CN113234820A

CN113234820A - Methods and kits for identifying prostate cancer status

Info

Publication number: CN113234820A
Application number: CN202110475391.2A
Authority: CN
Inventors: 徐春叶; 李明明; 蒲珏
Original assignee: Beijing Aikelun Medical Technology Co ltd
Current assignee: Beijing Aikelun Medical Technology Co ltd
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-08-10

Abstract

Provided herein is a method of identifying a prostate cancer status in a subject, comprising: 1) detecting the methylation level of a biomarker gene in a biological sample from the subject, wherein the biomarker gene is selected from one or more of the following genes: APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF 1A; and 2) comparing the methylation level detected in step 1) with a normal methylation level of a corresponding biomarker gene in the population to determine the prostate cancer status in the subject. Also provided herein are kits for identifying prostate cancer status in a subject. The method and the kit provided by the application provide a new way for predicting, diagnosing and evaluating the prostatic cancer, which is rapid, reliable and accurate.

Description

Methods and kits for identifying prostate cancer status

Technical Field

The present invention relates to methods and kits for identifying prostate cancer status in a subject.

Background

Prostate cancer is the most common malignancy of the male reproductive system, and in the united states, prostate cancer is the second leading cause of cancer death in men. In recent years, the incidence rate of prostate cancer in China is obviously increased, the prostate cancer has leap the sixth common tumor of Chinese men, and the increase rate of the prostate cancer is the fastest among all male tumors. The cure rate of the prostate cancer found in the early stage is as high as 95%, the survival rate of the prostate cancer found in the late stage 5 years is less than 30%, and the prevention and treatment situation of the prostate cancer in China is very severe at present.

Currently, the diagnosis of prostate cancer mainly includes PSA blood test, digital rectal examination, ultrasonic wave, and the like. However, in recent years, studies have reported that the diagnosis of prostate cancer based on PSA is not as accurate as imagined, and the most prominent problem is misdiagnosis of many people and possible over-treatment. Digital rectal examination is a non-specific examination and is often found in advanced stages of prostate cancer. Ultrasound is a very valuable means of diagnosing prostate cancer and can help physicians examine a patient's prostate and surrounding tissue structures for suspicious lesions and can make an initial determination of the size of a tumor. However, ultrasound examination is less specific in prostate cancer diagnosis, and finding a prostate hypoechoic lesion is to be identified with a variety of diseases. Therefore, there is no effective method for diagnosing prostate cancer, especially for early diagnosis of prostate cancer.

With the gradual exploration of the molecular mechanism of prostate cancer occurrence, the development of molecular markers for early diagnosis of prostate cancer is highly concerned, and unfortunately, no molecular marker with high sensitivity and specificity has been found so far. In recent years, the research on the epigenetics of the prostate cancer is relatively fast, particularly the DNA methylation, and the research finds that a plurality of specific tumor-related genes have methylation state changes to different degrees in the early stage of the prostate cancer, thereby providing a new opportunity for the search of early diagnosis markers of the prostate cancer.

Genomic DNA methylation abnormality and tumor occurrence are always one of the hot spots of medical research, and methylation of related genes is involved in cell cycle, DNA repair, angiogenesis, apoptosis and the like. The most likely regulatory role of DNA hypermethylation is to determine the fate of the cell by inhibiting the expression of key genes, as studies of DNA methylation abnormalities in tumor cells have made a number of major advances in a variety of tumors. In mammals, methylation only affects cytosine (CpG) before guanine on a DNA chain, methylation distribution of CpG dinucleotides in normal cells is not uniform, approximately 50% of genes have CpG islands with concentrated CpG distribution in a promoter region, the lengths of the CpG islands are different from 0.5 kb to 2kb, and the region has close relation with transcription regulation of the genes. CpG island methylation of certain gene regulation areas of human bodies frequently appears in related cancer cell tissues, and shows to be related to the pathogenesis, disease course progression and prognosis, drug sensitivity and the like of certain tumors. To date, abnormalities in gene methylation have been found in most human tumors, and studies have found that epigenetic codes are disturbed in cancer cells, first manifested by a disturbance in the level of DNA methylation, also known as methylation rearrangement. Since the local hypermethylation of CpG islands of tumor suppressor genes precedes the malignant proliferation of cells, the detection of DNA methylation can be used for early diagnosis of tumorigenesis. Cancer-associated gene methylation is also an early event in prostate cancer development, and therefore the methylation state of the associated gene can be an effective index for predicting the risk of early prostate cancer. Nevertheless, means for effectively detecting the methylation status of these cancer-associated genes and processing the detection results are still lacking.

