CN110551821B - Primers, probe and kit for detecting MEF2D gene rearrangement by using fluorescent quantitative PCR - Google Patents
Primers, probe and kit for detecting MEF2D gene rearrangement by using fluorescent quantitative PCR Download PDFInfo
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Abstract
The invention discloses a kit for detecting MEF2D gene rearrangement by using fluorescence quantitative PCR, which is characterized by comprising: the RNA extraction reagent, the reverse transcription reagent, the detection system PCR reaction solution, the positive reference substance and the negative reference substance can detect MEF2D gene rearrangement in human B-ALL, can effectively save detection time, improve detection precision, are beneficial to clinically detecting MEF2D gene rearrangement in a B-ALL patient body, and have important significance for typing diagnosis, adjusting treatment schemes, evaluating treatment effects, predicting prognosis and preventing clinical relapse.
Description
Technical Field
The invention belongs to the fields of life science and biotechnology, and relates to a gene detection method for clinical examination, which can detect MEF2D gene rearrangement in human B-ALL by using a probe real-time fluorescent PCR technology, can effectively save detection time and improve detection precision.
Background
Leukemia is a group of highly heterogeneous hematopoietic malignancies, the etiology and pathogenesis of which have not been elucidated. Cytogenetic alterations have been found to play an important role in the development of leukemia, and common cytogenetic abnormalities such as chromosome number alterations, chromosome translocations, and abnormal expression of proto-oncogenes are common. They lead to the transformation of hematopoietic stem cells or committed progenitors into leukemia cells by altering the self-renewal, proliferation and differentiation mechanisms of hematopoietic cells. With the rapid development of immunology, molecular biology and molecular cytogenetics, people have deeper understanding on the molecular mechanism of leukemia occurrence, and find specific gene changes which are closely related to disease diagnosis and prognosis, such as acute promyelocytic leukemia accompanied by t (15. Besides common chromosome translocation types, some chromosome abnormalities have low incidence rate, but have important value for the differential diagnosis and prognosis of leukemia.
The Myocyte enhancer factor 2D (MEF 2D) gene is located at 1q22, and is a gene rearrangement identified in ALL in recent years, and there are mainly 5 gene fusions: BCL9 (1 q 21), HNRNPUL1 (19q13.2), DAZAP1 (19p13.3), CSF1R (5 q 32), and SS18 (18q11.2). MEF 2D-related fusion genes occur mainly in B-ALL in children and adolescents, with an incidence of about 2.4%. MEF2D is an important transcription factor in the body and can be involved in regulating the differentiation and development of muscle and nerve cells. MEF2D gene rearrangement has been found to enhance its transcriptional activity and lymphoid turnover, and thus may lead to a high risk leukemia subtype.
Although the incidence of MEF2D gene rearrangement is far less than that of common types such as BCL-ABL and the like, the incidence rate is only about 2 percent, however, the pathogenesis of leukemia carrying different types of gene rearrangement is different, the diagnosis standards of cytogenetics and molecular biology are different, and the treatment scheme and prognosis are also different, so the detection of MEF2D gene rearrangement plays an important role in the typing diagnosis, the treatment scheme and the prognosis evaluation of leukemia, particularly B-ALL.
The common techniques for detecting MEF2D gene rearrangement include Fluorescence In Situ Hybridization (FISH) and RQ-PCR. The FISH detection result is visual, but the test process is complicated, the related reagents are various, time and labor are wasted, the result needs to be interpreted by professionals with rich experience, and the result interpretation has larger subjectivity. RQ-PCR adopts Taqman probe fluorescence quantitative technology, integrates biology, enzymology and fluorescence chemistry into a whole, and amplification and result analysis are all carried out in a PCR reaction tube closed state, so that the problem of false positive caused by PCR product pollution is solved, meanwhile, the sensitivity is also improved, the result is expressed by copy number, the accurate quantification of the PCR product is realized, and the standard is easy to unify. The detection method can meet the requirement of MEF2D gene rearrangement, is considered to be the first detection method at present, and is used for evaluating treatment effect and predicting prognosis. Common methods in real-time fluorescent quantitative PCR include SYBR GreenI dye method, double-probe hybridization method, taqman technology and the like. Wherein, SYBR GreenI is unsaturated dye, so the specificity is not as good as that of a double-probe hybridization method and a Taqman method, and the specificity is judged by observing a dissolution curve; the two-probe hybridization method is expensive. Therefore, the real-time fluorescence PCR technology and the Taqman probe method are combined for gene rearrangement detection of MEF2D in the research.
