Primer group, kit and method for detecting UBTF-ATXN7L3 fusion gene

Abstract

The invention discloses a primer group for detecting a UBTF-ATXN7L3 fusion genotype, the primer group comprises a pair of detection primers and a detection probe, a super-quenching probe labeling technology is adopted at a specific base position of the detection probe, the length of the probe is controlled to be 35-40bp and is about 50% longer than that of a conventional probe, and the detection sensitivity is high. The sensitivity is also improved to a certain extent while the specificity is remarkably improved, the signal-to-noise ratio reaches the lowest in similar products, background signals are basically eliminated, the detection sensitivity reaches 1 * 10 < 1 > copies / mL, and the requirements of clinical low signal-to-noise ratio and low copy template detection on molecular diagnostic reagents can be completely met. Differential diagnosis analysis can not be carried out in a typing stage at the early stage of a disease course, and detection of the UBTF-ATXN7L3 fusion gene is realized when the expression quantity is low, so that the clinical diagnosis and prognosis judgment requirements of leukemia patients are met.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and more specifically, to a primer set, a kit and a method for detecting the UBTF-ATXN7L3 fusion gene in patients with precursor B-cell acute lymphoblastic leukemia (BCP-ALL). Background Art

[0002] Leukemia, also known as blood cancer, is a malignant tumor of the hematopoietic system. The source of the disease is the abnormal functioning of the hematopoietic tissue in the bone marrow due to the mutation of intracellular deoxyribonucleic acid. Stem cells in the bone marrow can produce thousands of red blood cells and white blood cells every day. Leukemia patients overproduce immature white blood cells, which interferes with other work of the bone marrow, which reduces the function of the bone marrow to produce other blood cells. Leukemia can spread to the lymph nodes, spleen, liver, central nervous system and other organs. Leukemia ranks first among malignant diseases in young people. There are many types of leukemia. Clinically, it is generally divided into acute leukemia and chronic leukemia. Including acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), young myelomonocytic leukemia (JML), and adult T-cell lymphocytic leukemia (ATL). The most common types of leukemia in adults are acute myeloid leukemia and chronic myeloid leukemia, while the most common type of leukemia in children is acute lymphocytic leukemia.

[0003] There is an acute lymphoblastic leukemia called B-precursor ALL (BCP-ALL), which originates from the malignant transformation of certain white blood cells and leads to a rapid and progressive decrease in bone marrow function, thereby disrupting blood formation. If not treated in time, it can cause the patient to die in a short period of time. Recent studies have shown that in more than 90% of cases, the molecular mechanism behind the disease is determined to be the gene fusion of two genes, UBTF and ATXN7L3, which leads to the occurrence of BCP-ALL.

[0004] Currently, the available detection methods for UBTF-ATXN7L3 fusion gene include high-throughput transcriptome sequencing analysis, Northern blot, fluorescence in situ hybridization (FISH), and real-time fluorescence quantitative PCR (qPCR). Compared with high-throughput transcriptome sequencing analysis, Northern blot, and fluorescence in situ hybridization (FISH), real-time fluorescence quantitative PCR (qPCR) has the advantages of simple operation, rapidity, and accuracy, and is currently a common method for detecting fusion genes. Since the precursor genes UBTF and ATXN7L3 of the UBTF-ATXN7L3 fusion gene are adjacent genes, nonspecific signals are easily generated during detection, resulting in false positive results; at the same time, the expression level of the UBTF-ATXN7L3 fusion gene is low in the early stage of the disease, less than 1×10 1 The expression of UBTF-ATXN7L3 is 2.344 million copies / mL, and the current qPCR technology is difficult to detect such a low level of expression, which can easily lead to false negatives. Therefore, it is urgent to develop a highly sensitive and specific molecular diagnostic product for detecting UBTF-ATXN7L3 fusion gene to lay the foundation for the early diagnosis of BCP-ALL. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a primer set for detecting the UBTF-ATXN7L3 genotype.

[0006] The second object of the present invention is to provide an application of the primer set for detecting the UBTF-ATXN7L3 genotype.

[0007] The third object of the present invention is to provide a kit for detecting the UBTF-ATXN7L3 genotype.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] A primer set for detecting the UBTF-ATXN7L3 genotype comprises a detection primer pair and a detection probe as shown below:

[0010] Upstream primer F: 5'-AAGCAGTACAAGGTGCACCT-3',

[0011] Downstream primer R: 5′-CATTTGTAAACTCTTGTGGAGAC-3′;

[0012] Probe: 5'-GCCGCCCGCTGTGCTATGAGCAGTCAGAGCGCCGTCTC-3', with the 5' end labeled with a fluorescent group, the 3' end labeled with a quencher group, and the middle of the sequence labeled with a built-in quencher group to form a double quencher.

