CN113308483A - DENND1A-SGTA fusion gene and application and detection kit thereof - Google Patents
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Abstract
The invention provides a DENND1A-SGTA fusion gene and application and a detection kit thereof, wherein the fusion gene sequence is shown as SEQ ID NO. 1. The invention firstly discovers and identifies the existence of a novel fusion gene DENND1A-SGTA in acute lymphocytic leukemia, evaluates that DENND1A-SGTA can be used as a specific molecular marker of the acute lymphocytic leukemia, and further develops a kit which can be clinically used for diagnosing and detecting Minimal Residual Disease (MRD) of a patient. The kit carries out absolute quantification on DENND1A-SGTA fusion genes in acute lymphocytic leukemia patients by using Taqman probe real-time fluorescent PCR, has good detection specificity, high sensitivity, simple, convenient and efficient method and low cost, and can meet the clinical diagnosis and treatment requirements.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an acute lymphoblastic leukemia fusion gene and application and a detection kit thereof.
Background
Acute B lymphocytic leukemia (B-ALL) is a hematologic malignancy, accounting for 80-85% of ALL acute lymphoblastic leukemias, and is characterized by proliferation and extensive infiltration of B lymphoblasts in the bone marrow, peripheral blood and lymphoid organs. In the past decades, B-ALL survival has been significantly improved by monitoring bone marrow cytology, immunophenotyping, molecular residues (especially the discovery of BCR-ABL fusion genes), etc., while the 5-year Overall Survival (OS) of adults and elderly patients is still only 30% -40%. With the rapid development of molecular genetic diagnostics, dozens of fusion genes such as BCR-ABL1, E2A-PBX1, ETV6-RUNX1, MLL rearrangement and the like are reported in B-ALL, but a large proportion of B-ALL patients lack molecular markers, so that great difficulty is brought to clinical diagnosis and treatment prognosis. Therefore, the discovery of the novel fusion gene related to B-ALL has important clinical significance for disease diagnosis, treatment scheme formulation, curative effect evaluation, prognosis evaluation and disease recurrence monitoring.
The DENND1A gene, also known as DENN Domain containment 1A, has the main functions of promoting GDP conversion to GTP, regulating clathrin-mediated synaptic vesicle endocytosis and mediating early endosome withdrawal. SGTA gene, namely small glutamine rich tetratricopeptide repeat co-factor alpha, is ubiquitously expressed in various tissues, and encodes a protein which can participate in the regulation and control of protein catabolism processes of folding errors through the interaction with BAG 6. The two genes form a novel fusion gene DENND1A-SGTA in the B-ALL patient and are molecular markers which are not reported in the literature to date.
Disclosure of Invention
The first purpose of the invention is to provide an acute lymphoblastic leukemia fusion gene DENND 1A-SGTA.
The second purpose of the invention is to provide the application of the fusion gene DENND1A-SGTA in preparing a kit for diagnosing or monitoring acute lymphocytic leukemia.
The third purpose of the invention is to provide a kit for detecting DENND1A-SGTA fusion gene.
The fourth purpose of the invention is to provide a method for detecting DENND1A-SGTA fusion gene for non-disease diagnosis.
In order to realize the first object, the invention provides a DENND1A-SGTA fusion gene, and the sequence of the fusion gene is shown as SEQ ID NO. 1.
In order to realize the second purpose, the invention provides the application of the fusion gene DENND1A-SGTA in preparing a kit for diagnosing or monitoring acute lymphocytic leukemia. Part of acute lymphoblastic leukemia patients carry the fusion gene DENND1A-SGTA, which can be used as a patient-specific molecular marker and applied to clinical diagnosis and regular monitoring of minimal residual lesions of the patients.
In order to realize the third purpose, the invention provides a kit for detecting the fusion gene DENND1A-SGTA, which comprises an upstream primer, a downstream primer, a Taqman probe, an internal reference, a PCR reaction buffer solution, a positive control and a negative control;
the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the Taqman probe is shown in SEQ ID NO. 4.
Preferably, the 5 'end of the Taqman probe is labeled with FAM group, and the 3' end of the Taqman probe is labeled with TAMRA group. When the target DNA molecule does not exist in the sample to be detected, the fluorescence of FAM is quenched by TAMRA and does not emit fluorescence; when the target DNA molecule exists in the sample to be detected, the probe is combined with the target DNA molecule, and then the probe is degraded by the 5'→ 3' double-strand exonuclease activity of Taq enzyme to release the fluorescent group FAM, so that fluorescence is emitted.
