CN110724741A - Primer, probe and kit for detecting minimal residual leukemia related fusion gene - Google Patents
Primer, probe and kit for detecting minimal residual leukemia related fusion gene Download PDFInfo
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Abstract
The invention relates to a primer and a probe for detecting a minimal residual leukemia related fusion gene and a kit thereof. The method utilizes a digital PCR detection platform, and adds a nucleotide sequence in the kit as shown in SEQ ID No: 1-7, and amplifying the BCR-ABL1 fusion gene and a self-contrast simultaneously, namely taking the gene to be detected as the contrast, obviously reducing the signal interference caused by multi-primer multi-amplification, realizing the transcript copy number detection without a standard substance, and improving the detection precision by more than 3 times when the detection sensitivity reaches 0.001%.
Description
Technical Field
The invention belongs to the technical field of medical nucleic acid detection, and particularly relates to a primer, a probe and a kit for detecting a minimal residual leukemia related fusion gene.
Background
Minimal Residual Disease (MRD) leukemia refers to the state of Residual trace leukemia cells in a patient with leukemia that have been treated to achieve Complete Remission (CR) according to current therapeutic criteria. With the development of the medical level and the emergence of various new drugs, the complete remission rate of leukemia is greatly improved, but a considerable part of patients still have relapse after CR, the life cycle is shortened, and finally death is causedAnd (7) death. The most prominent reason is the persistent presence of MRD in the bone marrow. It was shown that if the number of MRDs is still 10 at 12 weeks after treatment-4In the above cases, the possibility of acute leukemia recurrence is very high, and consolidation and intensive treatment after remission are necessary to reduce the clinical recurrence risk and recurrence rate. Therefore, MRD can be used as a sensitive index for prognosis evaluation, and if the MRD is less than 10-4The recurrence probability is small and is more than 10-4The disease is suggested to have a high probability of recurrence, and the smaller the MRD value, the longer the recurrence-free survival. Currently, the main indicators for clinical judgment of whether leukemia patients relapse can be divided into two categories: the clinical symptoms such as the age, sex, white blood cell number and the occurrence of central nervous system leukemia and the time required for completely relieving the disease for the first time of the disease are the first, but the sensitivity of the morphological examination of bone marrow cells is low, and the number of the leukemia cells remained in the body of the patient during CR is difficult to accurately reflect; the other is the biological characteristics of leukemia cells, such as cytogenetics, immunology and the like, and the two indexes are indirect analysis although the two indexes have great help for judging the recurrence of prognosis. The diagnosis of MRD must rely on special detection means with higher sensitivity and specificity. Classical means of MRD detection include Flow Cytometry (FCM), karyotyping, FISH detection, and real-time fluorescent quantitative PCR detection. Meanwhile, the specific markers of leukemia cells of each patient are not completely the same, and the leukemia cells are also provided with a plurality of markers of normal cells, so that immune monitoring of the body can be easily escaped. Therefore, how to determine the specific immune characteristics of leukemia cells, detect trace leukemia cells mixed in normal hematopoietic cells, and establish a kit which has high detection sensitivity and can monitor the change of tiny residual content in real time has great social significance for early predicting leukemia relapse, prolonging the disease-free survival time of leukemia patients and providing treatment schemes for the patients according to the detection results.
