CN117230203B - Primer probe set for detecting MYOD1 gene mutation and kit thereof - Google Patents
Primer probe set for detecting MYOD1 gene mutation and kit thereof Download PDFInfo
- Publication number
- CN117230203B CN117230203B CN202311506946.0A CN202311506946A CN117230203B CN 117230203 B CN117230203 B CN 117230203B CN 202311506946 A CN202311506946 A CN 202311506946A CN 117230203 B CN117230203 B CN 117230203B
- Authority
- CN
- China
- Prior art keywords
- probe
- mutation
- myod1
- primer
- myod1 gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000523 sample Substances 0.000 title claims abstract description 80
- 101150013833 MYOD1 gene Proteins 0.000 title claims abstract description 40
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 19
- 230000035772 mutation Effects 0.000 claims abstract description 33
- 201000009410 rhabdomyosarcoma Diseases 0.000 claims description 15
- 108020004707 nucleic acids Proteins 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 13
- 150000007523 nucleic acids Chemical class 0.000 claims description 13
- 125000006853 reporter group Chemical group 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000010791 quenching Methods 0.000 claims description 7
- 230000000171 quenching effect Effects 0.000 claims description 7
- 230000004544 DNA amplification Effects 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 41
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000013461 design Methods 0.000 abstract description 3
- 101001023043 Homo sapiens Myoblast determination protein 1 Proteins 0.000 description 16
- 102100035077 Myoblast determination protein 1 Human genes 0.000 description 16
- 230000003321 amplification Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000007480 sanger sequencing Methods 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 4
- 210000003098 myoblast Anatomy 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 101100239683 Homo sapiens MYOD1 gene Proteins 0.000 description 2
- 238000009004 PCR Kit Methods 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000011269 treatment regimen Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010027344 Basic Helix-Loop-Helix Transcription Factors Proteins 0.000 description 1
- 102000018720 Basic Helix-Loop-Helix Transcription Factors Human genes 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000009561 Forkhead Box Protein O1 Human genes 0.000 description 1
- 108010009306 Forkhead Box Protein O1 Proteins 0.000 description 1
- 101001023028 Gallus gallus Myogenin Proteins 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 206010068771 Soft tissue neoplasm Diseases 0.000 description 1
- 101001023030 Toxoplasma gondii Myosin-D Proteins 0.000 description 1
- 102000005937 Tropomyosin Human genes 0.000 description 1
- 108010030743 Tropomyosin Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 208000030748 clear cell sarcoma of kidney Diseases 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 201000009409 embryonal rhabdomyosarcoma Diseases 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 201000007211 kidney clear cell sarcoma Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 230000009756 muscle regeneration Effects 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 108091005981 phosphorylated proteins Proteins 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000012502 risk assessment Methods 0.000 description 1
- 230000002784 sclerotic effect Effects 0.000 description 1
- 210000002363 skeletal muscle cell Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 208000028647 spindle cell neoplasm Diseases 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a primer probe set for detecting MYOD1 gene mutation and a kit thereof, belonging to the technical field of gene detection. According to the invention, the L122R mutation site on the MYOD1 gene is taken as a detection object, a specific primer is utilized to accurately amplify and amplify a mutation target sequence, and through proper probe design, the problems of lower sensitivity, poorer accuracy and longer detection time of the mutation detection of the MYOD1 gene in the prior art are overcome.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer probe set for detecting MYOD1 gene mutation and a kit thereof.
Background
The MYOD1 gene, located on human chromosome 11p15.1, consists of 3 exons, encodes a nuclear protein consisting of 320 amino acids, and belongs to bHLH transcription factor family and myogenic factor subfamily members. It regulates muscle cell differentiation by inducing cell cycle arrest. In addition, it is also possible to participate in muscle regeneration by transcriptional regulation. The encoded tropomyosin-regulating protein (MyoD 1) is a phosphorylated protein having a molecular weight of 45kDa, and is a protein that binds to tropomyosin in the cytoskeleton.
