CN116334180A - BCR-ABL1 fusion gene quantitative genome RNA standard substance and preparation method thereof - Google Patents
BCR-ABL1 fusion gene quantitative genome RNA standard substance and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a quantitative genome RNA standard substance of a BCR-ABL1 fusion gene and a preparation method thereof, wherein genome RNA containing two mutation forms of the BCR-ABL1P210 fusion genes b2a2 and b3a2 is extracted and obtained by the method, an RNA storage buffer solution and a quantitative standard substance solution are prepared, corresponding primers and probes are designed, a one-step reverse transcription digital PCR method is adopted, the BCR-ABL1P210 fusion mutant genome RNA is used as a template for PCR amplification, and fluorescent signals are collected to detect the expression of the BCR-ABL1P210 fusion genes b2a2 and b3a2, so that the copy number content of the BCR-ABL1P210 fusion genes b2a2, b3a2 and ABL-WT and the mutation gene abundance are used as the magnitudes.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a BCR-ABL1 fusion gene quantitative genome RNA standard substance and a preparation method thereof.
Background
The BCR-ABL1 fusion gene consists of balanced reciprocal translocation between the protooncogene ebb-sen mouse leukemia virus gene homolog 1 (abselson murine leukemia viraloncogene homolog 1, ABL 1) on chromosome 9 long arm (9 q 34) and the breakpoint cluster region gene (break point cluster region, BCR) on chromosome 22 long arm (22 q 11), and is a typical philadelphia chromosome (philadelphia chromosome, ph). Chromosomal abnormalities carrying the BCR-ABL1 fusion gene were found to be a key factor in chronic myeloid leukemia (chronic myeloid leukemia, CML), and in about 95% of CML patients, the fusion gene BCR-ABL1 is P210-type. Therefore, the BCR-ABL1P210 fusion gene becomes one of important indexes for diagnosing CML, and dynamic change of the transcription level of the BCR-ABL1P210 fusion gene also becomes a reliable index for judging the prognosis of leukemia, thereby laying a foundation for judging and predicting the prognosis of leukemia.
In recent years, the technical method for detecting leukemia specific fusion genes in China is gradually established and perfected. Current methods for detecting BCR-ABL1 are Immunohistochemistry (IHC), fluorescence In Situ Hybridization (FISH), PCR-based techniques, high throughput sequencing (Next Generation Sequencing, NGS), and the like.
Among the various techniques available, molecular monitoring of BCR-ABL1 by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become a necessary means for assessing patient response, monitoring minimal residual disease (monitor minimal residual disease, MRD) and detecting recurrence. However, the procedures of RT-qPCR from sample collection to reference selection to result reporting vary widely from laboratory to laboratory, lack of reproducibility, and the quality problem of part of BCR-ABL1 fluorescent quantitative PCR reagents is also one of the important reasons for "false negative" and "false positive".
CN108265117A discloses a candidate reference substance of a BCR-ABL1 fusion gene e14a2 subtype plasmid, a preparation method and application thereof. When the candidate reference substance is prepared, a primer is designed firstly, and a recombinant plasmid candidate reference substance containing a BCR-ABL1 fusion gene e14a2 subtype is constructed by adopting a molecular cloning technology; after enzyme digestion electrophoresis and sequencing verification, performing real-time fluorescence quantitative PCR for uniformity and stability evaluation; and evaluating the measurement uncertainty. Although the method provides candidate reference substances of the BCR-ABL1 fusion gene e14a2 subtype plasmid, the candidate reference substances cannot be used for quantifying the b2a2 subtype and the b3a2 subtype of the P210, the plasmid is in a DNA form, has very large difference from the genome size, cannot participate in the reverse transcription step, and the quantitative method is a fluorescence quantitative PCR method, has no high-grade traceability characteristic as the conventional detection method, so the quantitative detection and traceability of the BCR-ABL1 fusion gene have obvious defects.
