CN114015710A - CHST2-SLC9A9-AS2 fusion gene and application and detection kit thereof - Google Patents
CHST2-SLC9A9-AS2 fusion gene and application and detection kit thereof Download PDFInfo
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Abstract
The invention provides a CHST2-SLC9A9-AS2 fusion gene and application thereof and a detection kit, wherein the sequence of the fusion gene is shown AS SEQ ID NO. 1. The invention firstly discovers and identifies that a new fusion gene CHST2-SLC9A9-AS2 exists in acute lymphocytic leukemia, the abundance of the fusion gene is closely related to the disease progression stage, and the fusion gene can be used AS a specific molecular marker of the acute lymphocytic leukemia, thereby developing a kit which can be clinically used for diagnosing and monitoring Minimal Residual Disease (MRD) of a patient. The kit carries out absolute quantification on the CHST2-SLC9A9-AS2 fusion gene in the body of an acute lymphocytic leukemia patient 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 Lymphoblastic Leukemia (ALL) is one of the most common hematological malignancies resulting from malignant proliferation of B or T lineage lymphoid progenitor cells. Patients are also often afflicted with mediastinal lumps and the central nervous system due to a deficiency of normal peripheral blood cells, clinically characterized by fever, recurrent infections, bleeding of the skin/mucosa, bone pain and joint pain. Chemotherapy remission rates of childhood ALL patients can reach 80-90%, but the prognosis of adult ALL is poor, with overall survival rates of 35-55%, and only 30% in adults over 60 years of age.
To assess the risk of relapse and predict clinical prognosis, monitoring Minimal Residual Disease (MRD) is one of the most common clinical monitoring methods. Among them, Real-time fluorescence Quantitative polymerase chain reaction (Quantitative Real-time PCR) analysis of leukemia specific fusion gene is one of the methods for monitoring MRD, and the sensitivity is higher than that of flow cytometry. With the rapid development of molecular genetic diagnostics, fusion genes such as BCR-ABL, BCL11B-TLX3, STIL-TAL1, NUP214-ABL1 and the like are reported in ALL at present, but a large proportion of ALL patients lack molecular markers, so that great difficulty is brought to clinical diagnosis and treatment prognosis. Therefore, the discovery and monitoring of the novel fusion gene related to ALL has important clinical significance for disease diagnosis, treatment scheme formulation, efficacy evaluation, prognosis evaluation and lesion recurrence.
The CHST2 (aspartate transferase 2) gene encodes a sulfotransferase protein, can catalyze the sulfation of non-reducing N-acetylglucosamine residues, and is involved in the homing of lymphocytes at inflammatory sites. The SLC9A9-AS2 gene, namely the antisense RNA2 of SLC9A9(Sodium/hydrogen exchange 9), is known to play an important role in maintaining the ion balance of organelles at present, and the antisense RNA2 of the SLC9A9 gene is lncRNA, so that the function of the lncRNA is not deeply researched at present and the antisense RNA may participate in the expression regulation of the SLC9A9 gene. The two genes form a novel fusion gene CHST2-SLC9A9-AS2 in ALL patients, and the two genes are not reported in the literature so far.
Disclosure of Invention
The first purpose of the invention is to provide an acute lymphoblastic leukemia fusion gene CHST2-SLC9A9-AS 2.
The second purpose of the invention is to provide the application of the fusion gene CHST2-SLC9A9-AS2 in preparing a preparation or a kit for diagnosing and/or monitoring acute lymphocytic leukemia.
The third purpose of the invention is to provide an acute lymphocytic leukemia diagnosis and/or monitoring kit.
The fourth purpose of the invention is to provide a method for detecting the CHST2-SLC9A9-AS2 fusion gene for non-disease diagnosis.
In order to realize the first object, the invention provides a CHST2-SLC9A9-AS2 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 an application of the CHST2-SLC9A9-AS2 fusion gene in preparing a preparation or a kit for diagnosing and/or monitoring acute lymphocytic leukemia. Part of acute lymphoblastic leukemia patients carry a fusion gene CHST2-SLC9A9-AS2, 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 achieve the third object, the present invention provides an acute lymphocytic leukemia diagnosis and/or monitoring kit, which comprises a reagent for detecting the CHST2-SLC9A9-AS2 fusion gene or detecting the expression product thereof.
As a preferred scheme, the kit 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 plasmid standard with CHST2-SLC9A9-AS2 fusion gene and 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 invention provides a method for detecting CHST2-SLC9A9-AS2 fusion gene for non-disease diagnosis, which comprises the following steps:
(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 CHST2-SLC9A9-AS2, and then develops a fusion gene detection and quantification kit combining real-time fluorescence PCR and Taqman probe technology. The real-time fluorescent 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 trace fusion genes at present, so the kit adopts Taqman probe real-time fluorescent PCR to detect the CHST2-SLC9A9-AS2 fusion genes and monitors MRD of a patient.
