Primer group for quantitatively detecting MLL-PTD fusion gene, kit and use method

Abstract

The invention relates to the field of biotechnology detection and kits, in particular to the field of IPC C12Q 1, and more particularly relates to a primer group and a kit for quantitatively detecting an MLL-PTD fusion gene and a use method. The primer group comprises an MLL-PTD upstream primer and an MLL-PTD downstream primer; the nucleotide sequence of the MLL-PTD upstream primer comprises SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the MLL-PTD downstream primer comprises SEQ ID NO: 3. The MLL-PTD fusion gene detection can provide molecular biology evidence for diagnosis of acute myelogenous leukemia, and can provide help for establishment of a clinical treatment scheme and prognosis evaluation. In addition, MLL-PTD fusion gene quantitative detection is carried out on MLL-PTD fusion gene positive patients, so that the MLL-PTD fusion gene quantitative detection can be used for detecting minimal residual lesions of leukemia, and the MLL-PTD fusion gene quantitative detection method has important significance in monitoring a treatment process and formulating a treatment scheme.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology detection and kits, in particular to the field of IPC C12Q 1, and more specifically to a primer set, a kit and a method for using the quantitative detection of MLL-PTD fusion gene. Background Art

[0002] Acute myeloid leukemia (AML) is a type of acute leukemia with an incidence of 2.57 / 100,000 people in China and a mortality rate of about 1.57 / 100,000 people per year. AML accounts for about 25% of childhood leukemia and usually occurs in infancy. In addition, the incidence of AML increases with age and is also a common acute leukemia in adults, with a median age of onset of 50 years. Leukemia fusion genes can be used as molecular biological specific markers for diagnosing different types of leukemia and as a basis for determining diagnosis.

[0003] The mixed lineage leukemia (MLL) gene is located in region 2, band 3, long arm of chromosome 11 (11q23). MLL gene rearrangement is common in adults and children with acute leukemia, and the prognosis of such patients is worse than that of patients without MLL gene rearrangement. MLL gene rearrangement manifests in various forms, including chromosomal translocation fusion and partial tandem duplication of MLL gene (MLL-PTD). MLL-PTD gene mutation is caused by genetic abnormalities such as chromosomal translocation, deletion or insertion. It is one of the more common forms of MLL gene rearrangement. It is a product formed by the insertion of the 5' end sequence of MLL gene into the genome and self-fusion. The most common duplication sequences of MLL-PTD include exon 3-9 rearrangement, exon 3-10 rearrangement and exon 3-11 rearrangement.

[0004] Chinese invention patent 201380024896.0 discloses a method for diagnosing, predicting, treating and processing acute myeloid leukemia, which involves predicting the survival rate of patients with acute myeloid leukemia, analyzing whether there are cytogenetic abnormalities in genetic samples isolated from the patients, and whether there are mutations in at least one of the genes FLT3, NPMI, DNMT3A, NRAS, CEBPA, TET2, WTI, IDHI, IDH2, KIT, RUNXI, MLL-PTD, ASXLI, PHF6, KRAS, PTEN, P53, HRAS and EZH2. The method involves a wide variety of genes and a complex detection method, which needs further improvement. Summary of the invention

[0005] The first aspect of the present invention provides a primer set for quantitatively detecting the MLL-PTD fusion gene, the primer set comprising an MLL-PTD upstream primer and an MLL-PTD downstream primer; the nucleotide sequence of the MLL-PTD upstream primer comprises SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the MLL-PTD downstream primer comprises SEQ ID NO: 3.

[0006] The primer with the nucleotide sequence of SEQ ID NO: 1 was named MLL-PTD-F1, the primer with the nucleotide sequence of SEQ ID NO: 2 was named MLL-PTD-F2, and the primer with the nucleotide sequence of SEQ ID NO: 3 was named MLL-PTD-R.

[0007] The concentrations of the MLL-PTD upstream primer and the MLL-PTD downstream primer are both 30-70 μmol / L.

[0008] Preferably, the concentrations of the MLL-PTD upstream primer and the MLL-PTD downstream primer are both 40-60 μmol / L.

