Primer and kit for detecting KMT2A:: ARCN1 fusion gene as well as use method and application

Abstract

The invention relates to the technical field of gene detection (IPC classification number: C12Q), in particular to a primer for detecting a KMT2A:: ARCN1 fusion gene and a kit thereof. The invention provides a primer aiming at the KMT2A:: ARCN1 fusion gene, a kit and a use method thereof, which are used for effectively detecting rare gene fusion and providing key molecular biology evidence for diagnosis of acute lymphoblastic leukemia. Besides, the kit can also be used for quantitatively detecting KMT2A:: ARCN1 gene fusion positive patients, assists leukemia minimal residual focus monitoring, and has important significance on formulation of a treatment scheme and prognosis evaluation.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology (IPC classification number: C12Q), and in particular to a primer for detecting a KMT2A::ARCN1 fusion gene and a kit thereof. Background Art

[0002] Leukemia fusion genes can be used as molecular biological specific markers for diagnosing different types of leukemia and as an important basis for determining the diagnosis.

[0003] KMT2A, also known as the MLL gene, is located on the short arm of chromosome 11, encoding histone H3 lysine 4 methyltransferase. The KMT2A gene is a common fusion gene in leukemia. The fusion partners of this gene that have been discovered include AF1p, AF4, AF9CBP, ENL, etc. There are also tests for the KMT2A fusion gene. For example, Chinese patent CN118240941A discloses a method and kit for quantitative detection of MLL-AF4 fusion gene based on digital PCR; Chinese patent CN101955991A discloses a combined detection method and diagnostic kit for multiple leukemia fusion genes such as MLL-AF4. KMT2A rearrangement is an independent factor in determining the prognostic risk of leukemia and is of great significance in risk assessment and treatment selection.

[0004] The ARCN1 gene is located on the short arm 23 of chromosome 11, which includes the mixed lineage leukemia and Friend leukemia virus integration 1 genes. Multiple disease-related chromosomal translocations occur in this region. The KMT2A-ARCN1 fusion gene has been found in patients with acute myeloid leukemia, but there is no technical concept in the existing technology that focuses on the precise detection of KMT2A::ARCN1 gene fusion.

[0005] Real-time fluorescence quantitative PCR technology refers to adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally using the standard curve to perform quantitative analysis on unknown templates. It has the advantages of simple operation, rapidity, and low cost. The accurate detection of KMT2A::ARCN1 gene fusion by real-time fluorescence quantitative PCR technology has positive research significance. Summary of the invention

[0006] The present invention provides a primer, a kit and a method of using the KMT2A::ARCN1 fusion gene, which can effectively detect the rare gene fusion and provide key molecular biological evidence for the diagnosis of acute lymphoblastic leukemia (ALL). In addition, the kit can also quantitatively detect patients with positive KMT2A::ARCN1 gene fusion, assist in monitoring the micro-residual lesions of leukemia, and is of great significance for the formulation of treatment plans and prognosis evaluation.

[0007] In a first aspect, the present invention provides a primer for detecting a KMT2A::ARCN1 fusion gene, wherein the primer comprises a KMT2A::ARCN1 primer probe sequence and an ABL1 internal reference primer probe sequence.

[0008] Preferably, the KMT2A::ARCN1 primer probe sequence comprises:

[0009] KMT2A::ARCN1-F:5'-AATTCCAGCAGATGGAGTCC A-3'

[0010] KMT2A::ARCN1-R:5'-GAGCTTTGGAAAAGCTGCT-3'

[0011] KMT2A::ARCN1-P:5'-FAM-GTTTGTGGAAATGACCCGAACTCGG-BHQ1-3'

[0012] Preferably, the ABL1 internal reference primer probe sequence includes:

[0013] ABL1 internal reference-F: 5'-AATTGCTACCTATGGCATGT-3'

[0014] ABL1 internal reference-R: 5'-CTTTTCCACTTCGTCTGAGA-3'

[0015] ABL1 internal control-P: 5'-FAM-GACCTGTCCCAGGTGTATGAGCT-BHQ1-3'.

