CN110551820A - bladder cancer detection kit - Google Patents
bladder cancer detection kit Download PDFInfo
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- CN110551820A CN110551820A CN201910834976.1A CN201910834976A CN110551820A CN 110551820 A CN110551820 A CN 110551820A CN 201910834976 A CN201910834976 A CN 201910834976A CN 110551820 A CN110551820 A CN 110551820A
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- bladder cancer
- gene
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- detection kit
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention belongs to the technical field of gene detection, and particularly discloses a bladder cancer detection kit. The bladder cancer detection kit detects bladder cancer by using the SIX2 gene and/or HSPG2 gene as markers. The bladder cancer detection kit comprises a detection primer for detecting the SIX2 gene and a detection primer for detecting the HSPG2 gene; the sequence of the detection primer of the SIX2 gene is shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; the sequence of the detection primer of the HSPG2 gene is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The invention uses SIX2 gene and/or HSPG2 as markers for bladder cancer detection for the first time, provides a new detection approach and enriches the detection products of bladder cancer. In addition, the kit provided by the invention can greatly improve the specificity of bladder cancer detection and improve the accuracy of bladder cancer detection.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a bladder cancer detection kit.
Background
Bladder cancer refers to malignant tumor occurring on the mucous membrane of bladder, is the most common malignant tumor of urinary system, and is one of ten common tumors of the whole body. In the screening of bladder cancer, the first difficulty lies in determining which gene mutations are related to bladder cancer, and further designing a primer by using the gene as a marker to detect the gene, thereby realizing the screening of bladder cancer. However, in the prior art, the research on the markers of bladder cancer is not deep enough, and the markers capable of being used for predicting bladder cancer are not many, so that more markers for predicting bladder cancer are urgently needed to be researched.
In addition, although some marker gene mutations in the prior art are related to bladder cancer, the mutations may also be related to other diseases; namely, the detected mutation of a certain gene is not necessarily bladder cancer, and can be finally determined by combining other detection indexes; this results in a reduction in the accuracy of the detection. Therefore, the development of a marker with high prediction accuracy and the further development of a bladder cancer detection kit with high detection accuracy have important significance for reducing the detection steps of bladder cancer and saving the screening cost of bladder cancer.
Disclosure of Invention
The invention aims to solve the primary technical problem of providing a bladder cancer detection kit. The invention discovers that the expression of the SIX2 gene and/or the HSPG2 gene is closely related to bladder cancer for the first time, and further develops a brand-new bladder cancer detection kit by taking the SIX2 gene and/or the HSPG2 gene as markers.
The technical problem to be solved by the invention is realized by the following technical scheme:
the invention provides a bladder cancer detection kit, which is used for detecting bladder cancer by taking SIX2 gene as a marker.
preferably, the bladder cancer detection kit detects bladder cancer by using the SIX2 gene and the HSPG2 gene as markers.
Preferably, the bladder cancer detection kit comprises a detection primer for detecting the SIX2 gene;
The sequence of the detection primer of the SIX2 gene is shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;
SEQ ID NO: 1 is as follows: 5'-GCCATGCTAATCCCAGACGAT-3', respectively;
SEQ ID NO: 2 is as follows: 5'-GTGCTCCAGTCAGCCGT-3' are provided.
Preferably, the bladder cancer detection kit comprises a detection primer for detecting the HSPG2 gene;
The sequence of the detection primer of the HSPG2 gene is shown as SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;
SEQ ID NO: 3 is as follows: 5'-GCCATAATTCCGGAATACGTA-3', respectively;
SEQ ID NO: 4 is as follows: 5'-GTGCCGAAGGTGTCAGT-3' are provided.
Preferably, the bladder cancer detection kit further comprises RT-qPCR special premix and ddH 2O.
preferably, the dosage of each component in the kit is 15 ~ 30 ul of RT-qPCR special premix, 0.4 ~ 0.8.8 ul of primers shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 respectively, and 0.8 ul of ddH 2 O20 ~ 40 ul.
Most preferably, the dosage of each component in the kit is 20 ul of the RT-qPCR special premix, 0.5 ul of primers shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 respectively, and 26 ul of ddH 2 O.
Preferably, the concentration of SEQ ID NO 1, 2, 3 and 4 is 400 ~ 600 nM.
Further preferably, the concentration of SEQ ID NO 1, 2, 3 and 4 is 500 ~ 600 nM.
most preferably, SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 was at a concentration of 500 nM.
the special premix for RT-qPCR adopts HiScript II Q RT Supermix for qPCR.