[0009] Further preferably, the concentrations of the MLL-PTD upstream primer and the MLL-PTD downstream primer are both 50 μmol / L.

[0010] The second aspect of the present invention provides a kit comprising the primer set.

[0011] The kit comprises a nucleic acid amplification reagent, which comprises an internal reference gene MIX reaction solution, an MLL-PTD MIX reaction solution and a PCR MIX3, wherein the MLL-PTD MIX reaction solution comprises an MLL-PTD upstream primer and an MLL-PTD downstream primer.

[0012] The MLL-PTD MIX reaction solution also includes an MLL-PTD probe, and the nucleotide sequence of the MLL-PTD probe includes SEQ ID NO:4.

[0013] The concentration of the MLL-PTD probe is 30-70 μmol / L.

[0014] Preferably, the concentration of the MLL-PTD probe is 40-60 μmol / L.

[0015] Further preferably, the concentration of the MLL-PTD probe is 50 μmol / L.

[0016] The internal reference gene MIX reaction solution includes ABL1 internal reference primers, which include ABL1 internal reference upstream primers and ABL1 internal reference downstream primers; the nucleotide sequence of the ABL1 internal reference upstream primer includes SEQ ID NO: 5, and the nucleotide sequence of the ABL1 internal reference downstream primer includes SEQ ID NO: 6.

[0017] The internal reference gene MIX reaction solution also includes an ABL1 internal reference probe, and the nucleotide sequence of the ABL1 internal reference probe includes SEQ ID NO:7.

[0018] The probe with the nucleotide sequence SEQ ID NO: 4 was named MLL-PTD-P, the primer with the nucleotide sequence SEQ ID NO: 5 was named ABL1 internal reference-F, the primer with the nucleotide sequence SEQ ID NO: 6 was named ABL1 internal reference-R, and the probe with the nucleotide sequence SEQ ID NO: 7 was named ABL1 internal reference-P.

[0019] The preparation method of the MLL-PTD MIX reaction solution includes: centrifuging dry powders of an MLL-PTD upstream primer, an MLL-PTD downstream primer and an MLL-PTD probe respectively, adding process water to dilute, and obtaining diluted MLL-PTD-F1, MLL-PTD-F2, MLL-PTD-R and MLL-PTD-P after centrifugation, and then adding MLL-PTD-F1, MLL-PTD-F2, MLL-PTD-R and MLL-PTD-P to process water, and oscillating and mixing to obtain the reaction solution.

[0020] The molar ratio of MLL-PTD-F1, MLL-PTD-F2, MLL-PTD-R and MLL-PTD-P is 1:(0.5-1.5):(0.5-1.5):(0.2-0.8).

[0021] Preferably, the molar ratio of MLL-PTD-F1, MLL-PTD-F2, MLL-PTD-R and MLL-PTD-P is 1:(0.8-1.2):(0.8-1.2):(0.3-0.6).

[0022] Further preferably, the molar ratio of MLL-PTD-F1, MLL-PTD-F2, MLL-PTD-R and MLL-PTD-P is 1:1:1:0.5.

[0023] The preparation method of the internal reference gene MIX reaction solution comprises: centrifuging dry powders of an ABL1 internal reference upstream primer, an ABL1 internal reference downstream primer and an ABL1 internal reference probe respectively, adding process water to dilute, centrifuging to obtain diluted ABL1 internal reference-F, ABL1 internal reference-R and ABL1 internal reference-P, then adding ABL1 internal reference-F, ABL1 internal reference-R and ABL1 internal reference-P to process water, and oscillating and mixing to obtain the reaction solution.

[0024] The molar ratio of the ABL1 internal reference-F, ABL1 internal reference-R and ABL1 internal reference-P is 1:(0.5-1.5):(0.5-1.5):(0.2-0.8).

[0025] Preferably, the molar ratio of ABL1 internal reference-F, ABL1 internal reference-R and ABL1 internal reference-P is 1:(0.8-1.2):(0.3-0.6).

[0026] Further preferably, the molar ratio of ABL1 internal reference-F, ABL1 internal reference-R and ABL1 internal reference-P is 1:1:0.5.