Therefore, there is an urgent need to develop a BCR-ABL1 fusion gene quantitative genomic RNA standard substance with high accuracy and traceability for verifying the sensitivity and accuracy of each analysis method, related reagent verification and evaluation, and laboratory quality control.
Disclosure of Invention
The invention aims to provide a BCR-ABL1 fusion gene quantitative genome RNA standard substance and a preparation method thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a preparation method of a BCR-ABL1 fusion gene quantitative genome RNA standard substance comprises the following steps:
(1) Extracting genome RNA of a cell line containing BCR-ABL1P210 type mutation, and detecting the concentration and purity of the genome RNA;
(2) The genome RNA of the BCR-ABL1P210 type mutant cell line is used as a template, a reverse transcription kit and PCR amplification are adopted to obtain a gene fragment with target fusion mutation, and the correctness of a target gene sequence is verified through Sanger sequencing PCR products;
(3) Adding a certain yeast RNA solution into RNA solution to prepare RNA storage buffer;
(4) Adding the genomic RNA of the cell line into the RNA storage buffer solution to dilute the genomic RNA into a standard solution with a certain concentration;
(5) Designing and obtaining a BCR-ABL1P210 type fusion gene b2a2 specific primer 1, a primer 2 and a specific probe 1, wherein the nucleotide sequences of the primer 1 and the primer 2 are shown as SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of the probe 1 is shown as SEQ ID No. 3;
designing and obtaining a BCR-ABL1P210 fusion gene b3a2 specific primer 3 and a primer 4 and a specific probe 2; the nucleotide sequences of the primer 3 and the primer 4 are shown as SEQ ID No.4 and SEQ ID No.5, and the nucleotide sequence of the probe 2 is shown as SEQ ID No. 6;
designing and obtaining ABL-WT gene specific primers 5, primers 6 and specific probes 3; the nucleotide sequences of the primer 5 and the primer 6 are shown as SEQ ID No.7 and SEQ ID No.8, and the nucleotide sequence of the probe 3 is shown as SEQ ID No. 9;
(6) The one-step reverse transcription digital PCR method is adopted, the BCR-ABL1P210 type fusion mutant genome RNA is used as a template for PCR amplification, fluorescent signals are collected to detect the expression of the BCR-ABL1P210 type fusion genes b2a2 and b3a2, and therefore the copy number content and the mutant gene abundance of the BCR-ABL1P210 type fusion genes b2a2, b3a2 and ABL-WT are obtained as the magnitudes.
In the step (3) and the step (4), the RNA storage buffer solution and the standard solution having a predetermined concentration are weighed, diluted and mixed by a balance weighing method.
Further, in the step (3), the yeast RNA concentration is 5-500 ng/. Mu.L.
Further, in the step (5), the 5 '-end of the probe 1, the probe 2 and the probe 3 is marked with a FAM fluorescent group, and the 3' -end is marked with a BHQ1 fluorescent group.
Further, in the step (5), the reaction concentration of the primer 1 and the primer 2 is 500nM, and the reaction concentration of the probe 1 is 400nM; the reaction concentration of the primer 3 and the primer 4 is 400nM, and the reaction concentration of the probe 2 is 300nM; the reaction concentration of the primer 5 and the primer 6 is 500nM, and the reaction concentration of the probe 3 is 400nM.
Further, the one-step reverse transcription digital PCR method comprises the following steps: 0 to 100ng,2 XOne-step Supermix (Bio-Rad) 5. Mu.L, reverse transcriptase. Mu.L, 300mM DTT 1. Mu.L, primer 1 and primer 2:1.0 μl, probe 1: 0.8. Mu.L; or primer 3 and primer 4:0.8 μl, probe 2: 0.6. Mu.L; or primer 5 and primer 6:1.0 μl, probe 3: 0.8. Mu.L.
Further, the PCR reaction conditions were: reverse transcription at 45℃for 60min and denaturation at 95℃for 10min; then denaturation at 94℃for 30sec,58℃for 60sec and annealing at 60℃for 40 cycles; finally, the mixture is extended for 10min at 98 ℃ and stored at 4 ℃.
