality control peak height is not higher than 1 / 4.

[0109] (2) Interpretation of sample validity: For the detection results of the test samples, in the amplification of the first to fourth groups, the target peak must appear at the quality control bin position, and the peak height is not lower than 300 RFU. This indicates that the sample cDNA template is normal.

[0110] (3)Result interpretation: On the premise of valid experimental operations and samples, analyze the amplification results of the 4 groups of samples. If a target peak higher than 100 RFU appears on the target bin except for the fusion types of quality control and monitoring expression levels: Hox11, HOX11L2, and EVI positions, it is determined that the fusion gene on the corresponding bin of the sample is positive; for the fusion types of monitoring expression levels: Hox11, HOX11L2, and EVI bin positions where a target peak appears, the peak height ratio needs to be calculated. 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.

[0111] Example 4 Specificity Detection of Primer Combinations and Kits

[0112] Select the HAL-01 cell line determined to contain the TCF3-HLF gene fusion, the MV-4-11 cell line of human acute B-lymphoblastic leukemia cell line containing the KMT2A-AF4 gene fusion, and the HL60 negative cell line without the fusion gene to verify the specificity of the system. Among them, the HAL-01 cell line, MV-4-11 cell line, and HL60 cell line were all purchased from Hangzhou Hulk Biotechnology Co., Ltd.

[0113] The detection method is as follows:

[0114] 1. Cell culture

[0115] Culture the above-mentioned purchased cell lines according to the standard operation of cell culture, and place them in a 37°C, 5% CO2 incubator. After culturing to about 5×10^5 cells / ml of the medium, perform cell RNA extraction.

[0116] 2. Cell RNA extraction

[0117] Use the RNA Easy Fast Total RNA Extraction Kit for Animal Tissues and Cells from Tiangen Biotech to extract the RNA of the 3 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 the RNA. The concentration of the extracted RNA needs to reach more than 10 ng / μL, and the OD260 / OD280 is between 1.9 - 2.1.

[0118] 3. Reverse transcription

[0119] Use the Promega GoScript TM Reverse Transcriptase Kit and operate according to the kit instructions to obtain cDNA.

[0120] 4. Multiplex RT-PCR amplification

[0121] Use the primer mixture of the 4 large groups described in Example 1 to prepare the reaction solution according to the amplification system of Example 3; perform PCR amplification on the cDNA reverse-transcribed from each cell line in 4 tubes (tubes 1-4) for the total system preparation.

[0122] After the system is prepared, perform PCR amplification, and the amplification procedure is the same as that in Example 3.

[0123] 5. Capillary electrophoresis detection

[0124] Prepare a loading mixture containing a molecular weight internal standard and formamide (the mixing ratio of the molecular weight internal standard and formamide is 1:500). 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 genotyping reference standards. Use a pipette to dispense 9 μL of the mixture of the molecular weight internal standard and formamide into each detection well. Take 1 μL of the PCR amplification product or 1 μL of the positive genotyping reference standard and add it to the mixture. Cover with a sealing film and centrifuge briefly to remove the bubbles in the sample loading tube. Denature the loading plate at 95 °C for 5 minutes and then quickly ice-bath for 2 minutes to completely denature the PCR products into single-stranded nucleic acids. Perform capillary electrophoresis detection and load onto the machine according to the steps in the user manual of the genetic analyzer. The parameter settings are: injection time is set to 15 s, injection voltage is 3 kV, and running time is 2000 s.

[0125] 6. Data analysis

[0126] Import relevant files into the GeneMapper software, including Panel, Bin, the corresponding Analysis Method, and Orange600 internal standard. Input the sample source data (.fsa file), select the previously imported files in the relevant parameter selection column, and analyze the data.

[0127] The detection results of the TCF3-HLF gene fusion in the HAL-01 cell line are as Figure 1 shown; the detection results of the KMT2A-AF4 fusion in the MV-4-11 cell line are as Figure 2 shown; the detection results of the absence of fusion genes in the HL60 cell line are as Figure 3 shown.