As a preferred scheme, the internal reference comprises an internal reference upstream primer, an internal reference downstream primer and an internal reference probe;
the nucleotide sequence of the internal reference upstream primer is shown as SEQ ID NO. 5;
the nucleotide sequence of the internal reference downstream primer is shown as SEQ ID NO. 6;
the nucleotide sequence of the internal reference probe is shown as SEQ ID NO. 7.
The 5 'end of the probe of the internal reference gene ABL can be marked with FAM group, the 3' end of the probe can be marked with TAMRA group, the FAM group is a report group, and the TAMRA group is a quenching group.
The PCR reaction buffer is a buffer commonly used in the art, and generally comprises a first strand cDNA synthesis reagent TIANCcript II RT Kit (TIANGEN Corp.) and a real-time fluorescence PCR mixture (Pro Taq HS Premix Probe qPCR Kit, Acurrate Biology Corp., AG11704) whose main components comprise DNA polymerase, Mg2+dNTP, reverse transcriptase, DTT.
The positive control contains a plasmid standard with DENND1A-SGTA fusion gene and a plasmid with ABL gene. The negative control contained deionized water and cDNA from 10 healthy bone marrow donors.
In order to achieve the fourth object, the present invention provides a method for detecting a DENND1A-SGTA fusion gene for non-disease diagnostic purposes, comprising the steps of:
(1) extracting total RNA in a human blood sample, and inverting the RNA into cDNA serving as a sample to be detected;
(2) preparing PCR reaction liquid, and then respectively adding a sample to be detected, a positive control and a negative control;
(3) detecting on a real-time fluorescent PCR instrument, wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; reacting at 95 ℃ for 15s and 58 ℃ for 35s for 40 cycles, and collecting fluorescence signals at 60 ℃;
the PCR reaction solution comprises an upstream primer, a downstream primer, a Taqman probe, an internal reference, a PCR reaction buffer solution, a positive control and a negative control, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the Taqman probe is shown in SEQ ID NO. 4.
The conditions for detecting the establishment of the experimental result are as follows: if no target gene amplification signal curve exists, the result is negative when the internal reference, the positive control and the negative control are detected normally; if the target gene amplification signal curve and the internal reference, positive control and negative control detection are normal, the result is positive. If the internal reference, the positive control and the negative control are abnormal, the reason needs to be found out, and the detection is carried out again after adjustment.
The invention discovers and verifies that part of acute lymphoblastic leukemia carries the fusion gene DENND1A-SGTA, and then develops a fusion gene detection and quantification kit combining real-time fluorescence PCR and Taqman probe technology. The real-time fluorescence PCR result is expressed by Ct value, has the advantages of good specificity, high sensitivity, simple operation, more visual result and the like, and is the preferred method for detecting the trace fusion gene at present, so the kit adopts Taqman probe real-time fluorescence PCR to detect DENND1A-SGTA fusion gene to monitor the MRD of a patient.
The invention has the advantages that (1) the fusion gene DENND1A-SGTA carried by the patient with acute lymphocytic leukemia is screened and identified by applying bioinformatics technology, which proves that the fusion gene DENND is a novel fusion gene which is not reported so far and can be used as a molecular marker of the patient to be applied to clinical diagnosis, selection of a proper treatment scheme and periodic monitoring of MRD of the patient.
(2) The DENND1A-SGTA fusion gene detection kit provided by the invention has the following advantages: the accuracy is high: and meanwhile, the probe and the primer are used for dual control, so that the specificity is good, and the false positive is low. ② the specificity is strong: the fusion gene sequence was recognized using a specific probe. ③ monitoring in the whole process: and monitoring the whole-course amplification signal in real time. Fourthly, safety, convenience and convenience are achieved: the operation is simple and safe, the automation degree is high, and the pollution is prevented. Fast: the detection completion time was 120 min.
(3) The invention adopts the real-time fluorescence PCR technology and the Taqman probe to detect the expression conditions of DENND1A-SGTA fusion gene and reference gene ABL in a tested body, can distinguish low-abundance gene signals from a complex background, and has great application potential in the monitoring of patient diagnosis, treatment scheme adjustment, treatment effect evaluation, prognosis prediction and clinical relapse prevention.
Drawings
FIG. 1 shows the structure of DENND1A-SGTA fusion gene and PCR, sanger verification.
FIG. 2 shows the construction results of plasmid standards for the DENND1A-SGTA fusion gene.
FIG. 3 is a graphical representation of the results of the detection of DENND1A-SGTA using the kit of the invention.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.