[0013] Furthermore, the built-in quenching group is marked at the 18th base "G" position from the 5' end.

[0014] When designing the above primer set, the present invention first analyzes and compares the homology of UBTF and ATXN7L3 genes, and then finds the corresponding breakpoints and the regions that are completely specific in the UBTF-ATXN7L3 genotype, without overlap and intersection, and designs upstream and downstream primers respectively. The probe is designed near the breakpoint of the UBTF-ATXN7L3 fusion gene, ensuring the high rigor of sequence specificity. Due to the high specificity and high sensitivity required for BCP-ALL to detect the UBTF-ATXN7L3 fusion gene, corresponding to the difficulty of detecting this fusion gene, the present invention adopts a new super quenching probe technology (Super Quencher, SQ), and through the improvement of the probe, on the one hand, it is to improve the detection signal-to-noise ratio, so that the result is more sensitive and specific; on the other hand, it is to enhance the flexibility of probe design and make its application range more extensive. The artificial modification of standard nucleotides has brought more possibilities. It can not only greatly broaden the application scope of oligonucleotide probes, but even open up new application areas for artificially synthesized oligonucleotides, and make their application direction more controllable. After the improvement, the specificity and sensitivity of low-copy UBTF-ATXN7L3 fusion gene amplification have been significantly improved.

[0015] Preferably, the fluorescent group is selected from Cy5, HEX, VIC or FAM.

[0016] Further preferably, the fluorescent group is FAM or VIC.

[0017] Preferably, the quencher group is selected from BHQ-1, IBFQ or Cy3.

[0018] Further preferably, the quencher group is BHQ-1.

[0019] Preferably, the built-in quencher group is ZEN.

[0020] The present invention also provides the use of any of the above primer sets for detecting UBTF-ATXN7L3 genotypes for non-disease diagnosis purposes or in preparing a kit for detecting UBTF-ATXN7L3 genotypes.

[0021] The present invention also provides a kit for detecting the UBTF-ATXN7L3 genotype, wherein the kit comprises any one of the above primer sets.

[0022] Preferably, the kit further comprises a PCR reaction solution, a PCR reaction enzyme system, a UBTF-ATXN7L3 genotype positive quality control product, a positive quality control product, and a negative quality control product.

[0023] A method for detecting UBTF-ATXN7L3 genotype for non-disease diagnosis purposes, comprising the following steps:

[0024] S1. Extract RNA from the sample to be tested;

[0025] S2. Reversely transcribe the RNA in step S1 into a cDNA template or directly use the RNA in step S1 as a template, and perform a one-step fluorescent quantitative PCR amplification reaction using any of the above primer sets;

[0026] S3. Result determination: Determine by the amplification curve. If the amplification curve shows an obvious single S-shaped curve, the UBTF-ATXN7L3 genotype is present in the sample to be tested. Otherwise, the UBTF-ATXN7L3 genotype is not present in the sample to be tested.

[0027] Preferably, the one-step fluorescent quantitative PCR amplification system is 1.2 μL of 20 μM primers, 0.6 μL of 20 μM probes, 5 μL of 5× one-step RT-PCR buffer, 2U of hot start enzyme, 100U of MMLV enzyme, and 0.6 μL of 25mM dNTPs.

[0028] Preferably, the one-step fluorescent quantitative PCR amplification program is 50°C for 15 min; 94°C for 10 min; 94°C for 15 sec, 64°C for 45 sec, and 45 cycles.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a detection primer set for detecting UBTF-ATXN7L3 genotype, comprising a pair of primers and a probe. The present invention designs upstream primers and probes in a completely specific region of the UBTF-ATXN7L3 genotype, and uses superquenching probe labeling technology at a specific base position of the detection probe to control the probe length to 35 to 40 bp, which is about 50% longer than the conventional probe length. While significantly improving the specificity, the sensitivity is also improved to a certain extent. The signal-to-noise ratio is the lowest among similar products, and the background signal is basically eliminated. The detection sensitivity reaches 1×10 1 copies / mL, which can fully meet the clinical requirements for low signal-to-noise ratio and low copy template detection of molecular diagnostic reagents. It can perform differential diagnosis analysis in the early stage of the disease when typing is not possible and detect the UBTF-ATXN7L3 fusion gene when the expression level is low, thus meeting the clinical diagnosis and prognosis needs of leukemia patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Results of the UBTF-ATXN7L3 fusion genotype amplification specificity test (each curve represents a sample tested). Green: VIC channel; blue: FAM channel.

[0032] Figure 2 The results of the RNA test of clinical samples with positive UBTF-ATXN7L3 fusion genotype. Green: VIC channel; blue: FAM channel.