[0016] The present invention innovatively designs specific primers to efficiently detect the rare KMT2A::ARCN1 gene fusion, providing a crucial molecular biological basis for the diagnosis of acute lymphoblastic leukemia. The research found that when the KMT2A::ARCN1 and ABL1 internal reference primer probe sequences composed of specific exon DNA chains are used, the target fusion gene and the internal reference gene can be accurately captured and identified. This discovery has opened up a new way to accurately detect the KMT2A::ARCN1 fusion gene and optimize the diagnosis and treatment of acute lymphoblastic leukemia.

[0017] The second aspect of the present invention provides a kit for detecting KMT2A::ARCN1 fusion gene; the kit comprises: a nucleic acid amplification reagent, a reverse transcription reagent, a control substance and a reference substance.

[0018] The nucleic acid amplification reagents include KMT2A::ARCN1 PCR reaction solution, ABL1 internal reference gene PCR reaction solution and PCRMIX.

[0019] The KMT2A::ARCN1 PCR reaction solution is the above-mentioned KMT2A::ARCN1 primer probe sequence.

[0020] The ABL1 internal reference gene PCR reaction solution is the above-mentioned ABL1 internal reference primer probe sequence.

[0021] Preferably, the reverse transcription reagent consists of MIX 1 and MIX 2; the volume ratio of MIX 1 to MIX 2 is (1-8):(2-5).

[0022] There is no specific limitation on the fluorescent labeling group of the primer probe, and any fluorescent group commonly used in the art can be selected, for example, any one of FAM, VIC, HEX, TexasRed, CY5, TET, JOE, CY3, TAMRA, ROX, LCRED640, and LCRED705.

[0023] In some preferred embodiments, different fluorescent groups may be used in the KMT2A::ARCN1 PCR reaction solution and the ABL1 internal reference gene PCR reaction solution, so that the two reaction solutions are combined into one tube for detection.

[0024] This test kit must be stored at -20℃±4℃ and away from light to ensure quality. It is sealed in a foam box with cold ice packs during transportation to ensure that the temperature does not exceed room temperature during the entire process and the transportation time does not exceed 4 days; after arrival, it must be frozen according to the storage conditions. The validity period is up to 12 months. Please note that the test kit should be avoided from repeated freezing and thawing. Please mix thoroughly after thawing before use to ensure the best effect.

[0025] The third aspect of the present invention provides a method for using the above-mentioned kit, which specifically comprises:

[0026] Step 1) Sample processing: extract RNA from the sample, mix and dissolve the extracted RNA with RNase-Free water to obtain a reverse transcription template for later use;

[0027] Step 2) reverse transcribing complementary DNA;

[0028] Step 3) preparing amplification reagents and adding samples;

[0029] Step 4) PCR amplification reaction;

[0030] Step 5) Data collection and analysis.

[0031] In step 1, the RNA in the sample to be tested can be extracted by using the extraction method commonly used in the art. During the test, the extracted RNA is mixed and dissolved with RNase-Free water, and 10 μL of the mixture is taken as the reverse transcription template; at the same time, a negative control sample is prepared, and the negative control sample is specifically the same volume of RNase-Free water.

[0032] Preferably, step 2 specifically comprises placing the reverse transcription template obtained in step 1 and the reverse transcription reagent in a PCR amplifier, mixing them and performing reverse transcription to synthesize complementary DNA (cDNA); the reverse transcription conditions are: reacting at 35-40° C. for 0.5-2 h, and then reacting at 65-75° C. for 5-20 min to obtain a complementary DNA template.

[0033] Preferably, the step 3 specifically includes:

[0034] Step 3-1) Prepare nucleic acid amplification reagents: Mix KMT2A::ARCN1 PCR reaction solution, ABL1 internal reference gene PCR reaction solution and PCR MIX, melt at room temperature and shake to mix, centrifuge at 1500-2800 rpm for 5-20 seconds to obtain nucleic acid amplification reagents;

[0035] Step 3-2) Configure the reaction system: Take each complementary DNA template to detect the KMT2A::ARCN1 fusion gene and the ABL1 internal reference gene, so configure the first reaction system and the second reaction system respectively:

[0036] Preferably, the first reaction system is as follows: for the KMT2A::ARCN1 fusion gene, the KMT2A::ARCN1 PCR reaction solution, PCR MIX and complementary DNA template are mixed; the volume ratio of the three is (3-8): (10-17): (2.5-10).

[0037] Preferably, the second reaction system is specifically as follows: for the ABL1 internal reference gene, the ratio of the ABL1 internal reference gene, the PCR MIX and the complementary DNA template is the same as that of the first reaction system, so as to form a control.