The invention has the advantages that (1) the fusion gene CHST2-SLC9A9-AS2 carried by the acute lymphocytic leukemia patient is screened and identified by applying bioinformatics technology, which proves that the fusion gene CHST2-SLC9A9-AS2 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 CHST2-SLC9A9-AS2 fusion gene detection kit 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 the CHST2-SLC9A9-AS2 fusion gene and the internal 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, adjustment of treatment schemes, evaluation of treatment effects, prognosis prediction and clinical relapse prevention.
Drawings
FIG. 1 shows the structure diagram of CHST2-SLC9A9-AS2 fusion gene and PCR, sanger test. A: the translocation of the 3q24 region resulted in the graphical display of the CHST2-SLC9A9-AS2 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 verification product of the patient carrying the fusion gene confirms the fusion of the CHST2 exon 2 and the SLC9A9-AS2 exon 2.
FIG. 2 shows the construction result of plasmid standard of CHST2-SLC9A9-AS2 fusion gene. A: the fusion gene standard quality particle pGEM T-easy-CHST2-SLC9A9-AS2 map; b: sequencing result of standard plasmid sanger.
FIG. 3 is a diagram showing the results of CHST2-SLC9A9-AS2 using the kit of the present invention. A: CHST2-SLC9A9-AS2 standard curve, positive sample and negative control; b: CHST2-SLC9A9-AS2 positive patients were treated for a change in mRNA level in CHST2-SLC9A9-AS 2.
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 fusion gene CHST2-SLC9A9-AS2
(1) The bioinformatics technology is applied to screen out the fusion gene of the acute lymphoblastic leukemia patient and a new CHST2-SLC9A9-AS2 fusion gene which is not reported in the literature.
(2) The novel CHST2-SLC9A9-AS2 fusion gene is formed by splicing the 1 st exon-2 of CHST2 gene with the 2 nd exon of SLC9A9-AS2, and the specific base sequence is shown AS SEQ ID NO. 1.
(3) By respectively applying PCR and carrying out sanger sequencing on PCR products, we confirm that the acute lymphoblastic leukemia patient carries the new fusion gene CHST2-SLC9A9-AS2, and the verification result is shown in figure 1. FIG. 1 shows the structure diagram of CHST2-SLC9A9-AS2 fusion gene and PCR, sanger test. A: the translocation of the 3q24 region resulted in the graphical display of the CHST2-SLC9A9-AS2 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 verification product of the patient carrying the fusion gene confirms the fusion of the CHST2 exon 2 and the SLC9A9-AS2 exon 2.
(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 result of plasmid standard of CHST2-SLC9A9-AS2 fusion gene. A: the fusion gene standard quality particle pGEM T-easy-CHST2-SLC9A9-AS2 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 condition of the fusion genes of the reference genes ABL and CHST2-SLC9A9-AS 2. 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, CHST2 Gene sequence, SLC9A9-AS2 Gene sequence are all from the nucleic acid database of the national center for Biotechnology information, ABL1 Gene Entrez Gene ID25, Gene reference NM-005157.5, CHST2 Gene Entrez Gene ID 9435, Gene reference ENST00000309575.5, SLC9A9-AS2 Gene Entrez Gene ID 106480356, Gene reference ENST 00000490153.1).
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 a CHST2-SLC9A9-AS2 fusion gene, an internal reference ABL primer and a probe corresponding to the primer, and specifically comprises the following steps:
CHST2-SLC9A9-AS2-F:ACACGGTATGTGGAGGTG(SEQ ID NO.2)；
CHST2-SLC9A9-AS2-R:GGCTGTTCCATGTTTGTC(SEQ ID NO.3)；
CHST2-SLC9A9-AS2-Probe:FAM-AAGCAGCGAAGTTTCATCTGAGAACCCA-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 CHST2-SLC9A9-AS2 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 the 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 mul cDNA into PCR reaction liquid of a 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. CHST2-SLC9A9-AS2 can be used AS a molecular marker specific to acute lymphocytic leukemia patients: the detection kit constructed above is used to perform MRD monitoring on 4 time node samples of clinical treatment of a patient positive to CHST2-SLC9A9-AS 2. FIG. 3 is a diagram showing the results of CHST2-SLC9A9-AS2 using the kit of the present invention. A: CHST2-SLC9A9-AS2 standard curve, positive sample and negative control; b: CHST2-SLC9A9-AS2 positive patients were treated for a change in mRNA level in CHST2-SLC9A9-AS 2. We found that the patient had a higher expression level of CHST2-SLC9A9-AS2 at the initial onset (20.2.21), and then after a period of chemotherapy, the patient had a slight remission than the previous disease condition, and the expression level of CHST2-SLC9A9-AS2 was reduced earlier (20.5.12), but soon the patient again reviewed cytology and flow showed primitive cytosis, tending to relapse again, and at the same time the expression level of CHST2-SLC9A9-AS2 was increased again (20.9.8), at this time, lymphocyte infusion was given to the donor, and the expression level of CHST2-SLC9A9-AS2 was monitored again (20.9.22), indicating that the expression of CHST2-SLC9A9-AS2 is consistent with the disease progression trend, but there was molecular residue and therefore the risk of relapse still needs to be monitored in real time. The results show that CHST2-SLC9A9-AS2 can be used AS a novel molecular marker for patients to monitor MRD, and the accuracy and reliability of the kit are proved.