-3 -4The kit has the beneficial effects that the bladder cancer is detected by taking the SIX2 gene and the HSPG2 gene as markers for the first time, the prior art does not mention that the expression abnormality of the SIX2 gene and the HSPG2 gene is related to the bladder cancer, the kit provides a new detection way for the detection of the bladder cancer, and detection products of the bladder cancer are enriched.
Detailed Description
The present invention is further explained below with reference to specific examples, which are not intended to limit the present invention in any way.
Example 1 bladder cancer detection kit
The bladder cancer detection kit comprises the following components:
HiScript II Q RT SuperMix for qPCR 10 ul；
SEQ ID NO: 1 is 0.5 ul respectively;
SEQ ID NO: 2 is 0.5 ul respectively;
SEQ ID NO: 3 is 0.5 ul respectively;
SEQ ID NO: 4, the primers are respectively 0.5 ul;
ddH2O 28 ul；
Wherein, SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 was at a concentration of 500 nM.
example 2 bladder cancer detection method
(1) collecting urine of a person to be screened, separating exosomes in the urine, and further extracting total nucleic acid from the exosomes;
(2) Taking 5 ul of total nucleic acid extracted in the step (1), and adopting the nucleotide sequence shown as SEQ ID NO: 1 and SEQ ID NO: 2, performing PCR amplification on the SIX2 gene under the following conditions: pre-denaturation at 95 ℃ for 4 min for 1 cycle; denaturation at 95 ℃ for 30 seconds, extension at 68 ℃ for 40 seconds, 40 cycles; cooling at 35 deg.C for 20 s; standing for 10min, and collecting Cq value of SIX2 gene;
(3) Taking 5 ul of total nucleic acid extracted in the step (1), and adopting the nucleotide sequence shown as SEQ ID NO: 3 and SEQ ID NO: 4, performing PCR amplification on the HSPG2 gene under the following amplification conditions: pre-denaturation at 95 ℃ for 4 min for 1 cycle; denaturation at 95 ℃ for 30 seconds, extension at 68 ℃ for 40 seconds, 40 cycles; cooling at 35 deg.C for 20 s; standing for 10min, and collecting Cq value of HSPG2 gene;
(4) respectively taking the artificially synthesized SIX2 gene and HSPG2 gene as templates, amplifying according to the steps (2) and (3), then respectively diluting the SIX2 gene and the HSPG2 gene into 10 -2, 10 -3, 10 -4, 10 -5, 10 -6 and 10 -7 umol/L, detecting Cq values of the SIX2 gene and the HSPG2 gene under different concentrations, and drawing a standard curve by taking the abscissa as concentration and the ordinate as the Cq value.
(5) Collecting urine of 100 patients diagnosed with bladder cancer and 100 persons without bladder cancer, detecting Cq values of SIX2 gene and HSPG2 gene in urine of the patients with bladder cancer and the persons without bladder cancer according to the method described in step (1) ~ (3), calculating contents of SIX2 gene and HSPG2 gene in urine according to the standard curve drawn in step (4), wherein according to statistical analysis, the contents of SIX2 gene and HSPG2 gene in urine of 100 patients with bladder cancer are higher than 2.0 x10 -3 umol/L and higher than 5.0 x10 -4 umol/L respectively, the SIX2 gene or HSPG2 gene in urine of 100 persons without bladder cancer is expressed, but the SIX2 gene and HSPG 36 gene are not expressed simultaneously in urine of 100 persons without bladder cancer, and the contents of SIX2 gene or HSPG 9634 umL 2 uml/360 x2 gene in urine of 100 persons without bladder cancer are lower than 360 x10 -4 uml/L respectively.
In conclusion, the invention can use the SIX2 gene and the HSPG2 gene as markers in urine to screen bladder cancer, and the invention can determine that a person to be detected has bladder cancer if the urine of the person to be detected simultaneously expresses the SIX2 gene and the HSPG2 gene by the detection of the kit, and further can definitely determine that the person to be detected has bladder cancer if the expression of the SIX2 gene and the HSPG2 gene in the urine of the person to be detected is higher than 2.0 x10 -3 umol/L and 5.4830 x 6 umol/L.
Sequence listing
<110> Guangzhou Zhongxin medical inspection technology Co., Ltd
<120> bladder cancer detection kit
<130> 2019
<141> 2019-09-05
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
gccatgctaa tcccagacga t 21
<210> 2
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gtgctccagt cagccgt 17
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
gccataattc cggaatacgt a 21
<210> 4
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
gtgccgaagg tgtcagt 17
Claims (10)
1. the bladder cancer detection kit is characterized by comprising
The SIX2 gene is used as a marker for detecting bladder cancer.