Rhabdomyosarcoma (Rhabdomyosarcoma), the most common soft tissue tumor in children, is a primitive malignancy with embryonic skeletal muscle cell phenotype and biological characteristics, and commonly expresses MyoD1 protein. The MYOD1 p.l122r mutation (nm_002478, np_002469) was first found in partially clinically invasive rhabdomyosarcoma. On one hand, the MYOD1 gene mutation transcription product can be competitively combined with a combining site of normal protein to cause myoblasts to be incapable of differentiating and keep the myoblasts in a proliferation state, and on the other hand, the myoblasts can be combined with MYC reaction genes to activate oncogenic transcription programs to promote tumorigenesis. MYOD1 mutations occur most frequently in spindle/sclerotic rhabdomyosarcoma, and occasionally in embryonal rhabdomyosarcoma, accounting for about 3% of all FOXO1 fusion-negative rhabdomyosarcomas. The prognosis of MYOD 1L 122R mutant patients is extremely poor, with long-term survival below 30%, an independent prognostic factor for patients, the presence or absence of which is independent of clinical stratification of patients. The detection of the mutation status of the MYOD1 gene has important significance for the risk classification and treatment strategy selection of patients.
Because of the important role of MYOD1 gene L122R mutation in rhabdomyosarcoma diagnosis, mutation detection has become one of the important matters for risk assessment and treatment regimen selection. The gold standard for detecting the gene mutation in clinic is Sanger sequencing method, and the method has the advantages of simple experimental method, visual and reliable result and lower cost. Through decades of development, the technology has been accepted by vast medical staff, but the technology has low detection sensitivity (the lower limit of detection of mutation content is about 25%), is easy to generate false positive due to baseline interference, and has a long detection period (24-48 hours).
Disclosure of Invention
The invention aims to provide a primer probe set for detecting MYOD1 gene mutation and a kit thereof, so as to solve the problems of lower sensitivity, poorer accuracy and longer detection time of MYOD1 gene mutation detection in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer probe group for detecting MYOD1 gene mutation, which comprises a MYOD1 gene amplification primer pair and/or a MYOD1 gene mutation site probe group;
the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
the MYOD1 gene mutation site probe set comprises a mutant probe and/or a wild probe, the nucleotide sequence of the mutant probe is shown as SEQ ID NO.3, and the nucleotide sequence of the wild probe is shown as SEQ ID NO. 4.
Preferably, the 5' end of the mutant probe is marked with a reporter group;
and/or, the 5' end of the wild type probe is marked with a reporter group;
and/or, the 3' end of the mutant probe is marked with a quenching group;
and/or, the 3' -end of the wild-type probe is marked with a quenching group.
Preferably, the reporter group is selected from one or more of FAM, VIC, ROX, HEX, TET, JOE, NED, cy and Cy 3;
the reporter group of the wild-type probe is different from the reporter group of the mutant probe.
Preferably, the quenching group is selected from one or more of NFQ-MGB, BQH1, BHQ2, BHQ3 and TAMARA.
Preferably, the mutant probe and/or wild type probe is modified with locked nucleic acid.
Preferably, the 4 th base of the mutant probe is modified by a locked nucleic acid from the 5' end of the probe sequence;
the 4 th base of the wild type probe is modified by nucleic acid locking.
The invention also provides application of the primer probe set in preparation of a reagent or a kit for detecting the mutation of the MYOD1 gene L122R.
The invention also provides a kit comprising the primer probe set.
Preferably, the concentration of the MYOD1 gene amplification primer pair is 1-50 mu mol/L;
the concentration of the MYOD1 gene mutation site probe set is 1-50 mu mol/L.
The invention also provides application of the primer probe set or the kit in preparing a rhabdomyosarcoma auxiliary diagnostic reagent or a prognostic reagent.