Taking the relation between leukemia and related gene variation, for example, the common Chronic Myelocytic Leukemia (CML) detection, more than 95% of cases of CML are caused by malignant transformation of cells due to the translation of new protein after the fusion of BCR-ABL gene, and the detection of BCR-ABL gene level is simple, accurate and clear, so that the detection is adopted as the detection index of MRD. The international clinical trial conclusion and the establishment of CML treatment guidelines are derived from BCR-ABL IS data, and BCR-ABL fusion gene detection has important clinical significance for early-stage CML disease auxiliary diagnosis, subsequent targeted medication guidance and residual disease monitoring. At present, the gold standard for clinically examining BCR-ABL fusion genes IS a fluorescent quantitative PCR technology, according to the current practice guidelines, a patient needs to carry out detection once every three months, and the result IS reported in% IS units, but firstly, the quantitative detection of the qPCR technology depends on a standard reference substance; secondly, because the detection sensitivity IS not high enough due to the limitation of the qPCR technology, the RT-PCR has limitation in LoD and LoQ, the detection sensitivity IS generally 0.01% IS (MR4.0), namely 1 leukemia cell can be detected in 10000 normal cells, and the effective monitoring and treatment detection sensitivity of the MRD needs to be less than 0.002% IS (MR 4.7). Therefore, the detection sensitivity of 0.01% IS (MR4.0) possessed by RT-PCR affects the formulation of the clinical treatment scheme for CML patients, if leukemia cells can be detected earlier in normal cells, i.e. a detection method with higher sensitivity than qPCR IS found, i.e. MRD detection technology with 0.001% IS (MR5.0) or even lower sensitivity IS reached, the leukemia relapse condition can be found earlier, the treatment scheme IS provided for patients earlier, the survival time of patients IS greatly prolonged, but the MRD detection technology aiming at 0.001% IS (MR5.0) and lower detection sensitivity and good detection repeatability IS still blank in China at present.
Disclosure of Invention
The invention aims to overcome the existing RT-PCR, seek to design a detection kit for minimal residual disease, and establish a novel detection kit for minimal residual disease with super sensitivity and good detection repeatability. The method can realize the detection of the structural variation abundance of the target gene without establishing a standard curve and internal references, can simply and accurately judge the micro-residual disease, can detect the copy number and the occurrence frequency of related variation genes, and can enable the detection sensitivity to reach 0.001% IS.
In order to achieve the purpose of the invention, the technical scheme is as follows:
a primer and a probe for detecting a minimal residual leukemia related fusion gene have nucleotide sequences shown as SEQ ID No: 1 to 7.
Wherein:
(1) the primers and probes for detecting the BCR-ABL1(e13a2 or e14a2) fusion gene are as follows: consisting of SEQ ID No: 1, and a forward primer BCR-F1 consisting of SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 6, wherein the probe P2 shown in SEQ ID No: 6, FAM mark is arranged at the 5 'end of the nucleotide sequence shown in the figure, and BHQ-MGB mark is arranged at the 3' end;
(2) the primers and probes used for detecting the BCR-ABL1(e1a2) fusion gene were: consisting of SEQ ID No: 2, and the forward primer BCR-F2 consisting of SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 6, wherein the probe P2 shown in SEQ ID No: 6, FAM mark is arranged at the 5 'end of the nucleotide sequence shown in the figure, and BHQ-MGB mark is arranged at the 3' end;
(3) the primers and probes used for detecting the BCR-ABL1(e19a2) fusion gene were: consisting of SEQ ID No: 3, and a forward primer BCR-F3 consisting of SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 6, wherein the probe P2 shown in SEQ ID No: 6, FAM mark is arranged at the 5 'end of the nucleotide sequence shown in the figure, and BHQ-MGB mark is arranged at the 3' end;
(4) primers and probes used to detect ABL1 self control genes were: consisting of SEQ ID No: 4, and the forward primer ABL1-F shown by SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 7, wherein the sequence shown in SEQ ID NO: 7, a 5 'end of the nucleotide sequence is marked by HEX, and a 3' end is marked by BHQ-MGB;
a kit for detecting primers and probes of a minimal residual leukemia related fusion gene comprises nucleic acid amplification reagents, wherein the nucleic acid amplification reagents comprise:
(1) the digital PCR reaction solution for detecting BCR-ABL1(e13a2 or e14a2) fusion gene, BCR-ABL1(e1a2) fusion gene and BCR-ABL1(e19a2) fusion gene comprises the main components of forward primer BCR-F1, BCR-F2, BCR-F3, ABL1-F, reverse primer ABL1-R, probes P1, P2 and ddH2O。
(2) Digital PCR premix solution, main componentThe main components are qScriptTMXLT One-Step RT-qPCRTrough Mix, fluorescein sodium salt and ddH2O。
Wherein, the internal reference of the kit is the self-control ABL1 gene: consisting of SEQ ID No: 4, and the forward primer ABL1-F shown by SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 7, wherein the probe P1 shown in SEQ ID No: 7 has HEX mark at 5 'end and BHQ-MGB mark at 3' end.