Disclosure of Invention

To address the above-mentioned problems, in one aspect, provided herein is a method of identifying a prostate cancer status in a subject, comprising the steps of: 1) detecting the methylation level of a biomarker gene in a biological sample from the subject, wherein the biomarker gene is selected from one or more of the following genes: APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF 1A; and 1) comparing the methylation level detected in step 2) with a normal methylation level of a corresponding biomarker gene in the population to determine the prostate cancer status in the subject.

In some embodiments, the method further comprises performing step 1) again after the subject receives medical treatment and comparing the methylation level measurements obtained in two times to determine a change in prostate cancer status in the subject.

In some embodiments, step 1) may comprise extracting DNA from the biological sample and treating with bisulfite, such that unmethylated cytosine residues in the DNA are deaminated, while methylated cytosine residues remain unchanged.

In some preferred embodiments, the bisulfite salt is sodium bisulfite.

In some preferred embodiments, the biomarker genes in step 1) are selected from 2 or more of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF 1A.

In a more preferred embodiment, the biomarker genes in step 1) are selected from 5 or more of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF 1A.

In some embodiments, the biomarker genes in step 1) are APC, CCND2, CDH1, PITX2, and RARB.

In some preferred embodiments, the prostate cancer status is stage I or stage II prostate cancer and the biomarker gene is PITX2 and/or RARB.

In some preferred embodiments, the prostate cancer status is adenocarcinoma and the biomarker genes are CDH1, PITX2, and/or RARB.

In some preferred embodiments, the prostate cancer status is transitional cell carcinoma and the biomarker gene is CCND2 and/or RARB.

In some preferred embodiments, the prostate cancer state is squamous carcinoma and the biomarker gene is PITX2 and/or RARB.

In some embodiments, step 1) comprises detecting the level of methylation of a target region within the biomarker gene, the target region being a nucleotide sequence of at least 15 bases in length within the biomarker gene, or a complement thereof, respectively.