Further, the mutant gene abundance calculating formula is as follows
Further, the cell line is a K562 cell line.
The BCR-ABL1 fusion gene prepared by the preparation method provided by the invention is used for quantifying a genome RNA standard substance.
Compared with the prior art, the invention has the outstanding effects that:
(1) The BCR-ABL1 fusion gene quantitative genome RNA standard substance obtained by the method has high accuracy, and the value-fixing method has good precision and high accuracy, and ensures the reliability and traceability of the measurement result.
(2) The method of the invention respectively extracts and obtains genome RNA containing two mutant forms of BCR-ABL1P210 fusion genes b2a2 and b3a2, and prepares RNA storage buffer solution and quantitative standard solution by mixing yeast RNA and RNA solution through a balance weighing method.
(3) The one-step reverse transcription digital PCR method established by the method has the advantages of good precision and high accuracy, does not depend on a standard curve and CT values, directly calculates according to poisson distribution to achieve absolute quantification and the like, and the tracing route has passed the acceptance of the International quality Consultation Committee (CCQM) nucleic acid analysis working group (NAWG), thereby ensuring the reliability and traceability of the measurement result.
(4) The BCR-ABL1 fusion gene quantitative genome RNA standard substance can be used as a quantitative standard. The BCR-ABL1 fusion gene detection quantitative standard comprises two common mutation forms, namely b2a2 and b3a2, and can be used for development of a BCR-ABL1 fusion gene detection method, verification and evaluation of related reagents and laboratory quality control.
The BCR-ABL1 fusion gene quantitative genomic RNA standard substance and the preparation method thereof are further described below with reference to the accompanying drawings and specific examples.
Drawings
FIG. 1 shows the detection result of the BCR-ABL1 fusion gene detection quantitative genomic RNA standard substance chip electrophoresis.
FIG. 2 shows the sequencing results of the BCR-ABL1 fusion gene detection quantitative genomic RNA standard substance.
FIG. 3 shows the digital PCR reaction annealing temperature optimization of the quantitative genomic RNA standard substance for detecting the BCR-ABL1 fusion gene.
FIG. 4 shows the optimization of the digital PCR reaction system for detecting and quantifying genomic RNA standard substances by using the BCR-ABL1 fusion gene.
FIG. 5 shows the stability of the copy number concentration of the b2a2 genomic RNA standard substance detected and quantified by the BCR-ABL1 fusion gene.
FIG. 6 shows the stability of the copy number concentration of the b3a2 genomic RNA standard substance detected and quantified by the BCR-ABL1 fusion gene.
FIG. 7 shows the stability of the copy number concentration of the genomic RNA standard substance ABL1-WT detected and quantified by the BCR-ABL1 fusion gene.
FIG. 8 shows the result of the detection and quantification of the abundance stability of the genomic RNA standard substance b2a2 mutant gene by the BCR-ABL1 fusion gene.
FIG. 9 shows the result of the stability of the abundance of the b3a2 mutant gene of the genomic RNA standard substance detected and quantified by the BCR-ABL1 fusion gene.
Detailed Description
1. Confirmation of purity and concentration of extracted genomic RNA of cell line
By Qiagen, germany
The Mini Kit extracts and purifies genome RNA in a large number of cultured K562 cell lines containing BCR-ABL1P210 type mutation to obtain BCR-ABL1 genome RNA standard substance candidates. The specific operation steps are as follows:
1. for suspension cells, the cell density reached 10 7 cells/mL, transferred to centrifuge tube to 300Xg centrifuging for 5min, carefully sucking out all supernatant; for adherent cells, after the cells were fully confluent, the cells were digested and counted (cell density reached 10 7 cells/mL), transferred to a centrifuge tube, centrifuged at 300 Xg for 5min, and all supernatant carefully aspirated.