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> CHST2-SLC9A9-AS2 fusion gene and application and detection kit thereof
<130> /
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 2978
<212> DNA
<213> Homo sapiens
<400> 1
atgagccgca gcccgcagcg agctctgccc ccgggcgcgc tccctcggct gctccaggct 60
gcgcctgcag ccgcgccgcg tgccctgctc ccgcagtggc cccggcgccc aggacgccgc 120
tggcccgcgt cccctctcgg aatgaaggtg ttccgtagga aggcgctggt gttgtgcgcg 180
ggctatgcac tgctgctggt gctcactatg ctcaacctcc tggactacaa gtggcacaag 240
gagccgctgc agcagtgcaa ccccgatggg ccgctgggtg ccgcagcggg ggcagccgga 300
ggcagctggg ggcgcccagg gccgcctccg gccgggccgc cccgtgctca tgcccgtttg 360
gacctccgca ctccttaccg ccctcccgct gccgccgtcg gggcggctcc tgcagccgcg 420
gcagggatgg cgggggttgc ggcccctcca ggcaatggca ctcggggcac cgggggcgtc 480
ggggacaagc ggcagctggt gtacgtgttc accacgtggc gctctggctc gtcgttcttc 540
ggcgagctat tcaaccagaa tcccgaggtg ttctttctct acgagccagt gtggcatgta 600
tggcaaaaac tgtatccggg ggacgccgtt tccctgcagg gggcagcgcg ggacatgctg 660
agcgctcttt accgctgcga cctctctgtc ttccagttgt atagccccgc gggcagcggg 720
gggcgcaacc tcaccacgct gggcatcttc ggcgcagcca ccaacaaggt ggtgtgctcg 780
tcaccactct gccccgccta ccgcaaggag gtcgtggggt tggtggacga ccgcgtgtgc 840
aagaagtgcc cgccacagcg cctggcgcgt ttcgaggagg agtgccgcaa gtaccgcaca 900
ctagtcataa agggtgtgcg cgtcttcgac gtggcggtct tggcgccact gctgcgagac 960
ccggccctgg acctcaaggt catccacttg gtgcgtgatc cccgcgcggt ggcgagttca 1020
cggatccgct cgcgccacgg cctcatccgt gagagcctac aggtggtgcg cagccgagac 1080
ccgcgagctc accgcatgcc cttcttggag gccgcgggcc acaagcttgg cgccaagaag 1140
gagggcgtgg gcggccccgc agactaccac gctctgggcg ctatggaggt catctgcaat 1200
agtatggcta agacgctgca gacagccctg cagccccctg actggctgca gggccactac 1260
ctggtggtgc ggtacgagga cctggtggga gaccccgtca agacactacg gagagtgtac 1320
gattttgtgg gactgttggt gagccccgaa atggagcagt ttgccctgaa catgaccagt 1380
ggctcgggct cctcctccaa gcctttcgtg gtatctgcac gcaatgccac gcaggccgcc 1440
aatgcctggc ggaccgccct caccttccag cagatcaaac aggtggagga gttttgctac 1500
cagcccatgg ccgtcctggg ctatgagcgg gtcaacagcc ctgaggaggt caaagacctc 1560
agcaagaccc tgcttcggaa gccccgtctc taaaaggggt tcccaggaga cctgattccc 1620
tgtggtgata cctataaaga ggatcgtagt gtgtttaaat aaacacagtc cagactcaaa 1680
cggaggaagc ccacatattc tattatagat atataaataa tcacacacac acttgctgtc 1740
aatgttttga gtcagtgcat ttcaaggaac agccacaaaa tacacacccc taagaaaagg 1800
caagacttga acgttctgac caggtgcccc tcttcttctt tgccttctct tgtcctcttt 1860
ctcctatttc ttaccctgtc ctccacctgc cttccatttt gaagtgggat gttaatgaaa 1920
tcaagttcca gtaacccaaa tcttgtttac aaaatattcg tggtatctgt gaacatgtta 1980
agagtaattt ggatgtgggg gtgggggtgg agaaagggga agtggtccag aaacaaaaag 2040
ccccattggg catgataagc cgaggaggca ttcttctaaa gcagactttt gtgtaaaaag 2100
caaaggttac atgtgagtat taataaagaa gataataaat aatattcttt ttaatatttg 2160
cctccattac tttggattca cctgtgttac ttgtcctcag cttcagatag aatctccttc 2220
ccctccctcg tttccatttt tccagtctct ttcctgaaag gcagctaaac tgggtgggag 2280
caatttttgc ctccttggta ataacctggg ggatgttttc tcacatgtaa atctcagcct 2340
gttaggtgtc caggagtgaa acacatcttt gtattctaaa ggcagaaacc aaacagggca 2400
gtggaagaaa aagcacacta ctctcttcaa ctttgaacac aggaagaagc aactccttca 2460
ggacttgaag gcatttggtt tttcattcct cttcagttaa aatggggaac cttgaagcag 2520
agaccaatgt tttggtgctg aggctggttc agaaaaagga tttttaaaaa aagtatgtaa 2580
tttttaaaag ttctgatgat tagaacacag acctcaggaa agtagcgtga acatactgct 2640
ggcgatggta gcagcttcgt tggtttagca aagtgacaga agtatctatt tggagtgttt 2700
ttctgaccct gacacggtat gtggaggtgg atgaaagcag cgaagtttca tctgagaacc 2760
cagaggaagc tccgtttaga aatagctcct tggtcacatg ctggacaaac atggaacagc 2820
ctatggaaca gaacaatcat atctctatat aatgtgcatt ggtttctttg acttgcctgg 2880
tgttgtgagg aggggtcctc cttcagattt tcatccaggt ccacgccaac tctgttaaac 2940
aaaaccaaaa actggtcaat cccctgctgc agaaggaa 2978
<210> 2
<211> 18
<212> DNA
<213> Artificial Synthesis
<400> 2
<210> 3
<211> 18
<212> DNA
<213> Artificial Synthesis
<400> 3
ggctgttcca tgtttgtc 18
<210> 4
<211> 28
<212> DNA
<213> Artificial Synthesis
<400> 4
aagcagcgaa gtttcatctg agaaccca 28
<210> 5
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 5
<210> 6
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 6
<210> 7
<211> 28
<212> DNA
<213> Artificial Synthesis
<400> 7
ccatttttgg tttgggcttc acaccatt 28
Claims (7)
1. A CHST2-SLC9A9-AS2 fusion gene is characterized in that the sequence of the fusion gene is shown in SEQ ID NO. 1.
2. The use of the CHST2-SLC9A9-AS2 fusion gene of claim 1 in the preparation of a diagnostic and/or monitoring agent or kit for acute lymphocytic leukemia.
3. A kit for diagnosing and/or monitoring acute lymphocytic leukemia, wherein the kit comprises a reagent for detecting the CHST2-SLC9A9-AS2 fusion gene or detecting its expression product according to claim 1.
4. The acute lymphocytic leukemia diagnosis and/or monitoring kit according to claim 3, 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.
5. The acute lymphoblastic leukemia diagnosis and/or monitoring kit according to claim 4, 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.
6. The acute lymphocytic leukemia diagnosis and/or monitoring kit according to claim 4, 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.
7. A method for detecting the CHST2-SLC9a9-AS2 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 ℃;
wherein 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;
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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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251031A (en) * | 2011-06-30 | 2011-11-23 | 北京思尔成生物技术有限公司 | TaqMan MGB probe real-time fluorescence PCR detection method for leukemia fusion genes, and special primers, probes and kit used by same |
| CN112210568A (en) * | 2020-10-30 | 2021-01-12 | 大连医科大学附属第二医院 | Ph-like acute lymphoblastic leukemia fusion gene and application and detection kit thereof |
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2021
- 2021-12-16 CN CN202111544687.1A patent/CN114015710A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251031A (en) * | 2011-06-30 | 2011-11-23 | 北京思尔成生物技术有限公司 | TaqMan MGB probe real-time fluorescence PCR detection method for leukemia fusion genes, and special primers, probes and kit used by same |
| CN112210568A (en) * | 2020-10-30 | 2021-01-12 | 大连医科大学附属第二医院 | Ph-like acute lymphoblastic leukemia fusion gene and application and detection kit thereof |
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