[0128] From Figure 1 it can be seen that in the detection of the TCF3-HLF gene fusion in the HAL-01 cell line, a specific peak appears at the bin position of the E16insE4 fusion form of TCF3-HLF in the second group of amplification maps. The positive internal reference quality control is also normal in the first to fourth group of amplification maps, and normal peak maps also appear at the positions of the quality control peaks, and there are no non-specific peaks at other positions.

[0129] From Figure 2It can be seen that for the detection result of the MV-4-11 cell line containing the KMT2A-AF4 fusion, a specific peak also appeared at the bin position of the E10A4 fusion form of the KMT2A-AFF1 fusion gene in the amplification map of the second group. The positive internal reference quality control in the amplification maps of groups 1-4 was also normal, and normal peak maps were present at the positions of the quality control peaks, with no non-specific peaks at other positions.

[0130] From Figure 3 It can be seen that for the detection result of the HL60 cell line not containing the fusion gene, in the amplification of groups 1-4, except for the normal positive internal reference quality control and normal peak maps at the positions of the quality control peaks, there were no non-specific peaks at other positions.

[0131] The results showed that the verification results of the three cell lines using the kit were 100% consistent with the actual fusion conditions of the samples, showing high specificity.

[0132] Example 5 Application of the Kit in Detecting Leukemia Fusion Genes

[0133] Using the primer combination and kit of the 121 leukemia fusion genes super multiplex in Example 1, 5 bone marrow samples with determined typing were detected using a fluorescence quantitative PCR detection platform.

[0134] The specific detection method is as follows:

[0135] 1. RNA extraction from bone marrow samples

[0136] Use the RNAprep Pure High Efficiency Total RNA Extraction Kit for Blood (DP443) to extract the RNA of 5 clinical samples according to the kit instructions. And measure the concentration of the RNA. The concentration of the extracted RNA needs to reach more than 50 ng / μL, and the OD260 / OD280 is between 1.9 and 2.1.

[0137] 2. Reverse transcription

[0138] The present invention uses the Promega GoScript TM Reverse Transcriptase Kit and operates according to the kit instructions to obtain cDNA.

[0139] 3. Multiplex RT-PCR amplification

[0140] Use the primer mixture of the 4 large groups described in Example 1 to configure the reaction solution according to the same amplification system as in Example 3. Configure the total PCR amplification system for 4 tubes (tubes 1-4) for the cDNA reverse transcribed from 5 samples with determined typing and a negative control sample.

[0141] After the system is configured, perform PCR amplification, and the amplification program is the same as in Example 3.

[0142] 4. Capillary electrophoresis detection

[0143] 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.

[0144] 5. Data Analysis

[0145] 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.

[0146] 6. Interpretation of results

[0147] According to the data analysis spectrum interpretation criteria, 5 clinical samples were analyzed.

[0148] The results were as follows: 3 BCR-ABL1 P210 positive, 1 PML-RARA L type positive and 1 negative sample.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] Clinically, a fluorescence quantitative kit with high sensitivity recognized at present is used for detection. The detection results in this example are consistent with the clinical results, and the coincidence rate is 100%.

[0153] Thus, it can be seen that the leukemia fusion genes detected by using the primer combination and kit for ultra-multiplex detection of 121 leukemia fusion genes of the present invention are consistent with the clinical diagnosis, indicating that the primer combination, kit and detection method for ultra-multiplex detection of leukemia fusion genes based on capillary electrophoresis analysis of the present invention have high specificity and can be fully applied to the comprehensive and qualitative screening detection of 291 fusion subtypes of 121 leukemia fusion genes clinically. The detection method of the present invention is simple to operate, highly specific, fast and efficient, has high throughput and low cost, and has extremely high clinical application value.

[0154] All the documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a reference. 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 by this 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 the positive fragments amplified using the primers in the four primer groups are mixed and then prepared.

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.

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