2. Cells were lysed by addition of 600. Mu.L Buffer RLT, vortexed or blown down evenly.
3. Adding 70% ethanol with equal amount into the cell lysate, and mixing thoroughly.
4. Add 700. Mu.L of cell lysate to RNeasy spin column, centrifuge at 8000 Xg (. Gtoreq.10000 rpm) for 15s, and pour out the waste liquid.
5. The remaining solution was repeated for the previous step.
6. mu.L of Buffer RW1 was added to RNeasy spin column, centrifuged at 8000 Xg (. Gtoreq.10000 rpm) for 15s, and the waste liquid was poured off.
7. 500. Mu.L Buffer RPE was added to a RNeasy spin column spin column. The cover is lightly covered, the centrifugation is carried out for 2min at the speed of more than or equal to 8000 Xg (at the speed of more than or equal to 10000 rpm), and the waste liquid is poured out.
8. RNeasy spin column is placed in a new 2mL collection tube and the old collection tube is discarded along with the waste liquid. The lid was gently covered and centrifuged at full speed for 1min.
9. RNeasy spin column was placed in a fresh 1.5mL collection tube and 30-50. Mu.L of enzyme-free water was added to the spin-column membrane. The cover is lightly covered, and the mixture is centrifuged for 1min at the speed of more than or equal to 8000 Xg (at the speed of more than or equal to 10000 rpm).
10. To increase the RNA yield, the elution was repeated using the eluent of operation 9 (no enzyme water) to obtain higher concentration of RNA.
11. The high concentration RNA solution was collected.
The purity and concentration of the genomic RNA obtained by the above preparation were measured and repeated 5 times, and the measurement results are shown in Table 1. Meanwhile, the integrity of the sample is detected by utilizing micro-fluidic chip electrophoresis, and the detection result of Agilent 2100 is shown in FIG. 1.
TABLE 1 results of BCR-ABL1P210 genomic RNA concentration and purity
2. Confirmation of the sequence of the extracted genomic RNA target mutant Gene of the cell line
The genome RNA is reversely transcribed into cDNA by a reverse transcription kit, a gene fragment of the target fusion mutation is obtained through common PCR amplification, and then the PCR product is sequenced by using a Sanger sequencing method. The sequencing results are shown in FIG. 2.
3. Design three pairs of primers and probes for specifically amplifying BCR-ABL1P210 type b2a2 and b3a2 and ABL-WT genes
Three pairs of specific primers and probes were designed using PrimerExpress3.0 according to NCBI database in combination with BCR-ABL1P210 type b2a2 and b3a2 fusion mutation information. And the Blast is adopted for comparison, so that the specificity is high, and no potential nonspecific amplification region exists. The specific primers and the probes comprise a specific primer 1 and a primer 2 for specifically amplifying BCR-ABL1P210 type b2a2 and a specific probe 1; and specific primers 3 and 4 and specific probe 2 for specifically amplifying BCR-ABL1P210 type b3a 2; amplifying specific primers 5 and 6 and specific probes 3 of ABL-WT; the nucleotide sequences of the primer 1 and the primer 2 are shown as SEQ ID No.1 and SEQ ID No.2, the nucleotide sequence of the probe 1 is shown as SEQ ID No.3, the nucleotide sequences of the primer 3 and the primer 4 are shown as SEQ ID No.4 and SEQ ID No.5, the nucleotide sequence of the probe 2 is shown as SEQ ID No.6, the nucleotide sequences of the primer 5 and the primer 6 are shown as SEQ ID No.7 and SEQ ID No.8, and the nucleotide sequence of the probe 3 is shown as SEQ ID No.9, see Table 2.