Preferably, the kit also comprises a quality control product, wherein the quality control product comprises a negative quality control product and a positive quality control product, and the negative quality control product is ddH2And O, the positive quality control product is plasmid DNA mixed liquor.
Further preferably, the digital PCR reaction solution in the kit for detecting the BCR-ABL1(e13a2 or e14a2) fusion gene and BCR-ABL1(e1a2) fusion gene and BCR-ABL1(e19a2) fusion gene contains: 10 μmol/L of SEQ ID No: 1-5, 1 μ L each; 10 μmol/L of SEQ ID No: 6 to 7, 0.5. mu.L each.
Further preferably, the specific composition of the kit is shown in table 1.
TABLE 1 composition of the kit
More preferably, the minimal residual leukemia positive quality control product contains a mixed solution of 5 plasmids, wherein the plasmid DNA is: BCR-ABL1-e13a2 plasmid DNA, BCR-ABL1-e14a2 plasmid DNA, BCR-ABL1-e1a2 plasmid DNA, BCR-ABL1-e19a2 plasmid DNA and ABL1 self-control plasmid DNA.
The invention will be further explained and illustrated below:
the invention firstly verifies the principle correctness, feasibility, accuracy and precision of the kit. The optimal PCR amplification system is determined through verification, and the transcripts of the minimal residual leukemia can be detected to the maximum extent in simple and complex samples.
The kit of the invention can mainly carry out relapse monitoring on chronic myelogenous leukemia: most leukemia patients are treated to relieve the symptoms according to the current therapeutic standard, but a considerable part of the patients still face the risk of relapse due to the presence of MRD and finally die. At present, the detection sensitivity of RT-PCR IS 0.01% IS (MR4.0), the kit of the invention IS a kit with higher detection sensitivity than qPCR, and can detect leukemia cells in normal cells earlier, namely 0.001% IS (MR5.0) MRD detection technology can discover the recurrence condition of leukemia earlier, provide a treatment scheme for patients earlier and greatly prolong the survival time of patients.
The kit comprises accuracy quality control samples of 4 positive plasmids including BCR-ABL1-e13a2, BCR-ABL1-e14a2, BCR-ABL1-e1a2, BCR-ABL1-e19a2, and the positive coincidence rate of the accuracy quality control samples is 100%.
The kit also comprises an ABL1 self-control plasmid, can realize the detection of the structural variation abundance of the target gene without establishing a standard curve and internal parameters, and can detect the copy number and the occurrence frequency of related variation genes while simply and accurately judging the micro residual disease.
Specifically, the method comprises the following steps: in the kit of the present invention:
1. see Table 2 for primer and probe sequences for detection of BCR-ABL1-e13a2(P210) and BCR-ABL1-e14a2(P210) fusion genes.
Table 2 primer and probe sequences for detection of BCR-ABL1-e13a2(P210) and BCR-ABL1-e14a2(P210) fusion genes
2. See Table 3 for primer and probe sequences for detection of the BCR-ABL1-e1a2(P190) fusion gene.
TABLE 3 primer and Probe sequences for detection of BCR-ABL1-e1a2(P190) fusion Gene
3. See Table 4 for primer and probe sequences for detection of the BCR-ABL1-e19a2(P230) fusion gene.
TABLE 4 primer and Probe sequences for detection of the BCR-ABL1-e19a2(P230) fusion Gene
4. See table 5 for primer and probe sequences detected from control ABL 1.
TABLE 5 primer and Probe sequences detected from control ABL1
The kit can detect related fusion genes from RNA transcription cDNA extracted from anticoagulated blood, and the minimum detection amount reaches 0.14 copy/mu L.
The kit can detect each precision quality control product in corresponding reaction liquid repeatedly for 3 times, and CV values are greatly lower than the detection result of the kit using non-self-control as an internal reference.