The invention has the beneficial effects that:
the primer probe set for detecting the mutation of the MYOD1 gene provided by the invention takes the L122R mutation site on the MYOD1 gene as a detection object, utilizes a specific primer to accurately amplify and amplify a mutation target sequence, performs qualitative detection and interpretation on the mutation directly through fluorescence signal collection through proper probe design, and only needs one-step PCR amplification, thereby realizing the effects of higher sensitivity (mutation with the lowest detectable abundance of 1% in tumor tissues), higher accuracy, simple steps, shorter detection time (the result can be obtained by 2 h), visual and convenient interpretation, and better presenting the prognostic evaluation value of MYOD1 gene mutation detection.
The kit provided by the invention can be used for detecting the mRNA expression of MYOD1 due to the fact that the kit contains a wild type probe for detecting the mutation site of the MYOD1 gene. Because the mRNA expression of MYOD1 only exists in most rhabdomyosarcoma and does not exist in other sarcomas with similar tissue morphological characteristics, the kit capable of realizing specific detection can also realize auxiliary molecular diagnosis of rhabdomyosarcoma, and has certain auxiliary diagnosis and differential diagnosis values.
Drawings
FIG. 1 is a graph of detection results of a MYOD1 mutation detection kit for different concentrations;
FIG. 2 is a graph showing the linear relationship of a MYOD1 mutation detection kit for detection of different concentrations;
FIG. 3 is a graph of the detection results of a MYOD1 mutation detection kit for different mutation abundances;
FIG. 4 is a graph of the linear relationship of a MYOD1 mutation detection kit to the detection of different mutation abundances;
FIG. 5 is a graph showing the detection result of a wild-type probe;
FIG. 6 is a graph showing the detection results of mutant probes.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 primer and Probe design
Specific primers and probes for detecting MYOD1 wild type and mutant were designed and synthesized, and the nucleotide sequences are as follows:
upstream Primer MYOD1-Fwd-Primer:5'-GCAAGGCCGCCACCAT-3', as shown in SEQ ID NO. 1;
downstream Primer MYOD1-Rev-Primer:5'-TTGGATTGCTCGACGTGCA-3', as shown in SEQ ID NO. 2;
mutant Probe MYOD1-mut-Probe:5'-FAM-GCC (+g) GAGCAAA-NFQ-MGB-3' as shown in SEQ ID NO. 3;
wild type Probe MYOD1-wt-Probe:5'-VIC-GCC (+t) GAGCAAAG-NFQ-MGB-3' as shown in SEQ ID NO. 4;
in the above probes, sequence brackets +represents locked nucleic acid modification;
preparing a PCR kit for detecting the mutation of the human MYOD1 gene, wherein the kit comprises a nucleic acid amplification reagent and a reference substance, and the nucleic acid amplification reagent comprises MYOD1 wild type reaction liquid and mutant reaction liquid; the reaction liquid contains the specific primers and probes for detecting the MYOD1 wild type and mutant type respectively; the MYOD1 wild type reaction solution and the mutant reaction solution both comprise a pair of upstream primer and downstream primer and TaqMan fluorescent probe, and the MYOD1 wild type reaction solution can also be used as an internal reference primer and a probe for quality control of sample nucleic acid.
Example 2 detection protocol
The detection sample is paraffin embedded tumor tissue sample, DNA/RNA in the tissue sample is purified by a nucleic acid extraction reagent, RNA is reversely transcribed into cDNA, and then the detection is carried out by configuring the following reaction system:
table 1 reaction system (20 μl system for example):
note that: wherein the 2 Xprobe method qPCR reaction buffer solution contains Taq enzyme and Mg 2+ PCR buffer, dNTPs and water.
The amplification procedure was set as follows:
TABLE 2 amplification step
The PCR kit for detecting the mutation of the human MYOD1 gene also comprises a positive control and a negative control, wherein the negative control is pure water, and the positive control is a mixture of mutant plasmid and wild type plasmid (the ratio of the mutant type is 5%).