TABLE 2 digital PCR primer probe sequences
| Sequence numbering | Name of the name | Nucleotide sequence |
| SEQ ID No.1 | b2a2-F | CAGATGCTGACCAACTCGTGT |
| SEQ ID No.2 | b2a2-R | TCCTTGGAGTTCCAACGAGC |
| SEQ ID No.3 | b2a2-P | FAM-TGAGCCTCAGGGTCTGAGTGAAGCC-BHQ1 |
| SEQ ID No.4 | b3a2-F | CACTCAGCCACTGGATTTAA |
| SEQ ID No.5 | b3a2-R | TCCTTGGAGTTCCAACGAGC |
| SEQ ID No.6 | b3a2-P | FAM-TGAGCCTCAGGGTCTGAGTGAAGCC-BHQ1 |
| SEQ ID No.7 | ABL-WT-F | CCTTCAGCGGCCAGTAGC |
| SEQ ID No.8 | ABL-WT-R | GGACACAGGCCCATGGTAC |
| SEQ ID No.9 | ABL-WT-P | FAM-TGAGCCTCAGGGTCTGAGTGAAGCC-BHQ1 |
4. Optimizing amplification conditions of three pairs of primers and probes for specifically amplifying BCR-ABL1P210 type b2a2 and b3a2 and ABL-WT gene
The final concentrations of the primers b2a2 and b3a2 of BCR-ABL1P210 type were fixed at 500nM, and the probe concentrations were fixed at 250nM (FIGS. 3a and 3 b), and annealing temperatures were set at 55℃at 56℃at 57℃at 58℃at 59℃at 60 ℃.
The final concentration of ABL-WT primer was fixed at 500nM and the probe concentration was fixed at 250nM (FIG. 3 c), and annealing temperatures were set at 55℃at 56℃at 57℃at 58℃at 59℃at 60 ℃.
From the one-dimensional scatter diagram of the amplification result, better amplification is obtained at 6 annealing temperatures of the BCR-ABL1P210 type b2a2 and b3a2, but from the repeatability of the quantitative result, the repeatability is the best at 60 ℃, so that the optimal annealing temperature of the BCR-ABL1P210 type b2a2 and b3a2 is determined to be 60 ℃; better amplification was obtained at 4 annealing temperatures for ABL-WT, the best dispersion of both positive and negative droplets at 58℃and the reproducibility of the quantitative results were higher, thus confirming that the optimal annealing temperature for ABL-WT was 58 ℃.
The primer probe concentrations were set to be 200-500nM, respectively, and according to the optimal conditions that the negative amplification signal was significantly separated from the positive amplification signal, the droplet count and the copy number were higher, the final determined b2a2 primer probe concentrations were 500nM and 400nM, the b3a2 primer probe concentrations were 400nM and 300nM, and the ABL-WT primer probe concentrations were 500nM and 400nM.
The system of the microdroplet digital PCR reaction is: genome: 0-100 ng,2X One-step Supermix (Bio-Rad Laboratories) 5. Mu.L, reverse transcriptase. Mu.L, 300mM DTT 1. Mu.L, V1 upstream and downstream primers: 0.8 μl, probe: 0.4. Mu.L, V3 upstream and downstream primer: 1.0 μl, probe: 0.6. Mu.L, ABL-WT upstream and downstream primer: 1.0 μl, probe: 0.8. Mu.L, 2. Mu.L of RNA template and ddH 2 O makes up 20. Mu.L.
PCR reaction procedure: reverse transcription at 45℃for 60min, denaturation at 95℃for 10min, denaturation at 94℃for 30s, annealing at 58℃for 60s,40 cycles, extension at 98℃for 10min, and preservation at 4 ℃. The digital PCR reaction system is optimized as shown in FIG. 4.
5. Preparation of BCR-ABL1 fusion genome RNA standard substance and fixed value
Through the processes of the step (I) and the step (II), preparing RNA solution containing 5 ng/. Mu.L yeast RNA as an RNA storage buffer solution according to a balance weighing method, adding the obtained BCR-ABL1 fusion gene cell line genome RNA serving as a standard substance into the RNA storage buffer solution according to a weight method, uniformly mixing, sub-packaging for freezing, and then carrying out fixed value according to the established one-step reverse transcription digital PCR.