Disclosure of Invention
Aiming at the defects of the prior art, the invention discloses a primer, a probe and a kit for detecting MEF2D gene rearrangement by utilizing fluorescent quantitative PCR, which can detect MEF2D gene rearrangement in human B-ALL, effectively save detection time and improve detection precision.
The primers and the probes for detecting MEF2D gene rearrangement by using fluorescent quantitative PCR are characterized in that the base sequences of the primers and the probes are as follows:
MEF2D-BCL9(5-6/9)-F：TAATAGAGAGTGGGTTCTGGGA
MEF2D-BCL9(5-6/9)-Probe：
FAM-AGACTTCCAGGGATATTCACAGAGGC-BHQ1
MEF2D-BCL9(5-6/9)-R：GCCAGCACTACAGAGGAACA
MEF2D-BCL9(5-6/10)-F：ATTGGGCTGAGGGTTGG
MEF2D-BCL9(5-6/10)-Probe：FAM-CGGAACCACGGGGTTTGGACC-BHQ1
MEF2D-BCL9(5-6/10)-R：GCCAGCACTACAGAGGAACA
MED2F-HNRNPUL1-F：ACGCCGAGTTTACTCAGCC
MED2F-HNRNPUL1/SS18-Probe：
FAM-TCCATGCCCACTGCCTACAACAC-BHQ1
MED2F-HNRNPUL1-R：CCAGGAAGTCCCCAACATCT
MED2F-SS18-F：TGGCAACGCCGAGTTTACT
MED2F-SS18-R：TTGACCCATCATTCCCATAGG
MED2F-DAZAP1-F TAACTTCCACTTTTTTGCCCAT
MED2F-DAZAP1-Probe：FAM-TGGTCCACTGATTGTTCGTCCTCG-BHQ1
MED2F-DAZAP1-R：AGCGGCCAGCTAGTGCG
MED2F-CSF1R-F：CCAGGCAGGAAAGGGGTT
MED2F-CSF1R-Probe：FAM-TGAAGCCCAAGTACCAGGTCCGC-BHQ1
MED2F-CSF1R-R：GCGTGGGGTCGATGAAAG。
further, the primers and the probes also comprise primers and probes for detecting ABL reference genes, and the primers and the probes are respectively as follows:
Abl-F:GATACGAAGGGAGGGTGTACCA
Abl-R:CTCGGCCAGGGTGTTGAA
Abl-Probe:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-BHQ1。
the invention also provides a kit for detecting MEF2D gene rearrangement by using fluorescent quantitative PCR, which is characterized by comprising: RNA extraction reagent, reverse transcription reagent, detection system PCR reaction liquid, positive reference substance and negative reference substance; wherein the PCR reaction solution of the detection system comprises:
(1) The upstream and downstream primers and the probe for detecting the target gene have the following sequences:
MEF2D-BCL9(5-6/9)-F：TAATAGAGAGTGGGTTCTGGGA
MEF2D-BCL9(5-6/9)-Probe：
FAM-AGACTTCCAGGGATATTCACAGAGGC-BHQ1
MEF2D-BCL9(5-6/9)-R：GCCAGCACTACAGAGGAACA
MEF2D-BCL9(5-6/10)-F：ATTGGGCTGAGGGTTGG
MEF2D-BCL9(5-6/10)-Probe：FAM-CGGAACCACGGGGTTTGGACC-BHQ1
MEF2D-BCL9(5-6/10)-R：GCCAGCACTACAGAGGAACA
MED2F-HNRNPUL1-F：ACGCCGAGTTTACTCAGCC
MED2F-HNRNPUL1/SS18-Probe：
FAM-TCCATGCCCACTGCCTACAACAC-BHQ1
MED2F-HNRNPUL1-R：CCAGGAAGTCCCCAACATCT
MED2F-SS18-F：TGGCAACGCCGAGTTTACT
MED2F-SS18-R：TTGACCCATCATTCCCATAGG
MED2F-DAZAP1-F TAACTTCCACTTTTTTGCCCAT
MED2F-DAZAP1-Probe：FAM-TGGTCCACTGATTGTTCGTCCTCG-BHQ1
MED2F-DAZAP1-R：AGCGGCCAGCTAGTGCG
MED2F-CSF1R-F：CCAGGCAGGAAAGGGGTT
MED2F-CSF1R-Probe：FAM-TGAAGCCCAAGTACCAGGTCCGC-BHQ1
MED2F-CSF1R-R：GCGTGGGGTCGATGAAAG；
(2) The upstream and downstream primers and the probes for detecting the ABL reference gene have the following sequences:
Abl-F:GATACGAAGGGAGGGTGTACCA
Abl-R:CTCGGCCAGGGTGTTGAA
Abl-Probe:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-BHQ1。
further, the kit also comprises a positive control substance and a negative control substance, wherein the positive control substance is a MEF2D fusion gene-containing solution; the negative control is a solution without MEF2D fusion gene.