Example 1 acute lymphocytic leukemia patients carry the novel DENND1A-SGTA fusion gene
(1) The bioinformatics technology is applied to screen out the fusion gene of the patient with acute lymphoblastic leukemia and a DENND1A-SGTA new fusion gene which is not reported in the literature.
(2) The DENND1A-SGTA new fusion gene is formed by splicing exons 1 to 11 of the DENND1A gene and exon 12 of SGTA, and the specific CDS sequence is shown as SEQ ID NO. 1.
(3) Using PCR and sanger sequencing of the PCR products, we confirmed that this example of acute lymphoblastic leukemia patients carried the DENND1A-SGTA new fusion gene, and the results are shown in FIG. 1. FIG. 1 shows the structure of DENND1A-SGTA fusion gene and PCR, sanger verification. A: the transfer of the 9q33.3 region and the 19p13.3 region resulted in the graphical display of the occurrence of the DENND1A-SGTA fusion gene; b: PCR verification of the fusion gene in the patient's initial and control samples; c: the sanger sequencing result chart of the PCR-verified product of the patient carrying the fusion gene confirms the fusion of the DENND1A exon 11 and the SGTA exon 12.
(4) Selecting a proper plasmid, cloning a section of sequence containing the fusion breakpoint into the plasmid, selecting positive clones from the plasmids to perform PCR amplification and sanger sequencing, and verifying the correctness of the sequence transferred into the plasmid, thereby obtaining the standard product. FIG. 2 shows the construction results of plasmid standards for the DENND1A-SGTA fusion gene. A: fusing gene standard quality particle pGEM T-easy-DENND1A-SGTA map; b: sequencing result of standard plasmid sanger.
(5) Primers and probes for detecting the reference/target genes are designed, and the real-time fluorescent PCR technology is adopted to detect the expression conditions of the reference gene ABL and the DENND1A-SGTA fusion genes. The kit can ensure that the amplification efficiency and the amplification rate are both optimal by adjusting the primer probe ratio of the internal reference/target gene and the PCR reaction conditions.
Example 2 preparation of the kit
1. Design of specific primers and probes
Specific probes and primers were designed based on the Gene sequences (ABL1 Gene sequence, DENND1A Gene sequence, SGTA Gene sequence all from the nucleic acid database of the national center for Biotechnology information, ABL1 Gene Entrez Gene ID25, Gene reference sequence NM-005157.5, DENND1A Gene Entrez Gene ID 57706, Gene reference sequence NM-001352964.2, SGTA Gene Entrez Gene ID 6449, Gene reference sequence NM-003021.4).
2. Reagent kit component dispensing system
First strand cDNA Synthesis reagents: TIANCcript II RT Kit (TIANGEN Co.), detection System PCR reaction: pro Taq HS Premix Probe qPCR Kit (Acurrate Biology, AG11704) comprising DNA polymerase, Mg2+dNTP, reverse transcriptase, DTT.
Primers and probes: the method comprises the steps of detecting DENND1A-SGTA fusion gene, internal reference ABL primers and probes corresponding to the primers, and specifically comprises the following steps:
DENND1A-SGTA-F:CGCATCTGCTGGAC(SEQ ID NO.2)
DENND1A-SGTA-R:GGGCTGTAAGGGAGT(SEQ ID NO.3)
DENND1A-SGTA-Probe:FAM-ATTTAAGTTTAATGGAGCTGCTCCCGGTG-TAMRA(SEQ ID NO.4)
ABL1-F:CTAAAGGTGAAAAGCTCCG(SEQ ID NO.5)
ABL1-R:GACTGTTGACTGGCGTGAT(SEQ ID NO.6)
ABL1-Probe:FAM-CCATTTTTGGTTTGGGCTTCACACCATT-TAMRA(SEQ ID NO.7)。
positive control: comprises a plasmid standard product with a DENND1A-SGTA fusion gene and a plasmid with an ABL gene; negative control: deionized water and cDNA from 10 healthy bone marrow donors.
Example 3 protocol for testing ALL patient samples with the kit
1. Taking the anticoagulated blood sample of the ALL patient to be detected, extracting 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 10 min; centrifuging at 5000rpm for 5min, discarding supernatant, and collecting cells at bottom; adding 0.5ml of erythrocyte lysate again, centrifuging at 5000rpm 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 for 5min at room temperature; adding 0.2ml of chloroform, and shaking uniformly; centrifuging at 14000rpm and 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 10 min; centrifuging at 14000rpm and 4 ℃ for 10min, removing the supernatant, adding 1ml of 75% ethanol, and slightly reversing the upper part and the lower part to wash the tube wall; centrifuging at 14000rpm and 4 ℃ for 5min, and removing ethanol; drying at room temperature for 10-15min, adding 20 μ l RNase-free water to dissolve precipitate.