The one-step reverse transcription digital PCR method is adopted, the BCR-ABL1P210 type fusion mutant genome RNA is used as a template for PCR amplification, fluorescent signals are collected to detect the expression of the BCR-ABL1P210 type fusion genes b2a2 and b3a2, and therefore the copy number content and the mutant gene abundance of the BCR-ABL1P210 type fusion genes b2a2, b3a2 and ABL-WT are obtained as the magnitudes.
The calculation formula of the abundance of the target mutant gene is as follows:
To ensure sufficient uniformity and stability of the standard substance providing a quality control reference during the detection process, the accuracy of the delivered quantity value is thus accurate. According to the technical requirements for evaluating the uniformity of standard substances in the national measurement technical specification JJF 1343-2012 general principles of standard substance constant value and statistical principles, randomly extracting 11 packaging units from each standard substance, performing constant value detection by adopting an established one-step reverse transcription digital PCR method, and repeatedly measuring each unit for 3 times to obtain data shown in tables 3 and 4. F testing the obtained data, calculating F value and critical F obtained by looking up table α Comparing if F<F α Then it is considered that there is no obvious difference between the group and the groupThe difference, uniformity of the samples was good.
And (3) carrying out uniformity measurement on the quantitative standard substance for detecting the BCR-ABL1 fusion gene by using the established one-step reverse transcription digital PCR method, and firstly adopting a Charpy-Wilker method to test the normalization of data for the measurement result, wherein the uniformity data of all the standard substances meet the normal distribution. And then the Dixon's and Grubbs's inspection is adopted to reject the suspicious value, and no suspicious value is found. After F-test, the results showed that F < F0.05 (10, 22) for all standard substances, i.e. there was no significant difference in internal homogeneity of the standard substance solutions.
TABLE 3 results of uniformity test of BCR-ABL1 b2a2 fusion genes in genomic RNA
TABLE 4 results of uniformity test of BCR-ABL1 b3a2 fusion genes in genomic RNA
7. Monitoring the stability of the quantitative genomic RNA Standard of the BCR-ABL1 fusion Gene
In order to ensure the stability under the standard substance transportation condition, two temperatures of-20 ℃ and 4 ℃ are examined, 3 tubes of samples are randomly selected at sampling time points of 3 days, 7 days and 14 days respectively, the measurement is repeated for 3 times for each tube of samples, the copy number of two mutants of the BCR-ABL1 fusion gene, the copy number of the ABL-WT gene and the mutation gene abundance are determined, and the standard substance under the preservation condition of-80 ℃ is taken as a reference temperature, as shown in figures 5-9.
Thus, it can be demonstrated that the copy number concentration of the mutant gene of the standard substance and the mutation abundance do not change significantly within 14 days under the preservation conditions of-20 ℃ and 4 ℃. Therefore, dry ice is added in transportation or transportation is carried out under the condition that the temperature is controlled to be not more than 4 ℃, and the quantity of the standard substance can be kept stable as long as the transportation time is not more than 14 days, which lays an important foundation for the application of the standard substance.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (9)
1. The preparation method of the BCR-ABL1 fusion gene quantitative genome RNA standard substance is characterized by comprising the following steps:
(1) Extracting genome RNA of a cell line containing BCR-ABL1P210 type mutation, and detecting the concentration and purity of the genome RNA;
(2) The genome RNA of the BCR-ABL1P210 type mutant cell line is used as a template, a reverse transcription kit and PCR amplification are adopted to obtain a gene fragment with target fusion mutation, and the correctness of a target gene sequence is verified through Sanger sequencing PCR products;
(3) Adding a certain yeast RNA solution into RNA solution to prepare RNA storage buffer;
(4) Adding the genomic RNA of the cell line into the RNA storage buffer solution to dilute the genomic RNA into a standard solution with a certain concentration;
(5) Designing and obtaining a BCR-ABL1P210 type fusion gene b2a2 specific primer 1, a primer 2 and a specific probe 1, wherein the nucleotide sequences of the primer 1 and the primer 2 are shown as SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of the probe 1 is shown as SEQ ID No. 3;
designing and obtaining a BCR-ABL1P210 fusion gene b3a2 specific primer 3 and a primer 4 and a specific probe 2; the nucleotide sequences of the primer 3 and the primer 4 are shown as SEQ ID No.4 and SEQ ID No.5, and the nucleotide sequence of the probe 2 is shown as SEQ ID No. 6;
designing and obtaining ABL-WT gene specific primers 5, primers 6 and specific probes 3; the nucleotide sequences of the primer 5 and the primer 6 are shown as SEQ ID No.7 and SEQ ID No.8, and the nucleotide sequence of the probe 3 is shown as SEQ ID No. 9;
(6) The one-step reverse transcription digital PCR method is adopted, the BCR-ABL1P210 type fusion mutant genome RNA is used as a template for PCR amplification, fluorescent signals are collected to detect the expression of the BCR-ABL1P210 type fusion genes b2a2 and b3a2, and therefore the copy number content and the mutant gene abundance of the BCR-ABL1P210 type fusion genes b2a2, b3a2 and ABL-WT are obtained as the magnitudes.