The invention combines the real-time fluorescence PCR technology with the Tapman probe and utilizes the method of double standard curves to respectively construct the quantitative standard curves of reference genes ABL and MEF2D target genes and detect the MEF2D gene rearrangement condition in a tested person. Compared with the traditional detection means such as FISH, delta-delta CT and the like, the method has the advantages of high accuracy, convenient result interpretation and the like. The method has good specificity, high sensitivity and simple operation. The method is beneficial to clinically detecting MEF2D gene rearrangement in B-ALL patients, and has important significance for typing diagnosis, adjusting treatment schemes, evaluating treatment effects, predicting prognosis and preventing clinical relapse.
Drawings
FIG. 1 is a representation of the MEF2D gene rearrangement positive plasmid amplification curve;
fig. 2 is a representation of the amplification curve for the clinical sample MEF2D gene rearrangement No. 1.
Detailed Description
Example 1
The invention is used for assisting the typing diagnosis of the B-ALL clinically and a method for establishing an individual treatment scheme. Mainly comprises the following reagents: a lysate of red blood cells; TRIzol; chloroform; isopropyl alcohol; absolute ethyl alcohol;
detection system PCR reaction solution: reverTra Ace qPCR RT Kit (TOYOBO Co.); primers and probes of THUNDERBIRD Probe qPCR Mix (2X), ABL reference gene and MEF2D target gene are all 10 μ M;
wherein, the primers and probes for detecting the reference gene ABL and the target gene MEF2D are respectively as follows:
positive control: a solution containing the MEF2D genome;
negative control: solution without MEF2D genome.
Example 2
The method comprises the following operation flows:
(1) Extraction of total RNA in blood: 1ml of erythrocyte lysate is added into a clean centrifugal tube with 1.5ml, and 0.5ml of anticoagulation blood is taken and mixed evenly. Standing at room temperature for 10min; centrifuging at 1500rpm for 5min, discarding the supernatant, and collecting the cells at the bottom; adding 0.5ml of erythrocyte lysate again, centrifuging at 1500rpm for 5min, discarding the supernatant, and collecting the cells at the bottom; adding 1ml of TRIzol into the cells, repeatedly blowing and beating until the precipitate is completely dissolved, and standing at room temperature for 5min; adding 0.2ml of chloroform, and shaking uniformly; centrifuging at 14000rpm at 4 ℃ for 10min, sucking the supernatant and transferring to another new centrifuge tube; adding isopropanol with the same volume, mixing thoroughly, standing at room temperature for 10min; centrifuging at 14000rpm for 10min at 4 ℃, discarding the supernatant, adding 1ml of 75% ethanol, and slightly turning upside down to wash the tube wall; centrifuging at 14000rpm at 4 ℃ for 5min, and removing ethanol; drying at room temperature for 10-15min, adding 20ul RNase-free water to dissolve the precipitate.
(2) RNA was inverted to cDNA with reference to the Rever Tra Ace qPCR RT Kit instruction manual from TOYOBO.
(3) Reagent preparation: preparing X mu L of PCR reaction liquid of a detection system according to the number of detected persons, and subpackaging 23 mu L of each person:
x =23 μ L reaction X (8 parts internal reference (standard curve) +8 parts target gene (standard curve) + n parts specimen +1 part positive control +1 part negative control +1 part blank control);
(4) Sample adding: adding 2 mu L of cDNA into PCR reaction liquid of a detection system; directly adding 2 mu L of positive control substance and negative control substance into the positive control substance and the negative control substance; blank control was supplemented with 2. Mu.L of physiological saline or nothing.