In the kit, the BCR-ABL1(e13a2 or e14a2) fusion gene, the BCR-ABL1(e1a2) fusion gene, the BCR-ABL1(e19a2) fusion gene and ABL1 self-control share a reverse primer, and the process of detecting the BCR-ABL1 fusion gene by using double probes is essentially performed, so that the specificity, the precision and the accuracy of detection are greatly improved. According to the invention, firstly, a forward primer F1-F3 which shares a reverse primer ABL1-R and a BCR-ABL1 fusion gene is combined to simultaneously amplify a plurality of BCR-ABL1 fusion genes and ABL1 self-control genes, and the ABL1 self-control gene is taken as a reference for data analysis, so that the problems of poor repeatability, strong background signal and the like caused by mutual interference or inevitable nonspecific amplification caused by multiple primers in the prior art are avoided; the method has simple and convenient detection process and high precision, accuracy and specificity, can ensure the correctness of the detection result, and can directly obtain the results of the copy number of the structural variation genome, the copy number of the normal genome, the occurrence frequency of the structural variation of the genome and the like.
Drawings
FIG. 1 is a schematic diagram of the basic principle of the kit according to the present invention. Taking four fusion types of ABL1 genes as an example, when ABL1 gene is respectively fused with certain exons of BCR gene, forward primers F1, F2 and F3 are respectively designed for multiple BCR-ABL1 fusion gene types, a reverse primer ABL1-R is used for amplification, and a molecular probe P2 (such as FAM blue signal labeling) is used for generating a fluorescence signal; fluorescent signals were generated from the control ABL1 gene using the forward primer ABL1-F, the common reverse primer ABL1-R, and the molecular probe P1 (as labeled with HEX green fluorescent signal).
FIG. 2 is a fluorescent signal diagram (Blue + Green) of BCR-ABL1 fusion gene detection of a sample using the kit of the present invention in example 1 according to the present invention.
FIG. 3 shows the result of digital PCR assay after the gradient dilution of BCR-ABL1(e13a2-P210 and e14a2-P210) in example 2 according to the present invention. FIG. 3A shows that the mixed liquid of NTC and e13A2 and e14a2 is detected to be 10%, 1%, 0.1%, 0.032%, 0.01%, 0.0032%, 0.002% and 0.001% respectively in eight gradients (FAM channels); FIG. 3B shows total ABL1 expression levels (HEX channels);
FIG. 4 is a correlation analysis of the test results and expected results in example 2 according to the present invention.
Detailed Description
In order to more clearly explain the present invention, the technical solutions of the present invention will be more fully described below with reference to the following detailed description and accompanying drawings. The present invention can be applied to various gene tests without being limited to the examples described herein, and the experimental methods of the following examples, in which specific conditions are not noted, are generally performed as described in the conventional conditions.
Example 1:
1. see table 6 for the composition of the kit.
TABLE 6 composition of the kit
Wherein, the digital PCR reaction solution contains: 1 μ L of 10 μmol/L BCR-F1, 1 μ L of 10 μmol/L BCR-F2, 1 μ L of 10 μmol/L BCR-F3, 1 μ L of 10 μmol/L ABL1-F, 1 μ L of 10 μmol/L ABL1-R, 0.5 μ L of 10 μmol/L BCR-ABL1-P2, 0.5 μ L of 10 μmol/L ABL 1-P1;
the digital PCR premix comprises: 2 xqScriptTMXLT One-Step RT-qPCR Tough Mix 12.5. mu.L, 10. mu.M fluorescein sodium salt 2.5. mu.L.
The positive quality control product contains 5 plasmid mixed solutions in Table 7.
TABLE 7 plasmid 5 mixtures in Positive quality controls
| Name of plasmid | Concentration of plasmid | Volume (μ L) |
| BCR-ABL1-e13a2-P210 plasmid DNA | 1ng/μL | 50 |
| BCR-ABL1-e14a2-P210 plasmid DNA | 1ng/μL | 50 |
| BCR-ABL1-e1a2-P190 plasmid DNA | 1ng/μL | 50 |
| BCR-ABL1-e19a2-P230 plasmid DNA | 1ng/μL | 50 |
| ABL1 self-control plasmid DNA | 1ng/μL | 50 |
2. The using method comprises the following steps:
2.1. the instrument comprises the following steps: is suitable for NaicaTMStilla digital PCR instrument.