[0027] The kit further comprises at least one of a reverse transcription reagent, a reference substance, and a standard substance.

[0028] The third aspect of the present invention provides a method for using the kit, comprising the following steps:

[0029] S1, reverse transcription of the extracted RNA nucleic acid to obtain a cDNA template;

[0030] S2, the cDNA template and PCR MIX3 are divided into two parts respectively, the MLL-PTD MIX reaction solution and PCR MIX3 are taken, and after oscillation and mixing, they are mixed with one of the cDNA templates to obtain a first detection reagent, and then the internal reference gene MIX reaction solution and PCR MIX3 are oscillated and mixed, and then mixed with the other cDNA template to obtain a second detection reagent;

[0031] S3, after PCR amplification of the first detection reagent and the second detection reagent, detection is performed simultaneously. The volume ratio of MLL-PTD MIX reaction solution, PCR MIX3 and cDNA template in the first detection reagent is 1: (1.5-2): (0.5-1.5); the volume ratio of internal reference gene MIX reaction solution, PCR MIX3 and cDNA template in the second detection reagent is 1: (1.5-2): (0.5-1.5).

[0032] Preferably, the volume ratio of MLL-PTD MIX reaction solution, PCR MIX3 and cDNA template in the first detection reagent is 1:(1.7-2):(0.5-1); the volume ratio of internal reference gene MIX reaction solution, PCR MIX3 and cDNA template in the second detection reagent is 1:(1.7-2):(0.5-1).

[0033] Further preferably, the volume ratio of MLL-PTD MIX reaction solution, PCR MIX3 and cDNA template in the first detection reagent is 7:13:5; the volume ratio of internal reference gene MIX reaction solution, PCR MIX3 and cDNA template in the second detection reagent is 7:13:5.

[0034] Beneficial Effects

[0035] 1. The primer set includes an MLL-PTD upstream primer and an MLL-PTD downstream primer; the nucleotide sequence of the MLL-PTD upstream primer includes SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the MLL-PTD downstream primer includes SEQ ID NO: 3, which can be used for the detection of mixed lineage leukemia, provide molecular biological evidence (qualitative detection) for the diagnosis of acute myeloid leukemia, and provide assistance for establishing clinical treatment plans and prognosis evaluation.

[0036] 2. Use ABL1 internal reference primers, including ABL1 internal reference upstream primer and ABL1

[0037] Internal reference downstream primer; the nucleotide sequence of the ABL1 internal reference upstream primer includes SEQ ID NO: 5, and the nucleotide sequence of the ABL1 internal reference downstream primer includes SEQ ID NO: 6, which can be used to detect minimal residual leukemia lesions, so as to achieve the purpose of quantitative detection of MLL-PTD fusion gene in patients with positive MLL-PTD fusion gene.

[0038] 3. The volume ratio of MLL-PTD MIX reaction solution, PCR MIX3 and cDNA template in the first detection reagent is 1:(1.7-2):(0.5-1); the volume ratio of internal reference gene MIX reaction solution, PCR MIX3 and cDNA template in the second detection reagent is 1:(1.7-2):(0.5-1), which can improve the PCR amplification efficiency and thus improve the detection efficiency.

[0039] 4. By selecting specific MLL-PTD primers, MLL-PTD probes, ABL1 internal reference primers and ABL1 probes in the kit, the specificity of the detection can be provided, thereby improving the accuracy of the detection.

[0040] 5. The detection limit of the kit prepared in this application can reach 12 copies / reaction, which improves the sensitivity of detection.

[0041] 6. The CV value of 10 test results of the kit prepared in this application is 0.60%, with good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the amplification curve of the negative sample internal reference gene MIX reaction solution, where the purple curve in the front is the positive control, the pink curve at the bottom is the negative control, and the others are samples to be tested.

[0043] Figure 2 This is the amplification curve of the negative sample MLL-PTD MIX reaction solution, where the purple curve in the front is the positive control, the pink curve at the bottom is the negative control, and the others are samples to be tested.

[0044] Figure 3 This is the amplification curve of different copy numbers of MLL-PTD MIX reaction solution.