[0038] Step 3-3) Transfer the configured first reaction system and second reaction system to a PCR amplification instrument for detection.

[0039] In order to further improve the accuracy and sensitivity of the kit in detecting the KMT2A::ARCN1 fusion gene, preferably, the specific conditions for PCR amplification in step 4 are: ① pre-reaction stage: maintain at 42°C for 5 minutes; then increase to 94°C and maintain for 3 minutes; ② PCR cycle stage: maintain at 94°C for 15 seconds; maintain at 60°C for 60 seconds; the number of cycles is 35-45 times; and the fluorescence signal is collected after the cycle is completed.

[0040] Note: When using a PCR amplification instrument for testing, the detection channel is set to: FAM, and the reference fluorescence is set to none.

[0041] The data collection in step 4 is achieved by the supporting software of the PCR amplification instrument.

[0042] The present invention ensures the accuracy of the test results by innovating the method of using the test kit and adopting a simple and efficient detection process. The specificity, reproducibility and sensitivity of the detection method are significantly improved through the carefully designed reverse transcription complementary DNA reaction conditions and nucleic acid amplification reagent formula. After multiple verifications, the detection kit of the present invention performs well in identifying the KMT2A::ARCN1 fusion gene, and the test results are both accurate and stable. It is particularly worth mentioning that the detection limit of the kit is extremely low, and it can sensitively capture trace samples as low as 10 copies of the KMT2A::ARCN1 fusion gene, thereby achieving effective detection of low-concentration samples and broadening the scope of application. This innovative achievement plays a vital role in the diagnosis of acute lymphoblastic leukemia.

[0043] Note:

[0044] 1) When collecting and analyzing data, the baseline and threshold can be determined by conventional techniques in the art.

[0045] 2) This kit is suitable for testing bone marrow or peripheral blood samples. Use EDTA anticoagulant tubes to collect samples.

[0046] 3) The PCR amplifier used in the PCR test using the kit is not specifically limited, and any PCR amplifier commonly used in the art can be used.

[0047] The fourth aspect of the present invention provides the use of the above-mentioned kit and the method of using the same in the detection of KMT2A::ARCN1 fusion gene of acute lymphoblastic leukemia in the peripheral blood or bone marrow of an organism.

[0048] Beneficial effects:

[0049] The present invention provides a primer, a kit and a method for using a KMT2A::ARCN1 fusion gene, which have the following advantages:

[0050] On the one hand, the present invention successfully achieves effective detection of the rare KMT2A::ARCN1 gene fusion by virtue of specific primer design, providing indispensable molecular biological evidence for the diagnosis of acute lymphoblastic leukemia.

[0051] Secondly, the kit integrates reverse transcription reagents, nucleic acid amplification reagents, control substances and reference substances, and uses PCR combined with real-time fluorescent probe technology to achieve accurate quantitative detection of the RNA content of the KMT2A::ARCN1 fusion gene in the sample. It is easy to operate and highly practical.

[0052] Thirdly, by optimizing the method of use, the kit of the present invention can obtain accurate results using simple detection steps. After multiple verifications, the kit has shown high specificity, high reproducibility and high accuracy for the KMT2A::ARCN1 fusion gene.

[0053] Fourthly, the detection limit of this kit is extremely low, and it can detect at least 1000 copies of the ABL1 reference gene and as low as 10 copies of the KMT2A::ARCN1 fusion gene, effectively identifying low-concentration samples and broadening the scope of application.

[0054] Fifthly, the CV value of the test kit when the same sample was tested 10 times was 0.49%. The test results of the KMT2A::ARCN1 fusion gene had good repeatability and the data results were highly reliable.

[0055] Sixthly, this technology can be used for quantitative detection of patients with positive KMT2A::ARCN1 gene fusion, helping to detect tiny residual leukemia lesions, which is of great significance for treatment monitoring and program formulation, and provides strong support for clinical treatment and prognosis evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 .PCR amplification curves of ABL1 internal reference gene references 1-4.

[0057] Figure 2 .PCR amplification curves of KMT2A::ARCN1 fusion gene references 1-4.

[0058] Figure 3 .Amplification curve of the second reaction system (ABL1 internal reference gene) during specificity test.