(5) And (3) detection: the detection was performed on a real-time fluorescent PCR instrument, and available instruments include ABI7300, 7500 (Applied Biosystems, USA), and the like. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1min;95 ℃ 15s,60 ℃ 40sec40 cycles, fluorescence signal at 60 ℃ 40sec acquisition.
(6) And (5) judging a result: the threshold line is adjusted to be above the background signal and the negative amplification line, and the system automatically calculates the copy number according to the standard curve and the CT value.
1) When the internal reference is positive, the detection result is considered to be effective;
2) Positive judgment standard: ct <36, positive; ct is more than or equal to 35 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative.
Example 3
The nucleic acid detection method of the invention is adopted to detect the health physical examination crowd samples
22 samples of the physical examination to be detected were taken, and the genome was extracted, the reagent was prepared, and the detection was carried out as described in example 2.
Each sample was added to 2. Mu.L of the detection system PCR reaction solution. And simultaneously, making positive, negative and blank controls, and respectively copying standard curves of reference genes/target genes. A96-well fluorescent PCR instrument can simultaneously detect 22 samples, each sample is repeated 2 times, one positive control and one negative control, and the detection time is only 70 minutes. The ABL lines for all of the 22 screened specimens appeared, but no specimen appeared in the MEF 2D. The results are shown in Table 1.
TABLE 1 MEF2D mRNA expression levels in 22 physical examination samples
Example 4
Clinical specimens detected by the nucleic acid detection method of the invention
22 clinical samples to be detected are taken, and the genome is extracted, the reagent is prepared and detected according to the method described in the embodiment 2.
Each sample was added to 2. Mu.L of the detection system PCR reaction solution. And simultaneously, making positive, negative and blank controls, and respectively copying standard curves of reference genes/target genes. A96-well fluorescent PCR instrument can simultaneously detect 22 samples, each sample is repeated for 2 times, one positive control and one negative control, and the detection time is only 70 minutes. All of the 22 samples screened showed line-up in ABL, but no specimen in MEF 2D. The results of the experiment are shown in table 2 below:
TABLE 2 clinical samples MEF2D mRNA expression levels
FIG. 1 shows the expression of MEF2D gene rearrangement positive plasmid amplification curve.
Fig. 2 is a representation of the amplification curve of the clinical sample MEF2D gene rearrangement No. 1.
The primers, the probes and the kit for detecting MEF2D gene rearrangement by using fluorescent quantitative PCR can detect MEF2D gene rearrangement in human B-ALL, can effectively save detection time and improve detection precision.
Sequence listing
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Claims (4)
1. The primers and the probes for detecting MEF2D gene rearrangement by using fluorescent quantitative PCR are characterized in that the base sequences of the primers and the probes are as follows:
MEF2D-BCL9(5-6/9)-F：TAATAGAGAGTGGGTTCTGGGA
MEF2D-BCL9(5-6/9)-Probe：FAM-AGACTTCCAGGGATATTCACAGAGGC-BHQ1
MEF2D-BCL9(5-6/9)-R：GCCAGCACTACAGAGGAACA
MEF2D-BCL9(5-6/10)-F：ATTGGGCTGAGGGTTGG
MEF2D-BCL9(5-6/10)-Probe：FAM-CGGAACCACGGGGTTTGGACC-BHQ1
MEF2D-BCL9(5-6/10)-R：GCCAGCACTACAGAGGAACA
MED2F-HNRNPUL1-F：ACGCCGAGTTTACTCAGCC