2. The bladder cancer detection kit according to claim 1, wherein the bladder cancer detection kit is used for detecting bladder cancer by using the SIX2 gene and the HSPG2 gene as markers.
3. the bladder cancer detection kit according to claim 1 or 2, wherein the bladder cancer detection kit comprises a detection primer for detecting the SIX2 gene;
The sequence of the detection primer of the SIX2 gene is shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;
SEQ ID NO: 1 is as follows: 5'-GCCATGCTAATCCCAGACGAT-3', respectively;
SEQ ID NO: 2 is as follows: 5'-GTGCTCCAGTCAGCCGT-3' are provided.
4. The bladder cancer detection kit according to claim 2, wherein the bladder cancer detection kit comprises a detection primer for detecting HSPG2 gene;
the sequence of the detection primer of the HSPG2 gene is shown as SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;
SEQ ID NO: 3 is as follows: 5'-GCCATAATTCCGGAATACGTA-3', respectively;
SEQ ID NO: 4 is as follows: 5'-GTGCCGAAGGTGTCAGT-3' are provided.
5. The bladder cancer detection kit according to claim 2, further comprising RT-qPCR specific premix, ddH 2O.
6. The bladder cancer detection kit according to claim 5, wherein the amounts of the components in the kit are 15 ~ 30 ul of RT-qPCR special premix, 0.4 ~ 0.8 ul of primers shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, and 0.4 ~ ul of ddH 2 O20 ~ 40 ul of primers shown in SEQ ID NO. 4.
7. The bladder cancer detection kit according to claim 6, wherein the amount of each component in the kit is 20 ul of RT-qPCR special premix, 0.5 ul of primers shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, and 26 ul of ddH 2 O.
8. the bladder cancer detection kit according to claim 6, wherein the concentration of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 is 400 ~ 600 nM.
9. The bladder cancer detection kit according to claim 6, wherein the concentration of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 is 500 ~ 600 nM.
10. The bladder cancer detection kit of claim 6, wherein the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 was at a concentration of 500 nM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910834976.1A CN110551820A (en) | 2019-09-05 | 2019-09-05 | bladder cancer detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910834976.1A CN110551820A (en) | 2019-09-05 | 2019-09-05 | bladder cancer detection kit |
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| Publication Number | Publication Date |
|---|---|
| CN110551820A true CN110551820A (en) | 2019-12-10 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910834976.1A Pending CN110551820A (en) | 2019-09-05 | 2019-09-05 | bladder cancer detection kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872631A (en) * | 2019-12-26 | 2020-03-10 | 广州市基准医疗有限责任公司 | DNA methylation biomarker combination, detection method and kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102912019A (en) * | 2007-11-30 | 2013-02-06 | 基因特力株式会社 | Diagnosis kit and chip for bladder cancer using bladder cancer specific methylation marker gene |
| CN108342484A (en) * | 2018-05-09 | 2018-07-31 | 苏州海苗生物科技有限公司 | A kind of primer and detection kit of carcinoma of urinary bladder PCDH17, TCF21 gene |
-
2019
- 2019-09-05 CN CN201910834976.1A patent/CN110551820A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102912019A (en) * | 2007-11-30 | 2013-02-06 | 基因特力株式会社 | Diagnosis kit and chip for bladder cancer using bladder cancer specific methylation marker gene |
| CN108342484A (en) * | 2018-05-09 | 2018-07-31 | 苏州海苗生物科技有限公司 | A kind of primer and detection kit of carcinoma of urinary bladder PCDH17, TCF21 gene |
Non-Patent Citations (4)
| Title |
|---|
| ANDREW J. MURPHY等: "SIX2 and CITED1, markers of nephronic progenitor self-renewal,remain active in primitive elements of Wilms" tumor", 《PUBLIC》 * |
| EVERSON ET AL.: "Identification of sonic hedgehog-regulated genes and biological processes in the cranial neural crest mesenchyme by comparative transcriptomics", 《BMC GENOMICS》 * |
| JIAN-WANG LI 等: "Six2 is negatively correlated with good prognosis and decreases 5-FU sensitivity via suppressing E-cadherin expression in hepatocellular carcinoma cells", 《BIOMEDICINE & PHARMACOTHERAPY》 * |
| 宋东建等: "SIX2基因在肾母细胞瘤患儿血液中的表达及其甲基化", 《中华医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872631A (en) * | 2019-12-26 | 2020-03-10 | 广州市基准医疗有限责任公司 | DNA methylation biomarker combination, detection method and kit |
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