2. The RNA was inverted to cDNA, according to the TIANSEN Kit instructions for TIANCcript II RT Kit.
3. Reagent preparation: preparing X mul of PCR reaction liquid of a detection system according to the number of detected persons, wherein X is 23 mul of reaction liquid X (n parts of specimen +1 part of positive control +1 part of negative control +1 part of blank control), and subpackaging 23 mul of each person.
4. Sample adding: adding 2 mu lcDNA into the PCR reaction solution of the detection system; directly adding 2 mul 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 1 min; the reaction was carried out at 95 ℃ for 15s and at 58 ℃ for 35s for 40 cycles, and the fluorescence signal was collected at 60 ℃.
6. And (5) judging a result: if no target gene amplification signal curve exists, the result is negative when the internal reference, the positive control and the negative control are detected normally; if the target gene amplification signal curve and the internal reference, positive control and negative control detection are normal, the result is positive. If the internal reference, the positive control and the negative control are abnormal, the reason needs to be found out, and the detection is carried out again after adjustment.
7. DENND1A-SGTA can be used as a molecular marker specific to acute lymphocytic leukemia patients: we performed MRD monitoring on 5 time node samples of clinical treatment of one example of DENND1A-SGTA positive patients using the test kit constructed above. FIG. 3 is a graphical representation of the results of the detection of DENND1A-SGTA using the kit of the invention. A: DENND1A-SGTA positive samples and negative controls; b: DENND1A-SGTA Positive patients changes at mRNA levels with treatment, DENND 1A-SGTA. Sample 1, initiating; sample 2 after chemotherapy; sample 3, 5 months after transplantation; sample4 recurrence; sample 5 remission after reinfusion. We found that (1) the expression level of DENND1A-SGTA in the patient is high in the initial stage (Sample 1), and the expression level of DENND1A-SGTA is obviously reduced along with the obvious relief of induction chemotherapy and transplantation (Sample 2 and Sample 3) of clinical administration; (2) the flow of the patient is quickly rechecked to show that the original cell is increased again (Sample 4), the expression level of DENND1A-SGTA is increased again, at the moment, the lymphocyte infusion is given to a donor, the expression level of DENND1A-SGTA is monitored to be reduced again (Sample 5), and the condition that the expression level of DENND1A-SGTA is consistent with the disease progression trend is shown, but the risk of vigilance and relapse still needs to be monitored in real time. The results show that DENND1A-SGTA can be used as a novel molecular marker of a patient to monitor MRD, and the kit is proved to be accurate and reliable.
The results show that the kit can detect samples quickly and accurately with high flux, has good specificity and repeatability, and can effectively avoid false positive and false negative results. The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.
SEQUENCE LISTING
<110> university of Dalian medical university affiliated second Hospital
Xinhua Hospital Affiliated to Medical College of Shanghai Jiaotong University
<120> DENND1A-SGTA fusion gene and application and detection kit thereof
<130> /
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1984
<212> DNA
<213> Artificial Synthesis
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atgggctcca ggatcaagca gaatccagag accacatttg aagtatatgt tgaagtggcc 60
tatcccagga caggtggcac tctttcagat cctgaggtgc agaggcaatt cccggaggac 120
tacagtgacc aggaagttct acagactttg accaagtttt gtttcccctt ctatgtggac 180
agcctcacag ttagccaagt tggccagaac ttcacattcg tgctcactga cattgacagc 240
aaacagagat tcgggttctg ccgcttatct tcaggagcga agagctgctt ctgtatctta 300
agctatctcc cctggttcga ggtattttat aagctgctta acatcctggc agattacacg 360
acaaaaagac aggaaaatca gtggaatgag cttcttgaaa ctctgcacaa acttcccatc 420
cctgacccag gagtgtctgt ccatctcagc gtgcattctt attttactgt gcctgatacc 480
agagaacttc ccagcatacc tgagaataga aatctgacag aatattttgt ggctgtggat 540
gttaacaaca tgttgcatct gtacgccagt atgctgtacg aacgccggat actcatcatt 600
tgcagcaaac tcagcactct gactgcctgc atccacgggt ctgcggcgat gctctacccc 660
atgtactggc agcacgtgta catccccgtg ctgccgccgc atctgctgga ctactgctgt 720
gctcccatgc cctacctcat aggaatccat ttaagtttaa tggagctgct cccggtgtga 780
ccgcgtcctt ccctggccga cccgaaggaa gccttctggt tgtctgccac ttcctcctgt 840
tggactgcct gagagagggg aagagagaga cctcggacct gcatgtcaag atggattttc 900
cccttttatc tctgccctcc tccactccct ttttgtaact cccttacagc ccccagaccc 960
ttcttgaaac gagagccagc aagctgagca cagaccagca gcgacctccc ttccagcccc 1020
cagaaagctc ggtcacttga gtgttttcta gaatcctggg gtgctcccgg gccgctctca 1080
gagaagtggc aggtttcacg ttcagccgtg tggcggatcg tgtggcttcc aaagcctttt 1140
acagcccccg ccccccatcc cgtggtctgt ctgcaggaac tctcccgtct gtgagaagcc 1200
tctttccgag tcgacctccc ggccaccccg gccctgtgcc tgctcggaag agctcactgc 1260
cagctgcggc ctgggcaccg cgggccatgt gtgtttgcat gaggaactct ttagtggcag 1320
acacctaaga gacggctgcg gtcaccccac gcctccgcgg ctcaggagcc gtcctgggtg 1380
cataggacca gtttctgtga cttttctcca gttgggcatg ttgacagaca tgtttcccct 1440
cctcccaccc tcattttctg gtcctcgcga ctgagagcca ggggcgacat catgaccttc 1500
tgtcccggcc gccttagccc cgggcacagg gaaggcagct gggccgtttc tgtctgtgtc 1560
ccatcctgct gtccttctgt cctggatgtt tcatgggccc ggggcccccc agggaagctt 1620
acccctcctg tgctgggtgg aggccacggg acacctcagg tgccacccac cttggcccta 1680
aaacagccac caggaaagca gccggagagc cggacagcag gcagcctgtc tgggttcctg 1740
aggcctgggg gtggcagacg agcccacggc gccgtggtcc cagcagcagg gttgtcagtc 1800
ggagcatcct ggggctccct ggctcctggc cgtctgtgag gtaggcgcag taccgtgtat 1860
cgtaggtagc agtaggaacg ggggccgccg cggccctgca gccgctcatg gcggtgaggt 1920
gtgtgccaag cccacccggg gtgcagggcg tgacgtgtgg ggaataaata ggcgttgtga 1980
cctc 1984
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ctaaaggtga aaagctccg 19
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Claims (6)
1. A DENND1A-SGTA fusion gene is characterized in that the sequence of the fusion gene is shown in SEQ ID NO. 1.
2. The use of the DENND1A-SGTA fusion gene of claim 1 in the preparation of a kit for the diagnosis or monitoring of acute lymphocytic leukemia.
3. A kit for detecting the DENND1A-SGTA fusion gene of claim 1, wherein the kit comprises an upstream primer, a downstream primer, a Taqman probe, an internal reference, a PCR reaction buffer, a positive control and a negative control;
the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the Taqman probe is shown in SEQ ID NO. 4.
4. The kit for detecting DENND1A-SGTA fusion gene as claimed in claim 3, wherein the 5 'end of the Taqman probe is labeled with FAM group and the 3' end of the probe is labeled with TAMRA group.
5. The kit for detecting DENND1A-SGTA fusion gene as claimed in claim 3, wherein the internal reference comprises an internal reference upstream primer, an internal reference downstream primer and an internal reference probe;
the nucleotide sequence of the internal reference upstream primer is shown as SEQ ID NO. 5;
the nucleotide sequence of the internal reference downstream primer is shown as SEQ ID NO. 6;
the nucleotide sequence of the internal reference probe is shown as SEQ ID NO. 7.
6. A method for detecting the DENND1A-SGTA fusion gene of claim 1 for non-disease diagnostic purposes, comprising the steps of:
(1) extracting total RNA in a human blood sample, and inverting the RNA into cDNA serving as a sample to be detected;
(2) preparing PCR reaction liquid, and then respectively adding a sample to be detected, a positive control and a negative control;
(3) detecting on a real-time fluorescent PCR instrument, wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; reacting at 95 ℃ for 15s and 60 ℃ for 1min for 40 cycles, and collecting a fluorescence signal at 60 ℃;
the PCR reaction solution comprises an upstream primer, a downstream primer, a Taqman probe, an internal reference, a PCR reaction buffer solution, a positive control and a negative control, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the Taqman probe is shown in SEQ ID NO. 4.
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