MED2F-HNRNPUL1 -Probe：FAM-TCCATGCCCACTGCCTACAACAC-BHQ1
MED2F-HNRNPUL1-R：CCAGGAAGTCCCCAACATCT
MED2F-SS18-F：TGGCAACGCCGAGTTTACT
MED2F- SS18-Probe：FAM-TCCATGCCCACTGCCTACAACAC-BHQ1
MED2F-SS18-R：TTGACCCATCATTCCCATAGG
MED2F-DAZAP1-F TAACTTCCACTTTTTTGCCCAT
MED2F-DAZAP1-Probe：FAM-TGGTCCACTGATTGTTCGTCCTCG-BHQ1
MED2F-DAZAP1-R：AGCGGCCAGCTAGTGCG
MED2F-CSF1R-F：CCAGGCAGGAAAGGGGTT
MED2F-CSF1R-Probe：FAM-TGAAGCCCAAGTACCAGGTCCGC-BHQ1
MED2F-CSF1R-R：GCGTGGGGTCGATGAAAG。
2. the primers and probes as claimed in claim 1, further comprising primers and probes for detecting ABL reference genes, respectively:
Abl-F: GATACGAAGGGAGGGTGTACCA
Abl-R: CTCGGCCAGGGTGTTGAA
Abl-Probe: FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-BHQ1。
3. the kit for detecting MEF2D gene rearrangement by using fluorescence quantitative PCR is characterized by comprising: RNA extraction reagent, reverse transcription reagent, detection system PCR reaction liquid, positive reference substance and negative reference substance; wherein the PCR reaction solution of the detection system comprises:
(1) The upstream and downstream primers and the probe for detecting the target gene have the following sequences:
MEF2D-BCL9(5-6/9)-F：TAATAGAGAGTGGGTTCTGGGA
MEF2D-BCL9(5-6/9)-Probe：FAM-AGACTTCCAGGGATATTCACAGAGGC-BHQ1
MEF2D-BCL9(5-6/9)-R：GCCAGCACTACAGAGGAACA
MEF2D-BCL9(5-6/10)-F：ATTGGGCTGAGGGTTGG
MEF2D-BCL9(5-6/10)-Probe：FAM-CGGAACCACGGGGTTTGGACC-BHQ1
MEF2D-BCL9(5-6/10)-R：GCCAGCACTACAGAGGAACA
MED2F-HNRNPUL1-F：ACGCCGAGTTTACTCAGCC
MED2F-HNRNPUL1 -Probe：FAM-TCCATGCCCACTGCCTACAACAC-BHQ1
MED2F-HNRNPUL1-R：CCAGGAAGTCCCCAACATCT
MED2F-SS18-F：TGGCAACGCCGAGTTTACT
MED2F- SS18-Probe：FAM-TCCATGCCCACTGCCTACAACAC-BHQ1
MED2F-SS18-R：TTGACCCATCATTCCCATAGG
MED2F-DAZAP1-F TAACTTCCACTTTTTTGCCCAT
MED2F-DAZAP1-Probe：FAM-TGGTCCACTGATTGTTCGTCCTCG-BHQ1
MED2F-DAZAP1-R：AGCGGCCAGCTAGTGCG
MED2F-CSF1R-F：CCAGGCAGGAAAGGGGTT
MED2F-CSF1R-Probe：FAM-TGAAGCCCAAGTACCAGGTCCGC-BHQ1
MED2F-CSF1R-R：GCGTGGGGTCGATGAAAG；
(2) The upstream and downstream primers and the probes for detecting the ABL reference gene have the following sequences:
Abl-F: GATACGAAGGGAGGGTGTACCA
Abl-R: CTCGGCCAGGGTGTTGAA
Abl-Probe: FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-BHQ1。
4. the kit of claim 3, wherein the positive control is a solution containing MEF2D fusion gene; the negative control is a solution without MEF2D fusion gene.
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| CN109593861A (en) * | 2019-02-18 | 2019-04-09 | 南方医科大学 | The detection method and detection kit of different loci leukaemia MEF2D-BCL9 fusion oligonucleotides |
| CN110093421A (en) * | 2019-05-09 | 2019-08-06 | 首都医科大学附属北京儿童医院 | Leukaemia MEF2D gene break probe in detecting kit |
| WO2022178185A1 (en) * | 2021-02-17 | 2022-08-25 | Act Genomics (Ip) Co., Ltd. | Dna fragment joining detecting method and kit thereof |
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2019
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593861A (en) * | 2019-02-18 | 2019-04-09 | 南方医科大学 | The detection method and detection kit of different loci leukaemia MEF2D-BCL9 fusion oligonucleotides |
| CN110093421A (en) * | 2019-05-09 | 2019-08-06 | 首都医科大学附属北京儿童医院 | Leukaemia MEF2D gene break probe in detecting kit |
| WO2022178185A1 (en) * | 2021-02-17 | 2022-08-25 | Act Genomics (Ip) Co., Ltd. | Dna fragment joining detecting method and kit thereof |
Non-Patent Citations (2)
| Title |
|---|
| Genomic analyses identify recurrent MEF2D fusions in acute lymphoblastic leukaemia;Zhaohui Gu;《nature communications》;20161108;第1-10页 * |
| Ph阴性青少年与年轻成人急性淋巴细胞白血病预后及治疗综述;高泓浩;《解放军医学院学报》;20170516;第38卷(第4期);第389-392页 * |
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