The nucleotide sequence of the MYOD1 gene fragment contained in the mutant plasmid is as follows:
5'-CTGAAACCCGAAGAGCACTCGCACTTCCCCGCGGCGGTGCACCCGGCCCCGGGCGCACGTGAGGACGAGCATGTGCGCGCGCCCAGCGGGCACCACCAGGCGGGCCGCTGCCTACTGTGGGCCTGCAAGGCGTGCAAGCGCAAGACCACCAACGCCGACCGCCGCAAGGCCGCCACCATGCGCGAGCGGCGCCGCCGGAGCAAAGTAAATGAGGCCTTTGAGACACTCAAGCGCTGCACGTCGAGCAATCCAAACCAGCGGTTGCCCAAGGTGGAGATCCTGCGCAACGCCATCCGCTATATCGAGGGCCTGCAGGCTCTGCTGCGCG-3', as shown in SEQ ID NO. 5;
and verifying the performance of the MYOD1 mutation detection kit:
use of insertion mutant MYOD1 Gene fragmentsThe plasmids are used as standard substances and respectively take 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 And 10 -7 The ng/. Mu.L solution is used as a template to test the detection performance of the kit, and the result shows that the amplification curve of the kit is good in the range, as shown in FIG. 1.
At 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 And 10 -7 The Ct value of the amplification curve of the mutant plasmid standard substance with the concentration of ng/mu L is used as a standard curve, and the result shows that the detection system is 10 -2 ~10 -7 The ng/. Mu.L concentration interval has a good linear relationship as shown in FIG. 2.
The detection performance of the kit was tested using mutant and wild-type MYOD1 gene fragment plasmids as standards and mutant plasmid contents of 50%, 10%, 5%, 2%, 1%, 0.1%, 0.01% and 0% as templates, and the results showed that the kit can detect 1% mutation at the lowest, as shown in fig. 3.
The Ct values of the mutation ratio of 100%, 50%, 20%, 5%, 2% and 1% solution amplification results are used for standard quantitative curves, and the results show that the kit has good linear relationship in the mutation abundance of 1% -100%, as shown in FIG. 4.
By adopting the detection scheme, 62 cases of rhabdomyosarcoma samples are detected by using the kit provided by the invention, wherein 11 cases are mutant types, and meanwhile, sanger sequencing is used as a control.
Sanger sequencing method:
62 tumor samples with a tumor content of greater than 50% were selected for paraffin sections (3 μm,5-10 pieces/case), nucleic acid extraction was performed using a paraffin sample DNA extraction kit, nucleic acid amplification was performed using MYOD1 gene upstream primer 5'-CTGAAACCCGAAGAGCACTCGC-3' (shown in SEQ ID No. 6) and downstream primer 5'-GGATCTCCACCTTGGGCAACC-3' (shown in SEQ ID No. 7), sanger sequencing was performed on the PCR products, and comparison with wild-type MYOD1 gene sequences was performed to analyze whether the L122R mutation occurred.
The detection results are shown in Table 3 below, and examples of the sample detection results are shown in FIG. 5 and FIG. 6, wherein FIG. 5 is the detection result of a wild-type probe and FIG. 6 is the detection result of a mutant probe.
TABLE 3 detection statistics
The results show that compared with the gold standard Sanger sequencing, the sensitivity of the method can reach 100% { 11/(11+0) ×100%) }, the specificity can reach 100% { 51/(0+51) ×100% }, the accuracy can reach 100% { (11+51)/(11+0+0) ×100% }, and a remarkably better effect is achieved.
