CN111411156A - Kit and detection method for early detection of cancer - Google Patents
Kit and detection method for early detection of cancer Download PDFInfo
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- CN111411156A CN111411156A CN202010312263.1A CN202010312263A CN111411156A CN 111411156 A CN111411156 A CN 111411156A CN 202010312263 A CN202010312263 A CN 202010312263A CN 111411156 A CN111411156 A CN 111411156A
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- 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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- 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
- C12Q2600/154—Methylation markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention provides a kit and a detection method for early detection of cancer, wherein the kit comprises: a primer probe composition for detecting the DNA methylation level of PRKY, HOXD3b, HOXA7 or GSTP1 gene locus, DNA polymerase and a reaction system. By the invention, the DNA methylation level of the PRKY, HOXD3b, HOXA7 or GSTP1 gene target site can be detected quickly and with high specificity, the kit has the advantages of high sensitivity, strong specificity, simplicity and quickness in operation and the like, and can provide important reference for early diagnosis and differential diagnosis of prostate cancer, kidney cancer or bladder cancer for clinicians.
Description
Technical Field
The invention relates to the field of molecular detection, in particular to a kit and a detection method for early detection of cancer.
Background
Prostate cancer is one of the high-incidence and major lethal cancers worldwide.
Early diagnosis and early treatment of tumors are very important and are key determinants of survival rate after treatment. Early stage tumors, due to small tumor cell burden, often have insignificant changes in their characteristic molecules, which are difficult to distinguish from chronic disease. However, molecular changes are generally earlier than clinical imaging changes, and molecular detection remains the most important and most promising means for early diagnosis and differential diagnosis of tumors.
After search, the applicant finds that Chinese patent with publication number CN109182518A discloses a GSTP1 gene promoter methylation sequencing method based on pyrosequencing technology. The methylation site of the target gene of the invention is different from CN109182518A, and the claim coverage also has almost no same point. Clinical tests have considerable requirements on testing performance parameters, and a testing method available for scientific research is not always applicable to clinical tests, and a large amount of optimization and clinical sample verification are required. The key point of the invention is the clinical application value of the detection of methylation level of methylation sites specific to the combined genes or single genes rather than the pyrosequencing method per se.
Disclosure of Invention
The invention aims to disclose a kit and a detection method for early detection of cancer, and realize early screening and diagnosis of prostate cancer, kidney cancer or bladder cancer.
To achieve the first object, the present invention provides a kit for early detection of cancer, comprising:
a primer probe composition for detecting the DNA methylation level of PRKY, HOXD3b, HOXA7 or GSTP1 gene locus, DNA polymerase and a reaction system.
As a further improvement of the present invention, the primer probe composition comprises:
the primer probe composition is used for detecting the DNA methylation level of the PRKY gene locus and has a nucleotide sequence from SEQ NO.1 to SEQ NO. 3;
a primer probe composition which is used for detecting the DNA methylation level of the locus of the HOXD3b gene and has a nucleotide sequence from SEQ NO.4 to SEQ NO. 6;
a primer probe composition for detecting DNA methylation level of HOXA7 gene locus, and having a nucleotide sequence of SEQ NO.7 to SEQ NO. 9;
a primer probe composition which is used for detecting the DNA methylation level of GSTP1 gene locus and has a nucleotide sequence of SEQ NO.10 to SEQ NO. 12;
the primer probe composition is used for detecting reference gene beta-actin and has the nucleotide sequence of SEQ NO. 13-SEQ NO. 15.
As a further improvement of the present invention, the methylation target detection genes can be used in various combinations.
As a further improvement of the present invention, the methylation target detection gene can be used alone.
As a further improvement of the present invention, the methylated region of the target detection gene remains unchanged, but the amplification primer and/or probe sequences are partially or completely altered.
As a further improvement of the invention, the detection result of the kit provides reference for early diagnosis and differential diagnosis of cancer.
As a further improvement of the present invention, the cancer is prostate cancer, renal cancer or bladder cancer.
Meanwhile, the application also discloses a detection method based on the kit for early cancer detection disclosed by any invention, and a detection system is used for detecting detection materials.
As a further improvement of the invention, the detection material is human peripheral blood free DNA or isolated circulating tumor cells.
As a further improvement of the invention, the detection material is human urine or DNA extracted from urine after prostate massage or cell sediment of prostate massage liquid or biopsy tissue.
As a further improvement of the present invention, the methylated region of the target detection gene remains unchanged, but the amplification primer and/or probe sequences are partially or completely altered.
As a further improvement of the invention, the detection result provides a reference for early diagnosis and differential diagnosis of cancer.
As a further improvement of the present invention, the cancer is prostate cancer, renal cancer or bladder cancer.
As a further improvement of the present invention, the detection system comprises: qPCR detection, pyrosequencing detection, chip detection, mass spectrometry detection or high throughput sequencing detection.
Compared with the prior art, the invention has the beneficial effects that:
by the invention, the DNA methylation level of one or more gene sites of PRKY, HOXD3b, HOXA7 or GSTP1 can be detected quickly and with high specificity, the kit has the advantages of high sensitivity, strong specificity, simplicity and quickness in operation and the like, and can provide important medical reference for early diagnosis and differential diagnosis of prostate cancer, kidney cancer or bladder cancer for clinicians.
Drawings
FIG. 1 is an amplification curve for detecting the DNA methylation level of PRKY gene locus by the detection method based on the cancer early detection kit disclosed by the invention;
FIG. 2 is an amplification curve for detecting the DNA methylation level of the HOXD3b gene locus by the detection method based on the cancer early detection kit disclosed by the invention;
FIG. 3 is an amplification curve for detecting the DNA methylation level at the HOXA7 gene locus by the detection method based on the cancer early detection kit disclosed in the present invention;
FIG. 4 is an amplification curve for detecting the DNA methylation level at GSTP1 gene locus by the detection method based on the cancer early detection kit disclosed in the present invention.
Detailed Description
The present invention is described in detail with reference to the embodiments shown in the drawings, but it should be understood that these embodiments are not intended to limit the present invention, and those skilled in the art should understand that functional, methodological, or structural equivalents or substitutions made by these embodiments are within the scope of the present invention. In this application, the term "sec" is a unit of time: second; the term "min" is a unit of time: and (3) minutes. The term "mM" is the unit of concentration: millimoles per liter.
The first embodiment is as follows:
kit for early detection of cancer (hereinafter referred to as "kit" for short) ") The method comprises the following steps: the primer probe composition, the DNA polymerase and the reaction system are used for detecting the DNA methylation level of one or more gene sites of PRKY, HOXD3b, HOXA7 or GSTP 1. The kit is used for specifically detecting the DNA methylation level of one or more gene loci, and early diagnosis is carried out on the prostate cancer, the renal cancer or the bladder cancer by the kit. The DNA polymerase is Taq enzyme, and the reaction system comprises PCR buffer solution, dNTPs and MgCl2And (4) forming.
The internal reference Gene beta-actin, as a housekeeping Gene (HKG), generally refers to a protein expressed by housekeeping Gene code for mammalian cell expression. Their expression in various tissues and cells is relatively constant and is commonly used as a reference when detecting changes in the expression level of a protein. Therefore, the method provides accurate reference basis for detecting the DNA methylation level of PRKY, HOXD3b, HOXA7 and GSTP1 gene loci so as to detect the DNA methylation of one or more target gene loci. During specific analysis, the ratio of the target gene amplification Ct value to the sample actin amplification Ct value is compared and analyzed.
In this example, the specific probes are labeled with fluorescence quenching groups at the 3 'ends, and labeled with fluorescence reporter groups at the 5' ends, wherein the fluorescence quenching groups are selected from any one of DABCY L, MGB, BHQ-1, BHQ-2, BHQ-3 or thiophosphate, and the fluorescence reporter groups are selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA.
The sequence design of the primer probe compositions disclosed herein is shown in Table 1 below, primer and probe sequences
E is a fluorescent group, and Q is a quenching group.
Example two:
referring to fig. 1 to 4, the present example discloses a detection method based on a cancer early detection kit (hereinafter referred to as "detection method"), which includes the following steps (1) to (4). The detection method is realized based on the kit disclosed in the first embodiment.
And (1) extracting a biological sample. The biological sample is human peripheral blood or human urine cell sediment. Specifically, 10ml of peripheral blood of early stage prostate cancer confirmed patients (initial diagnosis, and prostate cancer does not transfer) is collected by using a STREK tube, and is subjected to uniform mixing and then is subjected to inspection; meanwhile, peripheral blood of a prostate benign hyperplasia patient of a normal person was collected as a control group.
And (2) extracting sample DNA from the biological sample, and adjusting the sample DNA to standard purity and standard concentration. Extraction of sample DNA ctDNA (Cell-freemolecular DNA) was extracted using QIAamp Circulating Nucleic Acid kit.
Step (3) converting the sample DNA with bisulfite. And fixing the DNA methylation change level of the sample DNA at the target gene locus. Specifically, in step (3), the sample DNA may be converted using a Qiagen Rapid bisulfite conversion kit. The amount of ctDNA before transformation is not less than 10 ng.
And (4) carrying out methylation level detection on the target gene and the reference gene by using a detection system. The detection system comprises: a qPCR detection system, a pyrosequencing detection system, a chip detection system, a mass spectrometry detection system, or a high-throughput detection system. Prostate, renal, or bladder cancer. In this embodiment, the applicant exemplifies that the qPCR detection system is selected as the detection system, and it is known to those skilled in the art that the same or similar detection systems such as pyrosequencing detection system, chip detection system, etc. can also specifically detect the DNA methylation level of the gene sites of PRKY, HOXD3b, HOXA7 or GSTP1 by using the kit disclosed in the first embodiment, so as to perform early screening and diagnosis on prostate cancer, kidney cancer or bladder cancer. The qPCR detection system adopts a qPCR amplification instrument with the model number ABI 7500.
The qPCR amplification system is shown in table 2 below:
TABLE 2 exemplary assay System
| Reagent | Amount used (ul) |
| 10 × |
2 |
| dNTP(10mM) | 1 |
| Mgcl2(50mM) | 1.6 |
| Taq polymerase (10U/ul) | 0.2 |
| Primer probe composition | 0.4 each |
| ddH2O | Complement |
| DNA template after transformation | 5 |
| |
20 |
Specifically, in this implementation, the reaction sequence of the amplification reaction performed using the qPCR detection system is:
and (5) analyzing the methylation level expression of the target gene and the reference gene.
As shown in fig. 1, when the qPCR detection system is used for the PRKY gene locus, the Ct value of the sample DNA is about 19, and the control group has no typical amplification curve.
As shown in fig. 2, when the qPCR detection system was used for the HOXD3b gene locus in a test sample of blood cfDNA of a prostate cancer patient (i.e., the aforementioned sample DNA) extracted from a diagnosed patient with early stage prostate cancer, the Ct value of the sample DNA was about 18, and there was no typical amplification curve in the control group.
As shown in fig. 3, when the qPCR detection system was used for HOXDA7 gene locus in a test sample of blood cfDNA of a prostate cancer patient (i.e., the aforementioned sample DNA) extracted from a patient diagnosed with early stage prostate cancer, the Ct value of the sample DNA was about 18, and there was no typical amplification curve in the control group.
As shown in fig. 4, when the qPCR detection system is used for GSTP1 gene locus in a detection sample of blood cfDNA of a prostate cancer patient (i.e., the aforementioned sample DNA) extracted from a patient diagnosed with early stage prostate cancer, the Ct value of the sample DNA is about 18, and there is no typical amplification curve in the control group.
In this example, a specific fluorescent probe, which is an oligonucleotide, is added simultaneously with a pair of primers during qPCR amplification, and a reporter fluorophore and a quencher fluorophore are labeled at both ends (i.e., 3 'end and 5' end), respectively. When the probe is complete, the fluorescent signal emitted by the reporter fluorophore is absorbed by the quenching fluorophore; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme degrades the probe enzyme digestion, so that the report fluorescent group and the quenching fluorescent group are separated, and a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the complete synchronization of the accumulation of the fluorescence signal and the formation of a qPCR product is realized.
The detection method disclosed in this example uses the kit disclosed in example one for qPCR amplification. The real-time fluorescent quantitative PCR detection of the amplified product by using the probe of the specific fluorescent marker can effectively detect the DNA methylation level of one or more gene sites of PRKY, HOXD3b, HOXA7 or GSTP1, and has the characteristics of stability, rapidness, high specificity, low detection cost and the like.
Example three:
this example discloses a method for early detection of cancer by detecting the DNA methylation level of one or more of the gene sites PRKY, HOXD3b, HOXA7 or GSTP 1. The cancer is prostate cancer, renal cancer or bladder cancer. The method for early detection of cancer disclosed in this example can use the kit disclosed in example one and can detect cancer by the detection method based on the kit disclosed in example two, and can perform early screening and specific detection on prostate cancer, renal cancer or bladder cancer.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
It will be clear to a person skilled in the art that the present invention is not limited to the details of the exemplary embodiments presented above, but that it can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should be able to make the description as a whole, and the embodiments may be appropriately combined to form other embodiments understood by those skilled in the art.
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Claims (14)
1. A kit for early detection of cancer, comprising:
a primer probe composition for detecting the DNA methylation level of PRKY, HOXD3b, HOXA7 or GSTP1 gene locus, DNA polymerase and a reaction system.
2. The kit of claim 1, wherein the primer probe composition comprises:
the primer probe composition is used for detecting the DNA methylation level of the PRKY gene locus and has a nucleotide sequence from SEQ NO.1 to SEQ NO. 3;
a primer probe composition which is used for detecting the DNA methylation level of the locus of the HOXD3b gene and has a nucleotide sequence from SEQ NO.4 to SEQ NO. 6;
a primer probe composition for detecting DNA methylation level of HOXA7 gene locus, and having a nucleotide sequence of SEQ NO.7 to SEQ NO. 9;
a primer probe composition which is used for detecting the DNA methylation level of GSTP1 gene locus and has a nucleotide sequence of SEQ NO. 10-SEQ NO. 12;
the primer probe composition is used for detecting reference gene beta-actin and has the nucleotide sequence of SEQ NO. 13-SEQ NO. 15.
3. The kit according to claim 1, wherein the methylation target detection genes can be used in various combinations.
4. The kit according to claim 1, wherein the methylation target detection gene can be used alone.
5. The kit of claim 1, wherein the methylated region of the target detection gene remains unchanged but the amplification primer and/or probe sequence is partially or completely altered.
6. The kit of claim 1, wherein the detection result of the kit provides reference for early diagnosis and differential diagnosis of cancer.
7. The kit of claim 6, wherein the cancer is prostate cancer, renal cancer, or bladder cancer.
8. A detection method based on the kit for early detection of cancer according to any one of claims 1 to 7, characterized in that a detection material is detected using a detection system.
9. The method of claim 8, wherein the test material is human peripheral blood free DNA or isolated circulating tumor cells.
10. The method of claim 8, wherein the detection material is human urine or post-prostate massage urine or prostate massage solution cell sediment or DNA extracted from a biopsy.
11. The method of claim 8, wherein the methylated region of the target test gene is maintained, but the amplification primer and/or probe sequence is partially or completely altered.
12. The method of claim 8, wherein the test results provide a reference for early and differential diagnosis of cancer.
13. The detection method according to claim 12, wherein the cancer is prostate cancer, kidney cancer, or bladder cancer.
14. The inspection method of claim 8, wherein the inspection system comprises: qPCR detection, pyrosequencing detection, chip detection, mass spectrometry detection or high throughput sequencing detection.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016067A (en) * | 2008-01-22 | 2011-04-13 | 维里德克斯有限责任公司 | Detection of GSTP1 hypermethylation in prostate cancer |
| CN109072310A (en) * | 2016-04-01 | 2018-12-21 | 纳诺梅德诊断私人有限公司 | Cancer is detected in urine |
| CN109182518A (en) * | 2018-09-12 | 2019-01-11 | 黄映辉 | GSTP1 gene promoter methylation detection method based on pyrosequencing techniques |
| CN110484625A (en) * | 2019-08-29 | 2019-11-22 | 无锡市申瑞生物制品有限公司 | For detecting primer combination of probe object, kit and the detection method of PRKY gene methylation |
| CN110964809A (en) * | 2018-09-29 | 2020-04-07 | 广州市康立明生物科技有限责任公司 | HOXA7 methylation detection reagent |
-
2020
- 2020-04-20 CN CN202010312263.1A patent/CN111411156A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016067A (en) * | 2008-01-22 | 2011-04-13 | 维里德克斯有限责任公司 | Detection of GSTP1 hypermethylation in prostate cancer |
| CN109072310A (en) * | 2016-04-01 | 2018-12-21 | 纳诺梅德诊断私人有限公司 | Cancer is detected in urine |
| CN109182518A (en) * | 2018-09-12 | 2019-01-11 | 黄映辉 | GSTP1 gene promoter methylation detection method based on pyrosequencing techniques |
| CN110964809A (en) * | 2018-09-29 | 2020-04-07 | 广州市康立明生物科技有限责任公司 | HOXA7 methylation detection reagent |
| CN110484625A (en) * | 2019-08-29 | 2019-11-22 | 无锡市申瑞生物制品有限公司 | For detecting primer combination of probe object, kit and the detection method of PRKY gene methylation |
Non-Patent Citations (1)
| Title |
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| IGOR BRIKUN等: "A panel of DNA methylation markers for the detection of prostate cancer from FV and DRE urine DNA", 《CLINICAL EPIGENETICS》 * |
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