[0059] Figure 4 .Amplification curve of the first reaction system (KMT2A::ARCN1) during specificity testing.

[0060] Figure 5 .PCR amplification curve of the ABL1 internal reference gene in the sample during the repeatability test.

[0061] Figure 6 .PCR amplification curve of KMT2A::ARCN1 fusion gene in samples during repeatability test.

[0062] Figure 7 .PCR amplification curve of the ABL1 internal reference gene in positive samples.

[0063] Figure 8 .PCR amplification curve of KMT2A::ARCN1 fusion gene in positive samples. DETAILED DESCRIPTION

[0064] Unless otherwise specified, the room temperature in the embodiments of the present invention is 25°C.

[0065] Example 1.

[0066] In a first aspect, the present embodiment provides a primer for detecting the KMT2A::ARCN1 fusion gene, wherein the primer includes a KMT2A::ARCN1 primer probe sequence and an ABL1 internal reference primer probe sequence.

[0067] The KMT2A::ARCN1 primer probe sequence includes:

[0068] KMT2A::ARCN1-F:5'-AATTCCAGCAGATGGAGTCC A-3'

[0069] KMT2A::ARCN1-R:5'-GAGCTTTGGAAAAGCTGCT-3'

[0070] KMT2A::ARCN1-P:5'-FAM-GTTTGTGGAAATGACCCGAACTCGG-BHQ1-3'

[0071] The ABL1 internal reference primer probe sequence includes:

[0072] ABL1 internal reference-F: 5'-AATTGCTACCTATGGCATGT-3'

[0073] ABL1 internal reference-R: 5'-CTTTTCCACTTCGTCTGAGA-3'

[0074] ABL1 internal reference-P:5'-FAM-GACCTGTCCCAGGTGTATGAGCT-BHQ1-3'

[0075] In a second aspect, the present embodiment provides a kit for detecting the KMT2A::ARCN1 fusion gene; the kit comprises: a nucleic acid amplification reagent, a reverse transcription reagent, a control substance and a reference substance.

[0076] The nucleic acid amplification reagents, reverse transcription reagents, control substances and reference substances are all from Shanghai Yuanqi Biotechnology Co., Ltd.

[0077] The nucleic acid amplification reagents include KMT2A::ARCN1 PCR reaction solution, ABL1 internal reference gene PCR reaction solution and PCRMIX.

[0078] The KMT2A::ARCN1 PCR reaction solution is the above-mentioned KMT2A::ARCN1 primer probe sequence.

[0079] The ABL1 internal reference gene PCR reaction solution is the above-mentioned ABL1 internal reference primer probe sequence.

[0080] The reverse transcription reagent consists of MIX 1 and MIX 2; the volume ratio of MIX 1 to MIX 2 is 3:2, with a total of 10 μL.

[0081] The third aspect of this embodiment provides a method for using the above-mentioned kit, which specifically includes:

[0082] Step 1) Sample processing: extract RNA from the sample to be tested, and mix the extracted RNA with RNase-Free water to dissolve it to obtain a reverse transcription template for later use;

[0083] Step 2) reverse transcribing complementary DNA;

[0084] Step 3) preparing amplification reagents and adding samples;

[0085] Step 4) PCR amplification reaction;

[0086] Step 5) Data collection and analysis.

[0087] Step 1 uses the Trizol method to extract RNA from the sample to be tested. During the test, the extracted RNA precipitate is mixed and dissolved with RNase-Free water (the volume of RNase-Free water is 50 μL), and 10 μL of the mixture is taken as the reverse transcription template; at the same time, a negative control sample is prepared, and the negative control sample is specifically the same volume of RNase-Free water. The selection of the sample to be tested is specifically described in the test method.

[0088] Specifically, step 2 includes placing the reverse transcription template obtained in step 1 and the reverse transcription reagent in a PCR amplifier, mixing them, and reverse transcribing and synthesizing complementary DNA; the reverse transcription conditions are: reacting at 37° C. for 1 hour, and then reacting at 70° C. for 10 minutes to obtain a complementary DNA template.

[0089] The step 3 specifically includes:

[0090] Step 3-1) Prepare nucleic acid amplification reagents: Mix the KMT2A::ARCN1 PCR reaction solution and the ABL1 internal reference gene PCR reaction solution with PCR MIX (volume ratio of 7:13), melt at room temperature and vibrate to mix, and centrifuge at 2000 rpm for 10 seconds to obtain nucleic acid amplification reagents;

[0091] Step 3-2) Configure the reaction system: Take each complementary DNA template to detect the KMT2A::ARCN1 fusion gene and the ABL1 internal reference gene, so configure the first reaction system and the second reaction system respectively:

[0092] The first reaction system is specifically as follows: for the KMT2A::ARCN1 fusion gene, the KMT2A::ARCN1 PCR reaction solution, PCR MIX and complementary DNA template are mixed; the volume ratio of the three is 7:13:5, and the volume of the first reaction system is 25 μL in total.

[0093] The second reaction system is specifically as follows: for the ABL1 internal reference gene, the ABL1 internal reference gene PCR reaction solution, PCR MIX and complementary DNA template are mixed; the volume ratio of the three is 7:13:5, and the volume of the second reaction system is 25 μL in total.

[0094] Step 3-3) Transfer the configured first reaction system and second reaction system to a PCR amplification instrument for detection.

[0095] The specific conditions of PCR amplification in step 4 are: 42°C for 5 min; then raised to 94°C for 3 min; ②PCR cycle stage: 94°C for 15 s; 60°C for 60 s; the number of cycles is 40. After the cycle is completed, the fluorescence signal is collected.

[0096] Note: When using a PCR amplification instrument for testing, the detection channel is set to: FAM, and the reference fluorescence is set to none.

[0097] The data collection in step 5 is achieved by the supporting software of the PCR amplification instrument.

[0098] The PCR amplification instrument used in this kit is the Bio-Rad CFX96 fluorescence PCR instrument.

[0099] Performance Testing Methods

[0100] 1. Determination of the effectiveness of the method:

[0101] The linear standard curves of KMT2A::ARCN1 fusion gene and ABL1 internal reference gene were used for PCR detection (refer to steps 2-5 in the method of using the kit in Example 1, wherein the information of each reference product is shown in Table 1 below).

[0102] Table 1

[0103] Reference product name Reference concentration KMT2A::ARCN1 fusion gene linear standard 1 <![CDATA[1.0×10 6 copies <!-- 5 -->]]> KMT2A::ARCN1 fusion gene linear standard 2 <![CDATA[1.0×10 5 copies]]> KMT2A::ARCN1 fusion gene linear standard 3 <![CDATA[1.0×10 4 copies]]> KMT2A::ARCN1 fusion gene linear standard 4 <![CDATA[1.0×10 3 copies]]> ABL1 internal reference gene linear standard 1 <![CDATA[1.0×10 6 copies]]> ABL1 internal reference gene linear standard 2 <![CDATA[1.0×10 5 copies]]> ABL1 internal reference gene linear standard 3 <![CDATA[1.0×10 4 copies]]> ABL1 internal reference gene linear standard 4 <![CDATA[1.0×10 3 copies]]>

[0104] C of linear standards 1-4 of KMT2A::ARCN1 fusion gene and ABL1 reference gene T The values ​​should be ≤36, and the absolute value of the standard curve fit should be ≥0.980. T The value should be ≥38 or display "Undet". The test results of the final reference and negative control meet the above requirements.

[0105] 2. Qualitative determination method: According to the reaction solution C T The basis for qualitative judgment of the value is shown in Table 1 below.

[0106] Table 1

[0107]

[0108]

[0109] a: After retesting, if the C T If the value is <38, the KMT2A::ARCN1 fusion gene is positive; if the C T Value ≥38, KMT2A::ARCN1 fusion gene RNA is below the minimum detection limit.

[0110] b: For internal reference reaction solution C T The value is >36 and the C T For samples with a value > 36, it is necessary to increase the sample volume, re-extract RNA and perform PCR detection (i.e., repeat steps S1 to S5 after increasing the sample volume), and refer to Table 1 for the test results. T Value>36 and KMT2A::ARCN1 reaction solution C T If the value is >36, the sample is judged to not meet the requirements.

[0111] 3. Quantitative determination method:

[0112] 1) Drawing a standard curve: Linear standards 1-4 of the KMT2A::ARCN1 fusion gene and the ABL1 internal reference gene were used for PCR detection (refer to steps S2 to S5 in the method for using the kit in Example 1). Specifically, the linear standards 1-4 were added to the two reaction systems in step S3 in the amounts shown in Table 1 for measurement.

[0113] The amount of KMT2A::ARCN1 fusion gene linear standards 1-4 added to the first reaction system (KMT2A::ARCN1 fusion gene) was set to: 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 The amount of ABL1 internal reference gene linear standard 1-4 added to the second reaction system (ABL1 internal reference gene) was set to: 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 After the PCR amplification reaction is completed, a standard curve can be drawn according to the added amount of linear standards 1-4 and the fluorescence signal test results.

[0114] 2) Substitute the test results of the samples into the two standard curves in step 1) to obtain the detection concentration of the KMT2A::ARCN1 fusion gene (A) and the detection concentration of the ABL1 internal reference gene (B) of each sample.

[0115] 3) Analysis of test results:

[0116] a. If the detection concentration of KMT2A::ARCN1 fusion gene (A)>1×10 7 copies, that is, they are not within the linear range and need to be appropriately diluted and retested.

[0117] b. If the detection concentration of ABL1 internal reference gene (B)>1×10 7 copies, that is, they are not within the linear range and need to be appropriately diluted and retested.

[0118] c. If the test result is 10 copies ≤ KMT2A::ARCN1 fusion gene detection concentration (A) ≤ 1×10 7 copies, and 1×10 3 copies≤ABL1 internal reference gene detection concentration (B)≤1×10 7 If there are 100 copies of the fusion gene, the output fusion gene ratio is the ratio of the KMT2A::ARCN1 fusion gene detection concentration (A) to the ABL1 reference gene detection concentration (B) (i.e. A / B) × 100%.

[0119] Among them, the PCR amplification curves of ABL1 internal reference gene linear standards 1-4 are shown in Figure 1 ; The corresponding standard curve parameters are: E = 94.7%, R 2 =99.9%, Slope=-3.457, y-int=42.249.

[0120] The PCR amplification curves of KMT2A::ARCN1 fusion gene linear standards 1-4 are shown in Figure 2 ; The corresponding standard curve parameters are: E = 94.8%, R 2 =100.0%, Slope=-3.454, y-int=41.835.

[0121] 4. Specificity Test Verification

[0122] Take 24 KMT2A::ARCN1 negative samples (source: Ruiang Gene), and perform PCR detection with reference to steps S1-S5 of the kit usage method in Example 1 to verify the specificity of this kit; the test results are shown in Figure 3 and Figure 4 .

[0123] The test results of each sample under the conditions of reaction solution B (ABL1 internal reference gene) are shown in Table 2 below. (N / A: No signal detected)

[0124] Table 2

[0125]

[0126]

[0127] The test results of each sample under the condition of reaction solution A (KMT2A::ARCN1 fusion gene) are specifically shown in Table 3 below.

[0128] (N / A: No signal detected)

[0129] Table 3

[0130] Sample name Detection of genes <![CDATA[C T Value]]> Sample name Detection of genes <![CDATA[C T Value]]> Sample 1 KMT2A::ARCN1 N / A Sample 13 KMT2A::ARCN1 N / A Sample 2 KMT2A::ARCN1 N / A Sample 14 KMT2A::ARCN1 N / A Sample 3 KMT2A::ARCN1 N / A Sample 15 KMT2A::ARCN1 N / A Sample 4 KMT2A::ARCN1 N / A Sample 16 KMT2A::ARCN1 N / A Sample 5 KMT2A::ARCN1 N / A Sample 17 KMT2A::ARCN1 N / A Sample 6 KMT2A::ARCN1 N / A Sample 18 KMT2A::ARCN1 N / A Sample 7 KMT2A::ARCN1 N / A Sample 19 KMT2A::ARCN1 N / A Sample 8 KMT2A::ARCN1 N / A Sample 20 KMT2A::ARCN1 N / A Sample 9 KMT2A::ARCN1 N / A Sample 21 KMT2A::ARCN1 N / A Sample 10 KMT2A::ARCN1 N / A Sample 22 KMT2A::ARCN1 N / A Sample 11 KMT2A::ARCN1 N / A Sample 23 KMT2A::ARCN1 N / A Sample 12 KMT2A::ARCN1 N / A Sample 24 KMT2A::ARCN1 N / A

[0131] Combining the test results in Table 2 and Table 3, it can be seen that the ABL1 reference gene test results of the 24 samples were normal, and the KMT2A::ARCN1 fusion gene results showed no signal detected. The above results show that the specificity of this kit is good.

[0132] 5. Determination of detection limit

[0133] By performing PCR detection on the detection limit reference (plasmid dilutions of different concentrations) (refer to steps S3 to S5 in the method of using the kit in Example 1), the detection limit of the kit of the present invention for the KMT2A::ARCN1 fusion gene and the ABL1 reference gene was determined. The test results are shown in the table below. According to the test results in Table 4, the minimum detection limit of the ABL1 reference gene of the kit is 1000 copies, and the minimum detection limit of the KMT2A::ARCN1 fusion gene of the kit is 10 copies.

[0134] Note: The negative control in Table 4 is high-purity water.

[0135] Table 4

[0136] Sample name Sample copy number Detection of genes <![CDATA[C T Value]]> ABL1 Gradient Sample 1 1.00E+06 ABL1 internal reference gene 20.94 ABL1 Gradient Sample 2 1.00E+05 ABL1 internal reference gene 24.11 ABL1 Gradient Sample 3 1.00E+04 ABL1 internal reference gene 27.51 ABL1 Gradient Sample 4 1.00E+03 ABL1 internal reference gene 31.15 Negative control N / A ABL1 internal reference gene N / A KMT2A::ARCN1 Gradient Sample 1 1.00E+06 KMT2A::ARCN1 fusion gene 21.18 KMT2A::ARCN1 gradient sample 2 1.00E+05 KMT2A::ARCN1 fusion gene 24.34 KMT2A::ARCN1 Gradient Sample 3 1.00E+04 KMT2A::ARCN1 fusion gene 27.60 KMT2A::ARCN1 gradient sample 4 1.00E+03 KMT2A::ARCN1 fusion gene 30.82 KMT2A::ARCN1 Gradient Sample 5 1.00E+02 KMT2A::ARCN1 fusion gene 34.21 KMT2A::ARCN1 Gradient Sample 6 1.00E+01 KMT2A::ARCN1 fusion gene 37.00 Negative control N / A KMT2A::ARCN1 fusion gene N / A

[0137] 6. Repeatability:

[0138] The reproducibility of the detection results of ABL1 internal reference gene and KMT2A::ARCN1 fusion gene were investigated respectively.

[0139] ABL1 internal reference gene: The same sample (specifically, the sample is RNA extracted from pseudovirus) was tested 11 times (corresponding to the second reaction system), and the PCR test curve is shown in Figure 5 The 11 test results are recorded in Table 5, and the CV value of the 11 test results is calculated to be 0.65%, indicating that the detection results of the detection kit for the ABL1 internal reference gene determination result are reproducible.

[0140] KMT2A::ARCN1 fusion gene: The same sample (specifically, RNA extracted from pseudovirus) was tested 11 times (corresponding to the first reaction system). The PCR test curve is shown in Figure 6 The 11 test results are recorded in Table 5, and the CV value of the 11 test results is calculated to be 0.49%, indicating that the detection results of the detection kit for the KMT2A::ARCN1 fusion gene have good repeatability.

[0141] Table 5

[0142] Sample name Detection of genes <![CDATA[C T Value]]> Sample name Detection of genes <![CDATA[C T Value]]> Repeat 1 ABL1 23.21 Repeat 1 KMT2A::ARCN1 23.34 Repeat 2 ABL1 23.25 Repeat 2 KMT2A::ARCN1 23.18 Repeat 3 ABL1 23.08 Repeat 3 KMT2A::ARCN1 23.14 Repeat 4 ABL1 23.28 Repeat 4 KMT2A::ARCN1 23.21 Repeat 5 ABL1 23.42 Repeat 5 KMT2A::ARCN1 23.44 Repeat 6 ABL1 23.14 Repeat 6 KMT2A::ARCN1 23.09 Repeat 7 ABL1 23.15 Repeat 7 KMT2A::ARCN1 23.30 Repeat 8 ABL1 23.31 Repeat 8 KMT2A::ARCN1 23.28 Repeat 9 ABL1 23.21 Repeat 9 KMT2A::ARCN1 23.23 Repeat 10 ABL1 23.45 Repeat 10 KMT2A::ARCN1 23.30 Repeat 11 ABL1 23.14 Repeat 11 KMT2A::ARCN1 23.46

[0143] 7. Sample test

[0144] According to the method of using the kit in Example 1, 3 positive samples from the Department of Hematology of the First Affiliated Hospital of Dalian Medical University (sample 1 is a bone marrow sample, and samples 2 and 3 are peripheral blood samples) were tested, and the fusion gene ratio was calculated according to the ratio of the detection concentration of the KMT2A::ARCN1 fusion gene to the detection concentration of the ABL1 internal reference gene; the PCR amplification curve of the positive sample is shown in Figure 7 , Figure 8 The test results of each positive sample are shown in Table 6 below.

[0145] Table 6

[0146]

Claims

1. A primer for detecting KMT2A::ARCN1 fusion gene, characterized in that: The primers include a KMT2A::ARCN1 primer probe sequence and an ABL1 internal reference primer probe sequence.

2. The primer for detecting KMT2A::ARCN1 fusion gene according to claim 1, characterized in that: The KMT2A::ARCN1 primer probe sequence includes: KMT2A::ARCN1-F:5'-AATTCCAGCAGATGGAGTCC A-3' KMT2A::ARCN1-R:5'-GAGCTTTGGAAAAGCTGCT-3' KMT2A::ARCN1-P:5'-FAM-GTTTGTGGAAATGACCCGAACTCGG-BHQ1-3'.

3. A kit for detecting KMT2A::ARCN1 fusion gene, characterized in that: The kit comprises: a nucleic acid amplification reagent, a reverse transcription reagent, a control substance and a reference substance; The nucleic acid amplification reagents include KMT2A::ARCN1 PCR reaction solution, ABL1 internal reference gene PCR reaction solution and PCRMIX; The KMT2A::ARCN1 PCR reaction solution is the KMT2A::ARCN1 primer probe sequence of claim 2. The ABL1 internal reference gene PCR reaction solution is the ABL1 internal reference primer probe sequence of claim 3.

4. The kit for detecting KMT2A::ARCN1 fusion gene according to claim 3, characterized in that: The minimum detection limit of the kit for the KMT2A::ARCN1 fusion gene is 10 copies.

5. A method for using the kit according to claim 3, characterized in that: The method for using the kit specifically includes: Step 1) Sample processing: RNA is extracted from the sample and mixed with RNase-Free water to be used as a reverse transcription template. Step 2) Reverse transcription generates complementary DNA; Step 3) preparing amplification reagents and adding samples; Step 4) performing a PCR amplification reaction; Step 5) Collect and analyze data.

6. The method for using the kit according to claim 5, characterized in that: The step 3 specifically includes: Step 3-1) Prepare nucleic acid amplification reagents: Mix KMT2A::ARCN1 PCR reaction solution, ABL1 internal reference gene PCR reaction solution and PCR MIX, melt at room temperature and shake to mix, centrifuge at 1500-2800 rpm for 5-20 seconds to obtain nucleic acid amplification reagents; Step 3-2) Configure the reaction system: Take each complementary DNA template to detect the KMT2A::ARCN1 fusion gene and the ABL1 internal reference gene, so configure the first reaction system and the second reaction system respectively: Step 3-3) Transfer the configured first reaction system and second reaction system to a PCR amplification instrument for detection.

7. The method for using the kit according to claim 6, characterized in that: The first reaction system is specifically as follows: for the KMT2A::ARCN1 fusion gene, the KMT2A::ARCN1 PCR reaction solution, PCR MIX and complementary DNA template are mixed; the volume ratio of the three is (3-8): (10-17): (2.5-10).

8. The method for using the kit according to claim 7, characterized in that: The specific conditions of PCR amplification in step 4 are as follows: ① pre-reaction stage: maintain at 42°C for 5 minutes; then increase to 94°C and maintain for 3 minutes; ② PCR cycle stage: maintain at 94°C for 15 seconds; maintain at 60°C for 60 seconds; the number of cycles is 35-45 times; and the fluorescence signal is collected after the cycle is completed.

9. The method for using the kit according to claim 8, characterized in that: The CV value of repeated detection of the KMT2A::ARCN1 fusion gene in the same sample using the method is less than 2%; the number of repeated detections is greater than 5.

10. Use of the kit according to claim 3 or the method for using the kit according to claims 5 to 8 in the detection of KMT2A::ARCN1 fusion gene of acute lymphoblastic leukemia in peripheral blood or bone marrow of an organism.

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