[0045] Figure 4 This is the amplification curve of the MIX reaction solution for repeatability detection of the internal reference gene.

[0046] Figure 5 This is the amplification curve diagram of the MLL-PTD MIX reaction solution for repeatability detection. DETAILED DESCRIPTION

[0047] Example 1

[0048] A primer set for quantitatively detecting an MLL-PTD fusion gene, the primer set consisting of an MLL-PTD upstream primer and an MLL-PTD downstream primer; the nucleotide sequences of the MLL-PTD upstream primers are SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the MLL-PTD downstream primer is SEQ ID NO: 3, as shown in Table 1.

[0049] The primer with the nucleotide sequence of SEQ ID NO: 1 was named MLL-PTD-F1, the primer with the nucleotide sequence of SEQ ID NO: 2 was named MLL-PTD-F2, and the primer with the nucleotide sequence of SEQ ID NO: 3 was named MLL-PTD-R.

[0050] A kit contains the primer set.

[0051] The kit is composed of the following: nucleic acid amplification reagent, reverse transcription reagent, reference substance and standard substance, wherein the nucleic acid amplification reagent is internal reference gene MIX reaction solution, MLL-PTD MIX reaction solution and PCRMIX3.

[0052] The MLL-PTD MIX reaction solution, as shown in Table 3, consists of an MLL-PTD upstream primer, an MLL-PTD downstream primer, an MLL-PTD probe and process water, wherein the nucleotide sequence of the MLL-PTD probe includes SEQ ID NO: 4, and is named MLL-PTD-P.

[0053] The internal reference gene MIX reaction solution contains internal reference primers for detecting ABL1, and the formula is shown in Table 3. The ABL1 internal reference primers consist of an ABL1 internal reference upstream primer, an ABL1 internal reference downstream primer, an ABL1 internal reference probe, and process water.

[0054] As shown in Table 1, the nucleotide sequence of the ABL1 internal reference upstream primer is SEQ ID NO: 5, the nucleotide sequence of the ABL1 internal reference downstream primer is SEQ ID NO: 6, and the nucleotide sequence of the ABL1 internal reference probe is SEQ ID NO: 7.

[0055] The probe with the nucleotide sequence SEQ ID NO: 4 was named MLL-PTD-P, the primer with the nucleotide sequence SEQ ID NO: 5 was named ABL1 internal reference-F, the primer with the nucleotide sequence SEQ ID NO: 6 was named ABL1 internal reference-R, and the probe with the nucleotide sequence SEQ ID NO: 7 was named ABL1 internal reference-P.

[0056] Table 1

[0057]

[0058] Table 2

[0059]

[0060] Table 3

[0061]

[0062] The preparation method of the MLL-PTD MIX reaction solution is as follows: the dry powders of the MLL-PTD upstream primer, the MLL-PTD downstream primer and the MLL-PTD probe are centrifuged at 15,000 rpm for 2 minutes, respectively, and process water is added for dilution. After centrifugation, MLL-PTD-F1, MLL-PTD-F2, MLL-PTD-R and MLL-PTD-P with concentrations shown in Table 2 are obtained, and then the solutions of MLL-PTD-F1, MLL-PTD-F2, MLL-PTD-R and MLL-PTD-P are added to the process water and shaken for 4 minutes to obtain.

[0063] The preparation method of the internal reference gene MIX reaction solution is as follows: the dry powders of the ABL1 internal reference upstream primer, the ABL1 internal reference downstream primer and the ABL1 internal reference probe are centrifuged at 15000 rpm for 2 minutes, respectively, and process water is added for dilution. After centrifugation, ABL1 internal reference-F, ABL1 internal reference-R and ABL1 internal reference-P with concentrations shown in Table 3 are obtained, and then the solutions of ABL1 internal reference-F, ABL1 internal reference-R and ABL1 internal reference-P are added to the process water, and the mixture is shaken and mixed for 4 minutes to obtain the reaction solution.

[0064] Among them, “ / ” in Table 2-2 means it has no practical meaning and has not been measured.