2. The method for preparing the BCR-ABL1 fusion gene quantitative genomic RNA standard substance according to claim 1, wherein the method comprises the following steps: in the step (3) and the step (4), when the RNA storage buffer solution and the standard solution with a certain concentration are prepared, the solution is weighed, diluted and mixed by a balance weighing method.
3. The method for preparing the BCR-ABL1 fusion gene quantitative genomic RNA standard substance according to claim 1, wherein the method comprises the following steps: in the step (3), the concentration of yeast RNA is 5-500 ng/. Mu.L.
4. The method for preparing the BCR-ABL1 fusion gene quantitative genomic RNA standard substance according to claim 1, wherein the method comprises the following steps: in the step (5), FAM fluorescent groups are marked at the 5 'ends of the probe 1, the probe 2 and the probe 3, and BHQ1 fluorescent groups are marked at the 3' ends.
5. The method for preparing the BCR-ABL1 fusion gene quantitative genomic RNA standard substance according to claim 1, wherein the method comprises the following steps: in the step (5), the reaction concentration of the primer 1 and the primer 2 is 500nM, and the reaction concentration of the probe 1 is 400nM; the reaction concentration of the primer 3 and the primer 4 is 400nM, and the reaction concentration of the probe 2 is 300nM; the reaction concentration of the primer 5 and the primer 6 was 500nM, and the reaction concentration of the probe 3 was 400nM.
6. The method for preparing the BCR-ABL1 fusion gene quantitative genomic RNA standard substance according to claim 1, wherein the method comprises the following steps: the one-step reverse transcription digital PCR method comprises the following steps: 0 to 100ng,2 XOne-step Supermix (Bio-Rad) 5. Mu.L, reverse transcriptase. Mu.L, 300mM DTT 1. Mu.L, primer 1 and primer 2:1.0 μl, probe 1: 0.8. Mu.L; or primer 3 and primer 4:0.8 μl, probe 2: 0.6. Mu.L; or primer 5 and primer 6:1.0 μl, probe 3: 0.8. Mu.L.
7. The method for preparing the BCR-ABL1 fusion gene quantitative genomic RNA standard substance according to claim 6, wherein the method comprises the following steps: the PCR reaction conditions were: reverse transcription at 45℃for 60min and denaturation at 95℃for 10min; then denaturation at 94℃for 30sec,58℃for 60sec and annealing at 60℃for 40 cycles; finally, the mixture is extended for 10min at 98 ℃ and stored at 4 ℃.
8. The method for preparing the BCR-ABL1 fusion gene quantitative genomic RNA standard substance according to claim 1, wherein the method comprises the following steps: the mutant gene abundance calculating formula is as follows
9. The BCR-ABL1 fusion gene quantitative genomic RNA standard substance prepared by the preparation method of any one of claims 1 to 8.
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