2.2. Sample preparation:
the kit is suitable for anticoagulated blood and anticoagulated bone marrow samples.
Sample preservation: the samples were stored in blood collection tubes suitable for RNA storage.
RNA extraction requirements: mRNA in a sample is extracted by using a Biomiga Blood RNA miniprep kit (Cat # R6411), the specific operation is referred to kit product instructions, and the extracted RNA is recommended to be detected immediately, or stored at-80 ℃ for later use.
Sample requirements: RNA extracted from the sample can be used after quality detection by using internal reference.
2.3 test methods
2.3.1 sample treatment
mRNA in the sample is extracted by using a Biomiga Blood RNA miniprep kit (Cat # R6411), and the specific operation is shown in the kit product instruction.
2.3.2 sample concentration determination: the concentration of the RNA-extracted sample was measured using Qubit4.0.
2.3.3 amplification reagent preparation:
taking out various digital PCR reaction solutions and digital PCR premix from the kit, melting at room temperature, uniformly oscillating, centrifuging for several seconds, and preparing each reaction of each digital PCR reaction system according to the table 8.
TABLE 8 digital PCR reaction System
| Reagent composition | Add volume (μ L) |
| qScriptTMXLT One-Step RT-qPCR Tough Mix(2×) | 12.5 |
| Fluorescein sodium salt | 2.5 |
| Digital PCR reaction solution | 7 |
| RNA template (simultaneously as negative quality control and positive quality control 1) | 3 |
| Total of | 25 |
2.3.4 sample addition and amplification:
the chip was removed, the white cap 1/4 was gently swirled and discarded, and 25. mu.L of the reaction solution from step (1) above was removed and added to the well of the chip and covered with a long white cap.
Putting the chip added with 25 mu L of digital PCR reaction solution into NaicaTMIn the Geode microdroplet generation amplification system, digital PCR reactions were performed according to the digital PCR reaction program set forth in table 9.
TABLE 9 digital PCR reaction procedure
3. And (3) detection:
3.1, information acquisition: after the digital PCR amplification is finished, the chip is placed in NaicaTMThe prism3 droplet reads the assay system, and the detection and interpretation of the fluorescent signal is performed.
3.2 judging the result:
as shown in FIG. 2, the results were judged by using a Crystal Miner, and the copy number of the BCR-ABL1 fusion gene, the total copy number of ABL1 and the ratio of the fusion gene in the sample were determined by detecting the fluorescence signals of FAM (blue) and HEX (Green). FAM + HEX (Blue + Green) dual-fluorescence signals positioned in quadrant I represent fluorescence signals of BCR-ABL1 fusion genes; the hex (Green) single fluorescence signal in quadrant II represents the fluorescence signal of the normal gene, i.e., ABL1 subtracts the fluorescence signal value of the BCR-ABL1 fusion gene (Blue + Green double fluorescence signal value) from the fluorescence signal value of the control gene (sum of Green single fluorescence signal values); the background value of the fluorescence signal in the amplification-free state is represented by no fluorescence signal in the third quadrant; FAM (blue) monofluorescent signal in quadrant IV represents the fluorescent signal generated by non-specific amplification or reagent contamination, and in the normal absence of non-specific amplification and contamination, the region should be free of fluorescent signal. In conclusion, the content of 4 BCR-ABL1 fusion genes, the content of normal genes and the proportion of 4 BCR-ABL1 fusion genes can be clearly and directly determined.