[0065] A method for using a kit comprises the following steps:

[0066] S1, using the extracted RNA nucleic acid as a template for reverse transcription (PCR amplification instrument, 37°C for 1 hour), the reverse transcription material composition is shown in Table 4, and a cDNA template is obtained;

[0067] S2, as shown in Table 5, cDNA template and PCR MIX3 were divided into two parts respectively, MLL-PTD MIX reaction solution and PCR MIX3 were taken, melted at 25°C and vibrated, centrifuged at 2000rpm for 10s, and mixed with one of the cDNA templates to obtain the first detection reagent, and then the internal reference gene MIX reaction solution and PCR MIX3 were vibrated and mixed with the other cDNA template to obtain the second detection reagent;

[0068] S3, after performing PCR amplification on the first detection reagent and the second detection reagent respectively, performing detection simultaneously.

[0069] In step S1, RNA nucleic acid is extracted using a commercial kit, and RNeas-Free water is added to mix and dissolve for later use.

[0070] Step S3 also includes a negative control and a positive control. The negative control is process water; the positive control is MLL-PTD plasmid DNA solution (45,000 copies / reaction).

[0071] Table 4

[0072] MIX1 (produced by our company) MIX2 (produced by our company) RNA nucleic acid Total volume / test formula 6μl 4μl 10μl 20μl

[0073] Table 5

[0074]

[0075] The specific PCR amplification conditions are: ABI 7500 fluorescence PCR detector, Bio-Rad CFX96 fluorescence PCR detector amplification conditions: 42℃, 5min; 95℃, 10min, 40 cycles. The reaction system is 25μl. The fluorescence signal is collected at 60℃ in the second step of the PCR cycle. The reaction system is 25ul. The fluorescence signal is collected at 60℃ in the second step of the PCR cycle. The detection channel is: FAM-none, and the reference fluorescence is set to none.

[0076] The detection is specifically:

[0077] (1) Determination of baseline: According to conventional operations in the field, the part of the curve with less fluctuation and more stability is selected as the baseline.

[0078] (2) Determination of the threshold value: In the case of no amplification in the negative control, the threshold value is set at the highest point of the sample with no amplification curve, that is, higher than the highest point of the no amplification growth curve (that is, no peak appears in the "Component" column of the result analysis), and the negative control is not detected as a principle to determine the starting threshold value.

[0079] Performance testing methods and data

[0080] Effectiveness judgment

[0081] Referring to the method of Example 1, when performing PCR amplification, four different copy numbers were set in the internal reference reaction: 3.3×10 5 , 3.3×10 4 , 3.3×10 3 , 3.3×10 2 The C of the four reference products T The value should be ≤36, and the absolute value of the standard curve fit should be ≥0.980, otherwise the quantification is considered invalid.

[0082] Negative control C T The value should be ≥38 or "Undet" will be displayed.

[0083] (1) Qualitative determination, see Table 6:

[0084] Table 6

[0085]

[0086] Among them, △C T Value = MLL-PTD MIX C T Value-reference gene MIX C T value.

[0087] For internal reference reaction solution C T Value>36 and MLL-PTD reaction solution does not meet the "C TValue ≤36 and △C T For samples with a value of ≤6", it is necessary to increase the sample volume, re-extract and perform PCR test, and the results shall be determined according to the above table. T Value>36 and MLL-PTD reaction solution does not meet the "C T Value ≤36 and △C T If the value is ≤6", the sample is judged as not meeting the requirements.

[0088] (2) Quantitative determination:

[0089] When the MLL-PTD fusion gene in the specimen is positive and the detection concentrations of MLL-PTD and the internal reference gene are both greater than or equal to 1×10 2 When the copies are obtained, the following quantitative results analysis is performed.

[0090] The copy numbers of reference products 1-4 in the internal reference reaction were set as: 3.3×10 5 , 3.3×10 4 , 3.3×10 3 , 3.3×10 2 After the amplification, the obtained standard curve was used to obtain the detection concentration of MLL-PTD (A) and the detection concentration of the internal reference gene (B) of each sample.