[0033] Figure 3 The sensitivity amplification results of the concentration gradient of UBTF-ATXN7L3 positive standard. Green: VIC channel; blue: FAM channel. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0036] Example 1 Design of primers and probe sets for detecting UBTF-ATXN7L3 fusion genotype

[0037] First, the homology of UBTF (SEQ ID NO.1) and ATXN7L3 (SEQ ID NO.2) genes was analyzed and compared, and then the breakpoints and characteristic sequences of UBTF-ATXN7L3 fusion gene UBTF-ATXN7L3 type were found. After confirming the position and sequence of the breakpoint, the corresponding breakpoint and the area that is completely specific in the UBTF-ATXN7L3 genotype, without overlap and intersection, were found to design upstream and downstream primers respectively, and the probe was designed near the breakpoint of the UBTF-ATXN7L3 fusion gene, ensuring the high rigor of sequence specificity. At the same time, the innovation of the probe design is also: the superquenching probe technology is applied to further upgrade the sequence design of the detection probe, and the probe length is appropriately extended to 35-40bp, which is about 50% longer than the conventional probe length. While the long sequence significantly improves the specificity of the probe detection, the sensitivity is also improved to a certain extent. The signal-to-noise ratio is the lowest among similar products, and the background signal is basically eliminated, so that the short fragment detection probe can achieve a very ideal specificity and sensitivity in the PCR system. Due to the high specificity and high sensitivity required by BCP-ALL to detect the UBTF-ATXN7L3 fusion gene, corresponding to the difficulty of detecting this fusion gene, the present invention adopts a new superquenching probe technology. Through the improvement of the probe, on the one hand, it is to improve the detection signal-to-noise ratio, so that the result is more sensitive and specific; on the other hand, it is to enhance the flexibility of probe design and make its application range more extensive. The artificial modification of standard nucleotides brings more possibilities to it, which can not only greatly broaden the application range of oligonucleotide probes, but also open up new application fields for artificially synthesized oligonucleotides, and make its application direction more controllable. After the improvement, the specificity and sensitivity of low-copy UBTF-ATXN7L3 fusion gene amplification are significantly improved. After the design is completed, the secondary structure of the above three oligonucleotides is analyzed by Oligo7.0 software, and the specificity of the sequence is determined by the BLAST function comparison analysis of NCBI. After the initial screening, the final screening and verification test is carried out, and the final screening obtains the following primer set:

[0038] Upstream primer F: 5'-AAGCAGTACAAGGTGCACCT-3',

[0039] Downstream primer R: 5′-CATTTGTAAACTCTTGTGGAGAC-3′;

[0040] Probe: 5'-GCCGCCCGCTGTGCTATGAGCAGTCAGAGCGCCGTCTC-3', its 5' end is labeled with the fluorescent group FAM, the 3' end is labeled with the quencher group BHQ-1, and the 18th base "G" position from the 5' end is labeled with a proprietary built-in quencher group ZEN to form a double quencher.

[0041] The amplification system was 1.2 μL of 20 μM primer, 0.6 μL of 20 μM probe, 5 μL of 5× one-step RT-PCR buffer, 2 U of hot start enzyme, 100 U of MMLV enzyme, and 0.6 μL of 25 mM dNTPs.

[0042] The amplification program was 50°C for 15 min; 94°C for 10 min; 94°C for 15 sec, 64°C for 45 sec, for 45 cycles.

[0043] Example 2 Specificity Test

[0044] 1. Lentivirus testing

[0045] Lentivirus samples identified as BCR-ABL1, AML1-ETO, PML-RARA, DEK-CAN, TLS-ERG, MLL-AF9, MLL-ENL, MLL-ELL, NUP98-NSD1, and TEL-AML1 by the virus culture method were taken as specific test samples. Each virus was purified by nucleic acid, and PCR fluorescence amplification was performed using the primers and probe sets, reaction system and reaction procedures for detecting the UBTF-ATXN7L3 fusion genotype described in Example 1, and the results were analyzed. Negative and positive quality control controls (UBTF-ATXN7L3 fusion genotype) were set.

[0046] The results of ten lentivirus tests are as follows Figure 1 As shown in the figure, all lentivirus-specific test samples have no specific amplification curve, which is a typical negative amplification (the blue curve in the figure is the UBTF-ATXN7L3 fusion genotype amplification curve, and the green curve is the internal reference amplification curve). The amplification curve of the positive quality control product is a normal S-type typical amplification, and the negative quality control product is a typical negative amplification. This shows that the primer and probe set of the present invention for detecting the UBTF-ATXN7L3 fusion genotype has strong specificity.

[0047] 2. Real sample test

[0048] A clinical real UBTF-ATXN7L3 fusion gene positive RNA sample was taken, and PCR fluorescence amplification was performed using the primers, probe set, reaction system and reaction procedure for detecting the UBTF-ATXN7L3 fusion genotype described in Example 1.

[0049] Test results such as Figure 2 As shown, the sample showed typical specific amplification (the blue curve in the figure is the positive amplification curve of the UBTF-ATXN7L3 fusion genotype, and the green curve is the internal reference amplification curve).

[0050] The above results show that the primer and probe set for detecting the UBTF-ATXN7L3 fusion genotype of the present invention can completely eliminate nonspecific amplification interference signals during actual detection and has very good specificity.

[0051] Example 3 Sensitivity Test

[0052] 1. Methods

[0053] The recombinant plasmid was constructed according to the UBTF-ATXN7L3 fusion genotype sequence, and the concentration was 1×10 6 Then, the number of copies / mL was gradually diluted to a lower copy number of 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL, 1×10 2 copies / mL, 1×10 1 The primers, system and procedure described in Example 1 were used to perform sensitivity amplification test of the concentration gradient of the positive standard of UBTF-ATXN7L3 fusion genotype and analyze the results, and negative and positive quality control samples (UBTF-ATXN7L3 fusion genotype) were set.

[0054] 2. Results

[0055] Sensitivity amplification test results Figure 3 As described above, the FAM (blue curve is the UBTF-ATXN7L3 fusion genotype amplification curve) and VIC (green curve is the internal reference amplification curve) dual-color amplification of each standard concentration gradient showed typical positive amplification in each concentration group, and the amplification gradient was obvious, and the detection sensitivity was as low as 1×10 1 copies / mL, since the number of white blood cells in leukemia patients is 1×10 7 / ml or more, 1×10 1 copies / mL is equivalent to 10 6 One copy is detected in each white blood cell, and there will be no false negatives, which can fully meet the clinical low copy template (10 6 The detection of fusion genes in 1 nucleated cell requires rapid detection, high precision and accuracy.

Claims

1. A primer set for detecting UBTF-ATXN7L3 fusion gene, characterized in that: Contains the following detection primer pairs and detection probes: Upstream primer F: 5'-AAGCAGTACAAGGTGCACCT-3', Downstream primer R: 5′-CATTTGTAAACTCTTGTGGAGAC-3′; Probe: 5'-GCCGCCCGCTGTGCTATGAGCAGTCAGAGCGCCGTCTC-3', with the 5' end labeled with a fluorescent group, the 3' end labeled with a quencher group, and the middle of the sequence labeled with a built-in quencher group to form a double quencher.

2. The primer set according to claim 1, characterized in that: The fluorescent group is Cy5, HEX, VIC or FAM.

3. The primer set according to claim 1, characterized in that: The quenching group is BHQ-1, IBFQ or Cy3, and the built-in quenching group is ZEN.

4. Use of the primer set according to any one of claims 1 to 3 in detecting UBTF-ATXN7L3 genotype for non-disease diagnosis purposes or in preparing a kit for detecting UBTF-ATXN7L3 genotype.

5. A kit for detecting UBTF-ATXN7L3 genotype, characterized in that: Comprising the primer set according to any one of claims 1 to 3.

6. The kit according to claim 5, characterized in that It also includes PCR reaction solution, PCR reaction enzyme system, UBTF-ATXN7L3 genotype positive quality control product and negative quality control product.

7. Use of the kit according to claim 5 or 6 in detecting UBTF-ATXN7L3 genotype for non-disease diagnosis purposes.

8. A method for detecting UBTF-ATXN7L3 genotype for non-disease diagnosis purposes, characterized in that: The steps include: S1. Extract RNA from the sample to be tested; S2. Reversely transcribe the RNA of step S1 into a cDNA template or directly use the RNA of step S1 as a template to perform a one-step fluorescent quantitative PCR amplification reaction using any one of the primer sets of claims 1 to 3; S3. Result determination: Determine by the amplification curve. If the amplification curve is an obvious single S-shaped curve, it is determined that the UBTF-ATXN7L3 genotype exists in the sample to be tested. If the amplification curve is not a single S-shaped curve, it is determined that the UBTF-ATXN7L3 genotype does not exist in the sample to be tested.

9. The method according to claim 8, characterized in that: The one-step fluorescent quantitative PCR amplification system comprises 1.2 μL of 20 μM primers, 0.6 μL of 20 μM probes, 5 μL of 5× one-step RT-PCR buffer, 2 U of hot start enzyme, 100 U of MMLV enzyme, and 0.6 μL of 25 mM dNTPs.

10. The method according to claim 8, characterized in that: The one-step fluorescent quantitative PCR amplification program is 50°C for 15 min; 94°C for 10 min; 94°C for 15 sec, 64°C for 45 sec, and 45 cycles.

Images