Since MyoD1 protein is expressed only in embryonic rhabdomyoblasts, normal myoblasts are not expressed, are specific markers of rhabdomyosarcoma, and are hardly expressed in other types of tumors. Other tumors including 24 neuroblastomas, 3 NTRK rearranged spindle cell tumors, 2 renal clear cell sarcomas, 1 CIC rearranged sarcoma, and 1 pancreatic solid pseudopapilloma were used as control samples, rhabdomyosarcoma was used as detection sample, and the results are shown in table 4 below, using the kit provided by the present invention:
TABLE 4 MYOD1 expression assay
The result shows that the detection scheme provided by the invention has strong specificity, and the kit can be used for detecting the expression of the MYOD1 gene of rhabdomyosarcoma, thereby assisting the diagnosis of rhabdomyosarcoma.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. The primer probe group for detecting the mutation of the MYOD1 gene is characterized by comprising a MYOD1 gene amplification primer pair and a MYOD1 gene mutation site probe group;
the nucleotide sequence of an upstream primer of the MYOD1 gene amplification primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2;
the MYOD1 gene mutation site probe set comprises a mutation type probe and a wild type probe, the nucleotide sequence of the mutation type probe is shown as SEQ ID NO.3, and the nucleotide sequence of the wild type probe is shown as SEQ ID NO. 4;
the mutant probe and the wild probe are subjected to nucleic acid locking modification;
starting from the 5' end of the probe sequence, the 4 th base of the mutant probe is subjected to nucleic acid locking modification;
the 4 th base of the wild type probe is modified by nucleic acid locking.
2. The primer probe set for detecting MYOD1 gene mutation according to claim 1, wherein a reporter group is marked at the 5' end of the mutant probe;
the 5' end of the wild type probe is marked with a reporter group;
the 3' end of the mutant probe is marked with a quenching group;
the 3' -end of the wild type probe is marked with a quenching group.
3. The primer probe set for detecting MYOD1 gene mutation according to claim 2, wherein the reporter group of the wild type probe and the reporter group of the mutant probe are respectively selected from one of FAM, VIC, ROX, HEX, TET, JOE, NED, cy and Cy 3;
the reporter group of the wild-type probe is different from the reporter group of the mutant probe.
4. The primer probe set for detecting MYOD1 gene mutation according to claim 2, wherein the quenching group of the wild type probe and the quenching group of the mutant probe are respectively selected from one of NFQ-MGB, BQH1, BHQ2, BHQ3, TAMARA.
5. The application of the primer probe set of any one of claims 1-4 in preparing a reagent or a kit for detecting the mutation of the MYOD1 gene L122R.
6. A kit comprising the primer probe set of any one of claims 1 to 4.
7. The kit of claim 6, wherein the concentration of the MYOD1 gene amplification primer pair is 1-50 μmol/L;
the concentration of the MYOD1 gene mutation site probe set is 1-50 mu mol/L.
8. The use of the primer probe set according to any one of claims 1 to 4 for preparing a rhabdomyosarcoma auxiliary diagnostic reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311506946.0A CN117230203B (en) | 2023-11-14 | 2023-11-14 | Primer probe set for detecting MYOD1 gene mutation and kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311506946.0A CN117230203B (en) | 2023-11-14 | 2023-11-14 | Primer probe set for detecting MYOD1 gene mutation and kit thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117230203A CN117230203A (en) | 2023-12-15 |
| CN117230203B true CN117230203B (en) | 2024-02-06 |
Family
ID=89084555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311506946.0A Active CN117230203B (en) | 2023-11-14 | 2023-11-14 | Primer probe set for detecting MYOD1 gene mutation and kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117230203B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795653A (en) * | 2021-02-08 | 2021-05-14 | 深圳市核子基因科技有限公司 | Primer probe set and kit for KRAS gene mutation detection and application thereof |