[0091] a. If the sample: MLL-PTD RNA detection concentration> 3.3×10 6 copies, should not be within the linear range and should be appropriately diluted and re-tested.

[0092] If the sample: internal reference RNA detection concentration> 3.3×10 6 copies, should not be within the linear range and should be appropriately diluted and re-tested.

[0093] b. If the specimen is 1×10 2 copies≤MLL-PTD RNA detection concentration (i.e. A)≤3.3×10 6 copies, and

[0094] 1×10 2 copies≤ internal reference RNA detection concentration (i.e. B)≤3.3×10 6 If there are no copies, it is reported as the ratio of the detection concentration of MLL-PTD (A) to the detection concentration of the internal reference gene (B), that is, (A / B)×100%.

[0095] 1. Specificity verification

[0096] According to the implementation method, cDNA of 27 MLL-PTD negative samples was tested to verify the specificity of the reagent. Figure 1 As shown in Table 7, the MLL-PTD reaction solution test results of 27 samples △C T The values ​​all meet the interpretation criteria, indicating that the reagent has good specificity, and the “ / ” value is close to the baseline and is not read.

[0097] Table 7

[0098] Sample name <![CDATA[MLL-PTDC T Value]]> <![CDATA[Reference gene C T value]]> <![CDATA[△C T Value]]> Interpretation of results Negative control / / / / Positive Control 23.39 23.66 / / Sample 1 38.87 27.94 10.93 Negative Sample 2 / 27.22 / Negative Sample 3 37.71 27.01 10.70 Negative Sample 4 38.93 25.37 13.56 Negative Sample 5 / 26.93 / Negative Sample 6 36.99 29.02 7.97 Negative Sample 7 / 28.31 / Negative Sample 8 38.38 28.31 10.07 Negative Sample 9 38.70 27.40 11.30 Negative Sample 10 40.00 25.07 14.93 Negative Sample 11 39.00 27.19 11.81 Negative Sample 12 37.71 26.26 11.45 Negative Sample 13 / 29.40 / Negative Sample 14 38.67 26.61 12.06 Negative Sample 15 38.49 27.90 10.59 Negative Sample 16 / 27.35 / Negative Sample 17 / 27.81 / Negative Sample 18 / 32.31 / Negative Sample 19 36.52 25.81 10.71 Negative Sample 20 37.75 25.34 12.41 Negative Sample 21 / 26.11 / Negative Sample 22 / 30.75 / Negative Sample 23 / 26.97 / Negative Sample 23 / 26.80 / Negative Sample 25 / 31.21 / Negative Sample 26 / 25.22 / Negative Sample 27 37.36 26.80 10.56 Negative

[0099] 2. Detection limit

[0100] Depend on Figure 3 As shown in Table 8, the minimum detection limit of this kit is 12 copies / reaction. The minimum detection limit sample was tested 20 times, and 19 times were detected, with a detection rate of ≥95%, indicating that this kit can detect samples with a minimum of 12 copies / reaction.

[0101] Table 8

[0102]

[0103] NTC stands for negative control; N / A stands for no C T value.

[0104] 3. Repeatability verification

[0105] From Table 9 and Figure 4-5 As shown, the same sample was tested 10 times, the CV value of the MLL-PTD fusion gene results 10 times was 0.60%, and the CV value of the internal reference gene results 10 times was 0.60%, indicating that the detection kit has good repeatability.

[0106] Calculate the C of 10 replicates of the sample T The C values ​​of 10 replicates were calculated. T The value is standard deviation, and the CV (coefficient of variation) value = (standard deviation / mean value) × 100%.

[0107] Table 9

[0108] Sample name <![CDATA[MLL-PTDC T Value]]> Sample name <![CDATA[Internal reference gene C T value]]> Positive samples - replicate 1 25.03 Sample 10 - Replicate 1 24.94 Positive samples - replicate 2 25.21 Sample 10 - Repeat 2 25.22 Positive samples - replicate 3 25.11 Sample 10 - Repeat 3 25.43 Positive samples - replicate 4 25.27 Sample 10 - Repeat 4 25.38 Positive samples - replicate 5 25.59 Sample 10 - Repeat 5 25.29 Positive samples - replicate 6 25.28 Sample 10 - Repeat 6 25.19 Positive samples - replicate 7 25.19 Sample 10 - Repeat 7 25.20 Positive samples - replicate 8 25.28 Sample 10 - Repeat 8 25.30 Positive samples - replicate 9 25.20 Sample 10 - Repeat 9 25.06 Positive samples - repeat 10 25.13 Sample 10 - Repeat 10 25.07

[0109] 4. Sample testing

[0110] According to the implementation method, 41 samples with clinical diagnosis results from the Hematological Oncology Research Center of Harbin No. 1 Hospital were tested. The results are shown in Table 10, where the “ / ” value is close to the baseline and is not read.

[0111] Table 10

[0112]

[0113]

[0114] After testing, the MLL-PTD fusion gene detection results of 41 samples were consistent with the clinical diagnosis results. As shown in Table 11, two samples were tested for MRD by quantitatively detecting the MLL-PTD fusion gene ratio, and the test results were also consistent with the clinical diagnosis results. The specific results are as follows, where " / " is not measured.

[0115] Table 11

[0116]

Claims

1. A primer set for quantitative detection of MLL-PTD fusion gene, characterized in that: The primer set includes an MLL-PTD upstream primer and an MLL-PTD downstream primer; the nucleotide sequence of the MLL-PTD upstream primer includes SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the MLL-PTD downstream primer includes SEQ ID NO:

3.

2. The primer set for quantitatively detecting the MLL-PTD fusion gene according to claim 1, characterized in that: The concentrations of the MLL-PTD upstream primer and the MLL-PTD downstream primer are both 30-70 μmol / L.

3. A kit, characterized in that: Contains the primer set according to any one of claims 1-2.

4. The kit according to claim 3, characterized in that The kit comprises a nucleic acid amplification reagent, which comprises an internal reference gene MIX reaction solution, an MLL-PTD MIX reaction solution and PCRMIX3, and the MLL-PTD MIX reaction solution comprises an MLL-PTD upstream primer and an MLL-PTD downstream primer.

5. The kit according to claim 4, characterized in that The MLL-PTD MIX reaction solution also includes an MLL-PTD probe, and the nucleotide sequence of the MLL-PTD probe includes SEQ ID NO:

4.

6. The kit according to claim 5, characterized in that The internal reference gene MIX reaction solution includes ABL1 internal reference primers, which include ABL1 internal reference upstream primers and ABL1 internal reference downstream primers; the nucleotide sequence of the ABL1 internal reference upstream primer includes SEQ ID NO: 5, and the nucleotide sequence of the ABL1 internal reference downstream primer includes SEQ ID NO:

6.

7. The kit according to claim 6, characterized in that The internal reference gene MIX reaction solution also includes an ABL1 internal reference probe, and the nucleotide sequence of the ABL1 internal reference probe includes SEQ ID NO:

7.

8. The kit according to claim 7, characterized in that The kit also includes at least one of a reverse transcription reagent reference substance and a standard substance.

9. A method for using the kit according to claim 8, characterized in that: The following steps are involved: S1, reverse transcription of the extracted RNA nucleic acid to obtain a cDNA template; S2, the cDNA template and PCR MIX3 are divided into two parts respectively, the MLL-PTD MIX reaction solution and PCR MIX3 are taken, and after oscillation and mixing, they are mixed with one of the cDNA templates to obtain a first detection reagent, and then the internal reference gene MIX reaction solution and PCR MIX3 are oscillated and mixed, and then mixed with the other cDNA template to obtain a second detection reagent; S3, performing PCR amplification on the first detection reagent and the second detection reagent respectively, and then performing detection simultaneously.

10. The method for using the kit according to claim 9, characterized in that: The volume ratio of MLL-PTDMIX reaction solution, PCR MIX3 and cDNA template in the first detection reagent is 1:(1.5-2): (0.5-1.5); the volume ratio of the internal reference gene MIX reaction solution, PCR MIX3 and cDNA template in the second detection reagent is 1: (1.5-2): (0.5-1.5).

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