Example 2:
sensitivity analysis of the kit of the invention on BCR-ABL1 fusion gene detection
(1) Using BCR-ABL1(e13a2, e14a2) fusion gene as an example, two plasmids e13a2 and e14a2 containing BCR-ABL1 fusion gene at an initial concentration of about 100 ng/. mu.L were mixed, and the mixed plasmid e13a2/e14a2 and the plasmid containing ABL1 were mixed in 10-1～10-10Carrying out gradient dilution;
(2) the above e13a2/e14a2 was mixed with ABL1 plasmid to obtain 8 kinds of plasmid mixtures of e13a2/e14a2 in the total ABL1 ratio of 10%, 1%, 0.1%, 0.032%, 0.01%, 0.0032%, 0.002%, 0.001%, which are respectively referred to as International Union names MR1, MR2, MR3, MR3.5, MR4, MR4.5, MR4.7, MR5 (Table 10).
TABLE 10BCR-ABL1 fusion Gene plasmid mixture types and names
| Ratio of e13a2/e14a2 to |
10％ | 1％ | 0.1％ | 0.032％ | 0.01％ | 0.0032％ | 0.002％ | 0.001％ |
| Name (R) | MR1 | MR2 | MR3 | MR3.5 | MR4 | MR4.5 | MR4.7 | MR5 |
(3) MR1, MR2, MR3, MR3.5, MR4, MR4.5, MR4.7 and MR5 were subjected to digital PCR detection using the kit and detection method of example 1 and the ratio of the fusion genes was calculated from the detection results (FIG. 3), the expected values and the actual measured values were counted and the correlation function was calculated (Table 11). The detection result shows the detection ratioThe lowest value of the case is 0.001%, i.e., MR5, and the coefficient R is determined in the detection range of 0.001% to 10%2Was 0.99 (FIG. 4).
TABLE 11 digital PCR test results for MR1, MR2, MR3, MR3.5, MR4, MR4.5, MR4.7, MR5 samples
In order to further determine the stability of the kit to the detection of low-concentration fusion genes, three low concentrations (BCR-ABL 1-e13a2/e14a2 to ABL1, respectively) of MR4, MR4.7 and MR5 close to the lowest detection limit are selected again, and three repeated detections are carried out, and the Coefficient of Variation (CV) is calculated, wherein the proportions of the BCR-ABL1-e13a2/e14a2 to the ABL1 are 0.01%, 0.002% and 0.001%. The test results showed that the CV of MR4 was 11.59%, that of MR4.7 was 13.15%, and that of MR5 was 3.53% (Table 12).
TABLE 12 summary of MR4, MR4.7, and MR5 three-gradient assay results of BCR-ABL1 fusion genes
Example 3:
8 chronic myelogenous leukemia samples are taken, the kit and the detection method in the embodiment 1 are adopted for detection, and the detection results are shown in a table 13;
table 138 Chronic myelogenous leukemia samples tested with the kit and test method of example 1
The detection result of the invention is consistent with the confirmed diagnosis result. Then, 5 out of the 8 chronic myelogenous leukemia samples were randomly sampled, and the BCR-ABL1 fusion gene was tested in triplicate using the kit (Table 14) and the non-self-control kit (Table 15), respectively.
Table 145 samples three-time repeated detection results of BCR-ABL1 fusion gene by using the kit
Results of three-time repeated detection of BCR-ABL1 fusion gene by adopting non-self-control method kit on 155 samples in table
The results in tables 14 and 15 show that, in the repeated detection of the same sample, the repeatability (low CV value) of the kit (table 14) for the detection method of the BCR-ABL1 fusion gene is significantly better than that (high CV value) of the detection method of the currently common kit (table 15), which indicates that the kit has good repeatability for the detection method of the BCR-ABL1 gene fusion gene and the precision is improved by more than 3 times (average CV value comparison).
In summary, the kit disclosed by the application has the advantages that the detection sensitivity of the kit on the BCR-ABL1 fusion gene reaches 0.001%, and the detection precision is improved by more than 3 times.
Sequence listing
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<120> primer, probe and kit for detecting minimal residual leukemia related fusion gene
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Claims (8)
1. A primer and a probe for detecting a minimal residual leukemia related fusion gene are characterized in that the nucleotide sequences of the primer and the probe are shown as SEQ ID No: 1 to 7.
2. The primer and the probe for detecting the minimal residual leukemia associated fusion gene as claimed in claim 1, wherein the nucleotide sequence of the primer and the nucleotide sequence of the probe are as follows:
(1) the primers and probes for detecting the BCR-ABL1(e13a2 or e14a2) fusion gene are as follows: consisting of SEQ ID No: 1, and a forward primer BCR-F1 consisting of SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 6, wherein the probe P2 shown in SEQ ID No: 6, FAM mark is arranged at the 5 'end of the nucleotide sequence shown in the figure, and BHQ-MGB mark is arranged at the 3' end;
(2) the primers and probes used for detecting the BCR-ABL1(e1a2) fusion gene were: consisting of SEQ ID No: 2, and the forward primer BCR-F2 consisting of SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 6, wherein SEQ ID No: 6, FAM mark is arranged at the 5 'end of the nucleotide sequence shown in the figure, and BHQ-MGB mark is arranged at the 3' end;
(3) the primers and probes used for detecting the BCR-ABL1(e19a2) fusion gene were: consisting of SEQ ID No: 3, and a forward primer BCR-F3 consisting of SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 6, wherein the probe P2 shown in SEQ ID No: 6, FAM mark is arranged at the 5 'end of the nucleotide sequence shown in the figure, and BHQ-MGB mark is arranged at the 3' end;
(4) primers and probes used to detect ABL1 self control genes were: consisting of SEQ ID No: 4, and the forward primer ABL1-F shown by SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 7, wherein the probe P1 shown in SEQ ID No: 7 has HEX mark at 5 'end and BHQ-MGB mark at 3' end.
3. A kit containing the primers and the probe for detecting the minimal residual leukemia associated fusion gene according to any one of claims 1 or 2, wherein the kit comprises nucleic acid amplification reagents, and the nucleic acid amplification reagents comprise:
(1) detection BThe main components of the digital PCR reaction solution of the CR-ABL1(e13a2 or e14a2) fusion gene, the BCR-ABL1(e1a2) fusion gene and the BCR-ABL1(e19a2) fusion gene are forward primer BCR-F1, BCR-F2, BCR-F3, ABL1-F, reverse primer ABL1-R, probes P1, P2 and ddH2O。
(2) The main component of the digital PCR premix solution is qScriptTMXLT One-Step RT-qPCR ToughMix, fluorescein sodium salt, and ddH2O。
4. The kit for detecting the primers and the probes for the minimal residual leukemia associated fusion gene according to claim 3, wherein the internal parameters of the kit are that the self-control ABL1 gene is: consisting of SEQ ID No: 4, and the forward primer ABL1-F shown by SEQ ID No: 5, and a reverse primer ABL1-R consisting of SEQ ID No: 7, wherein the sequence shown in SEQ ID NO: 7 has HEX mark at 5 'end and BHQ-MGB mark at 3' end.
5. The kit for detecting the primers and the probes for the minimal residual leukemia associated fusion gene as claimed in claim 3, wherein the kit further comprises a quality control substance, the quality control substance comprises a negative quality control substance and a positive quality control substance, and the negative quality control substance is ddH2And O, the positive quality control product is plasmid DNA mixed liquor.
6. The kit for detecting the primers and probes for the minimal residual leukemia associated fusion gene according to claim 3, wherein the digital PCR reaction solution for detecting the BCR-ABL1(e13a2 or e14a2) fusion gene, the BCR-ABL1(e1a2) fusion gene and the BCR-ABL1(e19a2) fusion gene comprises: 10 μmol/L of SEQ ID No: 1-5, 1 μ L each; 10 μmol/L of SEQ ID No: 6 to 7, 0.5. mu.L each.
7. The kit for detecting the primers and probes for minimal residual leukemia associated fusion gene according to claim 3,
8. the kit for detecting the primers and the probes for the minimal residual leukemia associated fusion gene according to claim 7, wherein the positive quality control of the minimal residual leukemia comprises a mixture of 5 plasmids, and the plasmid DNAs are as follows: BCR-ABL1-e13a2 plasmid DNA, BCR-ABL1-e14a2 plasmid DNA, BCR-ABL1-e1a2 plasmid DNA, BCR-ABL1-e19a2 plasmid DNA and ABL1 self-control plasmid DNA.
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