| CN114182023A (en) * | 2022-02-14 | 2022-03-15 | 首都医科大学附属北京朝阳医院 | Detection kit and method for F-circP3F in rhabdomyosarcoma |
| CN114480660A (en) * | 2022-04-02 | 2022-05-13 | 普瑞基准科技（北京）有限公司 | Gene Panel for detecting pan-cancer species, probe and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160080815A (en) * | 2013-08-28 | 2016-07-08 | 카리스 라이프 사이언스 스위스 홀딩스 게엠베하 | Oligonucleotide probes and uses thereof |
-
2023
- 2023-11-14 CN CN202311506946.0A patent/CN117230203B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795653A (en) * | 2021-02-08 | 2021-05-14 | 深圳市核子基因科技有限公司 | Primer probe set and kit for KRAS gene mutation detection and application thereof |
| CN114182023A (en) * | 2022-02-14 | 2022-03-15 | 首都医科大学附属北京朝阳医院 | Detection kit and method for F-circP3F in rhabdomyosarcoma |
| CN114480660A (en) * | 2022-04-02 | 2022-05-13 | 普瑞基准科技（北京）有限公司 | Gene Panel for detecting pan-cancer species, probe and application |
Non-Patent Citations (3)
| Title |
|---|
| Digital PCR analysis of circulating tumor DNA: a biomarker for chondrosarcoma diagnosis, prognostication, and residual disease detection;Alice Gutteridge, 等;Cancer Medicine;第6卷(第10期);第2194-2202页，第2195页右栏第5段-2196页左栏第4段，表S5 * |
| Recurrent MYOD1 Mutations in Pediatric and Adult Sclerosing and Spindle Cell Rhabdomyosarcomas: Evidence for a Common Pathogenesis;Narasimhan P. Agaram，等;GENES, CHROMOSOMES & CANCER;第53卷(第9期);第779-787页 * |
| 赵杰宏，等.《烤烟化学成分和适应性》.四川科学技术出版社,2016,第187-188页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117230203A (en) | 2023-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111235272B (en) | Composition for once detecting multiple gene mutation of lung cancer and application thereof | |
| CN105039514A (en) | Detection kit for human BRAF gene V600E mutation | |
| CN111206100A (en) | Kit and method for detecting mutation of T790M site of EGFR gene | |
| WO2023142619A1 (en) | Marker for dilated cardiomyopathy genetic detection and use thereof | |
| CN106498028B (en) | Diagnostic method and kit for T790M mutation of EGFR | |
| CN106498029B (en) | Method for increasing diagnostic efficiency of T790M mutation of EGFR | |
| CN110846408A (en) | Primer combination for detecting TTN gene mutation and application thereof | |
| CN113621704B (en) | Reagent and kit for detecting and diagnosing liver cancer | |
| CN112824535A (en) | Primer composition for gene mutation multiplex detection and kit thereof | |
| CN110846409A (en) | Primer combination for detecting TNNI3K gene mutation and application thereof | |
| CN111304329A (en) | Kit for detecting mutation of BRAF gene V600E site | |
| CN117230203B (en) | Primer probe set for detecting MYOD1 gene mutation and kit thereof | |
| CN113337608B (en) | Combined marker for early diagnosis of liver cancer and application thereof | |
| CN114807350A (en) | Biomarker for judging benign and malignant thyroid nodules, polygene joint inspection kit and application | |
| CN109355362B (en) | High-sensitivity SNPs detection system and application | |
| CN108165546A (en) | A kind of miRNA biomarker, composition and application thereof | |
| WO2012002447A1 (en) | Method for detection of mutated polynucleotide | |
| CN107312833B (en) | LSP primer and kit for detecting human BRCA1 gene mutation | |
| CN111690736A (en) | Warfarin medication gene detection kit and use method thereof | |
| CN112695083B (en) | Nucleic acid composition and kit for detecting gene polymorphism of medicine for hypertension | |
| CN116397027B (en) | Composite amplification system and kit for combined detection of KRAS/NRAS/BRAF genotyping | |
| CN113444791B (en) | ARMS-PCR kit for assisting in screening familial papillary thyroid carcinoma | |
| CN117402976B (en) | Rhabdomyosarcoma detection primer probe set, kit and application thereof | |
| CN110951849B (en) | PCR (polymerase chain reaction) kit for detecting human B-raf gene mutation and application thereof | |
| CN116426617B (en) | High-sensitivity mutation detection system based on hairpin structure and enzyme digestion mechanism and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |