阅读说明：本技术 用于癌症早期检测的试剂盒及检测方法 (Kit and detection method for early detection of cancer ) 是由 朱桂春 于垚垚 姚鲁帅 盛青松 于 2020-04-20 设计创作，主要内容包括：本发明提供了用于癌症早期检测的试剂盒及检测方法,该试剂盒包括：检测PRKY、HOXD3b、HOXA7或者GSTP1基因位点的DNA甲基化水平的引物探针组合物,DNA聚合酶及反应体系。通过发明,能够通过对PRKY、HOXD3b、HOXA7或者GSTP1基因目标位点的DNA甲基化水平予以快速、高特异性检测,具有灵敏度高,特异性强,操作简便快捷等优点,可为临床医生对前列腺癌、肾癌或者膀胱癌早期诊断及鉴别诊断提供重要参考。(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.)

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.

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.