In some embodiments, the detecting of the methylation level of the APC gene in step 1) comprises using a nucleic acid having the sequence of SEQ ID NO: 11 and 12 or a primer pair having the sequences shown in SEQ ID NOs: 15 and 16, and performing PCR amplification reaction by using the APC gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation level of CCND2 gene includes using a dna having SEQ ID NO: 19 and 20, a primer pair having the sequences shown in SEQ ID NOs: 23 and 24, or a primer pair having the sequences shown in SEQ ID NOs: 27 and 28, and carrying out PCR amplification reaction by using the CCND2 gene after bisulfite treatment of the biological sample or a fragment thereof as a template; detection of the methylation level of the CDH1 gene includes using a dna having the sequence of SEQ ID NO: 31 and 32 or a primer pair having the sequences shown in SEQ ID NOs: 35 and 36, and carrying out PCR amplification reaction by using the CDH1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation levels of the GSTP1 gene includes using a nucleic acid having the sequence of SEQ ID NO: 39 and 40 or a primer pair having the sequences shown in SEQ ID NOs: 43 and 44, and carrying out PCR amplification reaction by using the GSTP1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of the methylation level of the MCAM gene involves the use of a nucleic acid sequence having SEQ ID NO: 47 and 48, a primer pair having the sequences shown in SEQ ID NOs: 51 and 52, or a primer pair having the sequences shown in SEQ ID NOs: 55 and 56, and carrying out PCR amplification reaction by taking the MCAM gene or the segment thereof treated by the bisulfite in the biological sample as a template; detection of methylation levels of PENK genes involves the use of a dna having the sequence of SEQ ID NO: 59 and 60 or a primer pair having the sequences shown in SEQ ID NOs: 63 and 64, and carrying out PCR amplification reaction by using the hydrosulfite-treated PENK gene or the fragment thereof in the biological sample as a template; detection of methylation levels of PITX2 gene includes using a probe having the sequence of SEQ ID NO: 67 and 68 or a primer pair having the sequences shown in SEQ ID NOs: 71 and 72, and carrying out PCR amplification reaction by using the PITX2 gene or the fragment thereof after bisulfite treatment in the biological sample as a template; detection of methylation levels of the PTGS2 gene included the use of a dna having the sequence of SEQ ID NO: 75 and 76 or a primer pair having the sequences shown in SEQ ID NOs: 79 and 80, and carrying out PCR amplification reaction by using the PTGS2 gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation levels of the RARB gene involves the use of a dna having the sequence of SEQ ID NO: 83 and 84, a primer pair having the sequences shown in SEQ ID NOs: 87 and 88, or a primer pair having the sequences shown in SEQ ID NOs: 91 and 92, and performing PCR amplification reaction by using the RARB gene or the fragment thereof treated by the bisulfite in the biological sample as a template; and detecting the methylation level of the RASSF1A gene comprises using a nucleic acid sequence having the sequence of SEQ ID NO: 95 and 96, a primer pair having the sequences shown in SEQ ID NOs: 99 and 100, or a primer pair having the sequences shown in SEQ ID NOs: 103 and 104, and carrying out PCR amplification reaction by using the RASSF1A gene or the fragment thereof after bisulfite treatment in the biological sample as a template.

In some preferred embodiments, the detection of the methylation level of the APC gene in step 1) comprises using a nucleic acid having the sequence of SEQ ID NO: 11 and 12 and a primer pair having the sequences shown in SEQ ID NOs: 13, or a blocking primer having the sequence shown in SEQ ID NO: 15 and 16 and a primer pair having the sequences shown in SEQ ID NOs: 17, performing PCR amplification reaction by using the APC gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation level of CCND2 gene includes using a dna having SEQ ID NO: 19 and 20 and a primer pair having the sequences shown in SEQ ID NOs: 21 using a blocking primer having the sequence shown in SEQ ID NO: 23 and 24 and a primer pair having the sequences shown in SEQ ID NOs: 25, or a blocking primer having the sequence shown in SEQ ID NO: 27 and 28 and a primer pair having the sequences shown in SEQ ID NOs: 29, and carrying out PCR amplification reaction by using the CCND2 gene treated by bisulfite in the biological sample or a fragment thereof as a template; detection of the methylation level of the CDH1 gene includes using a dna having the sequence of SEQ ID NO: 31 and 32 and a primer pair having the sequences shown in SEQ ID NOs: 33, or a blocking primer having the sequence shown in SEQ ID NO: 35 and 36 and a primer pair having the sequences shown in SEQ ID NOs: 37, and carrying out PCR amplification reaction by using the CDH1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation levels of the GSTP1 gene includes using a nucleic acid having the sequence of SEQ ID NO: 39 and 40 and a primer pair having the sequences shown in SEQ ID NOs: 41, or a blocking primer having the sequence shown in SEQ ID NO: 43 and 44 and a primer pair having the sequences shown in SEQ ID NOs: 45, and carrying out PCR amplification reaction by using the GSTP1 gene treated by bisulfite in the biological sample or a fragment thereof as a template; detection of the methylation level of the MCAM gene involves the use of a nucleic acid sequence having SEQ ID NO: 47 and 48 and a primer pair having the sequences shown in SEQ ID NOs: 49 using a blocking primer having the sequence shown in SEQ ID NO: 51 and 52 and a primer pair having the sequences shown in SEQ ID NOs: 53, or a blocking primer having the sequence shown in SEQ ID NO: 55 and 56 and a primer pair having the sequences shown in SEQ ID NOs: 57, performing PCR amplification reaction by using the MCAM gene treated by the bisulfite in the biological sample or the fragment thereof as a template; detection of methylation levels of PENK genes involves the use of a dna having the sequence of SEQ ID NO: 59 and 60 and a primer pair having the sequences shown in SEQ ID NOs: 61, or a blocking primer having the sequence shown in SEQ ID NO: 63 and 64 and a primer pair having the sequences shown in SEQ ID NOs: 65, performing PCR amplification reaction by using the PENK gene treated by the bisulfite in the biological sample or the fragment thereof as a template; detection of methylation levels of PITX2 gene includes using a probe having the sequence of SEQ ID NO: 67 and 68 and a primer pair having the sequences shown in SEQ ID NOs: 69, or using a blocking primer having the sequence shown in SEQ ID NO: 71 and 72 and a primer pair having the sequences shown in SEQ ID NOs: 73, and carrying out PCR amplification reaction by using the PITX2 gene or the fragment thereof after bisulfite treatment in the biological sample as a template; detection of methylation levels of the PTGS2 gene included the use of a dna having the sequence of SEQ ID NO: 75 and 76 and a primer pair having the sequences shown in SEQ ID NOs: 77, or a blocking primer having the sequence shown in SEQ ID NO: 79 and 80 and a primer pair having the sequences shown in SEQ ID NOs: 81, and carrying out PCR amplification reaction by using the PTGS2 gene or the segment thereof treated by the bisulfite in the biological sample as a template; detection of methylation levels of the RARB gene involves the use of a dna having the sequence of SEQ ID NO: 83 and 84 and a primer pair having the sequences shown in SEQ ID NOs: 85, using a blocking primer having the sequence shown in SEQ ID NO: 87 and 88 and a primer pair having the sequence shown in SEQ ID NO: 89, or a blocking primer having the sequence shown in SEQ ID NO: 91 and 92 and a primer pair having the sequences shown in SEQ ID NOs: 93, and carrying out PCR amplification reaction by using the RARB gene or the fragment thereof treated by the bisulfite in the biological sample as a template; and detecting the methylation level of the RASSF1A gene comprises using a nucleic acid sequence having the sequence of SEQ ID NO: 95 and 96 and a primer pair having the sequences shown in SEQ ID NOs: 97 using a blocking primer having the sequence shown in SEQ ID NO: 99 and 100 and a primer pair having the sequences shown in SEQ ID NOs: 101, or a blocking primer having the sequence shown in SEQ ID NO: 103 and 104 and a primer pair having the sequences shown in SEQ ID NOs: 105, and performing a PCR amplification reaction using the bisulfite treated RASSF1A gene or fragment thereof as a template, wherein the blocking primer has a 3' end modification that is resistant to DNA polymerase extension amplification.

In a more preferred embodiment, the detection of the methylation level of the APC gene in step 1) comprises the use of a nucleic acid having the sequence of SEQ ID NO: 11 and 12, a primer pair having the sequences shown in SEQ ID NOs: 13 and a blocking primer having the sequence shown in SEQ ID NO: 14, or a probe having a sequence shown in SEQ ID NO: 15 and 16, a primer pair having the sequences shown in SEQ ID NOs: 17 and a blocking primer having the sequence shown in SEQ ID NO: 18, performing PCR amplification reaction by using the APC gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation level of CCND2 gene includes using a dna having SEQ ID NO: 19 and 20, a primer pair having the sequences shown in SEQ ID NOs: 21 and a blocking primer having the sequence shown in SEQ ID NO: 22, using a probe having the sequence shown in SEQ ID NO: 23 and 24, a primer pair having the sequences shown in SEQ ID NOs: 25 and a blocking primer having the sequence shown in SEQ ID NO: 26, or a probe having the sequence shown in SEQ ID NO: 27 and 28, a primer pair having the sequences shown in SEQ ID NOs: 29 and a blocking primer having the sequence shown in SEQ ID NO: 30, and performing PCR amplification reaction by using the CCND2 gene treated by bisulfite in the biological sample or a fragment thereof as a template; detection of the methylation level of the CDH1 gene includes using a dna having the sequence of SEQ ID NO: 31 and 32, a primer pair having the sequences shown in SEQ ID NOs: 33 and a blocking primer having the sequence shown in SEQ ID NO: 34, or a probe having the sequence shown in SEQ ID NO: 35 and 36, a primer pair having the sequences shown in SEQ ID NOs: 37 and a blocking primer having the sequence shown in SEQ ID NO: 38, and carrying out PCR amplification reaction by using the CDH1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation levels of the GSTP1 gene includes using a nucleic acid having the sequence of SEQ ID NO: 39 and 40, a primer pair having the sequences shown in SEQ ID NOs: 41 and a blocking primer having the sequence shown in SEQ ID NO: 42, or a probe having the sequence shown in SEQ ID NO: 43 and 44, a primer pair having the sequences shown in SEQ ID NOs: 45 and a blocking primer having the sequence shown in SEQ ID NO: 46, and carrying out PCR amplification reaction by using the GSTP1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of the methylation level of the MCAM gene involves the use of a nucleic acid sequence having SEQ ID NO: 47 and 48, a primer pair having the sequences shown in SEQ ID NOs: 49 and a blocking primer having the sequence shown in SEQ ID NO: 50, using a probe having the sequence shown in SEQ ID NO: 51 and 52, a primer pair having the sequences shown in SEQ ID NOs: 53 and a blocking primer having the sequence shown in SEQ ID NO: 54, or a probe having the sequence shown in SEQ ID NO: 55 and 56, a primer pair having the sequences shown in SEQ ID NOs: 57 and a blocking primer having the sequence shown in SEQ ID NO: 58, and performing PCR amplification reaction by using the MCAM gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation levels of PENK genes involves the use of a dna having the sequence of SEQ ID NO: 59 and 60, a primer pair having the sequences shown in SEQ ID NOs: 61 and a blocking primer having the sequence shown in SEQ ID NO: 62, or a probe having the sequence shown in SEQ ID NO: 63 and 64, a primer pair having the sequences shown in SEQ ID NOs: 65 and a blocking primer having the sequence shown in SEQ ID NO: 66, and carrying out PCR amplification reaction by using the hydrosulfite-treated PENK gene or the fragment thereof in the biological sample as a template; detection of methylation levels of PITX2 gene includes using a probe having the sequence of SEQ ID NO: 67 and 68, a primer pair having the sequences shown in SEQ ID NOs: 69 and a blocking primer having the sequence shown in SEQ ID NO: 70, or using a probe having the sequence shown in SEQ ID NO: 71 and 72, a primer pair having the sequences shown in SEQ ID NOs: 73 and a blocking primer having the sequence shown in SEQ ID NO: 74, and carrying out PCR amplification reaction by using the PITX2 gene or the fragment thereof after bisulfite treatment in the biological sample as a template; detection of methylation levels of the PTGS2 gene included the use of a dna having the sequence of SEQ ID NO: 75 and 76, a primer pair having the sequences shown in SEQ ID NOs: 77 and a blocking primer having the sequence shown in SEQ ID NO: 78, or a probe having the sequence shown in SEQ ID NO: 79 and 80, a primer pair having the sequences shown in SEQ ID NO: 81 and a blocking primer having the sequence shown in SEQ ID NO: 82, and carrying out PCR amplification reaction by using the PTGS2 gene or the fragment thereof treated by the bisulfite in the biological sample as a template; detection of methylation levels of the RARB gene involves the use of a dna having the sequence of SEQ ID NO: 83 and 84, a primer pair having the sequences shown in SEQ ID NOs: 85 and a blocking primer having the sequence shown in SEQ ID NO: 86, using a probe having the sequence shown in SEQ ID NO: 87 and 88, a primer pair having the sequences shown in SEQ ID NOs: 89 and a blocking primer having the sequence shown in SEQ ID NO: 90, or using a probe having the sequence shown in SEQ ID NO: 91 and 92, a primer pair having the sequences shown in SEQ ID NOs: 93 and a blocking primer having the sequence shown in SEQ ID NO: 94, and carrying out PCR amplification reaction by using the RARB gene or the fragment thereof treated by the bisulfite in the biological sample as a template; and detecting the methylation level of the RASSF1A gene comprises using a nucleic acid sequence having the sequence of SEQ ID NO: 95 and 96, a primer pair having the sequences shown in SEQ ID NOs: 97 and a blocking primer having the sequence shown in SEQ ID NO: 98, using a probe having the sequence shown in SEQ ID NO: 99 and 100, a primer pair having the sequences shown in SEQ ID NOs: 101 and a blocking primer having the sequence shown in SEQ ID NO: 102, or a probe having the sequence shown in SEQ ID NO: 103 and 104, a primer pair having the sequences shown in SEQ ID NOs: 105 and a blocking primer having the sequence shown in SEQ ID NO: 106 and a probe with a sequence shown in the specification, wherein the probe takes a bisulfite-treated RASSF1A gene or a fragment thereof of the biological sample as a template to carry out PCR amplification reaction, and the probe has a fluorescent group at one end and a fluorescence quenching group at the other end.

In some embodiments, step 1) further comprises using a peptide having SEQ ID NO: 107 and 108 and a primer pair having the sequences shown in SEQ ID NOs: 109, and carrying out PCR amplification reaction by using the ACTB gene or the segment thereof which is treated by the bisulfite and is used as the reference gene in the biological sample as a template.

In some embodiments, step 2) comprises determining the prostate cancer status in the subject based on logistic regression from the methylation levels of the biomarker genes.

In another aspect, provided herein is a kit for identifying a prostate cancer state in a subject, comprising a primer pair for detecting a methylation level of a biomarker gene in a biological sample from the subject, wherein the primer pair is used for performing a PCR amplification reaction with bisulfite-treated the biomarker gene or a fragment thereof as a template; the biomarker genes are selected from one or more of the following genes: APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF 1A.

In some preferred embodiments, the biomarker genes are selected from 2 or more of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF 1A.

In a more preferred embodiment, the biomarker genes are selected from 5 or more of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF 1A.

In some embodiments, the biomarker genes are APC, CCND2, CDH1, PITX2, and RARB.

In some embodiments, the prostate cancer status is stage I or stage II prostate cancer and the biomarker gene is PITX2 and/or RARB. In some embodiments, the prostate cancer status is adenocarcinoma and the biomarker genes are CDH1, PITX2, and/or RARB. In some embodiments, the prostate cancer status is transitional cell carcinoma and the biomarker gene is CCND2 and/or RARB. In some embodiments, the prostate cancer state is squamous carcinoma and the biomarker gene is PITX2 and/or RARB.

In some embodiments, the primer pair for APC methylation level detection in the kit has the sequence of SEQ ID NO: 11 and 12 or SEQ ID NO: 15 and 16; the primer pair for detecting the methylation level of CCND2 has the sequence shown in SEQ ID NO: 19 and 20, SEQ ID NO: 23 and 24 or SEQ ID NO: 27 and 28; the primer pair for detection of the level of methylation of CDH1 has the sequence of SEQ ID NO: 31 and 32 or SEQ ID NO: 35 and 36; the primer pair for detecting the methylation level of GSTP1 has the nucleotide sequence shown in SEQ ID NO: 39 and 40 or SEQ ID NO: 43 and 44; the primer pair for detecting the MCAM methylation level has the sequence of SEQ ID NO: 47 and 48, SEQ ID NO: 51 and 52 or the sequences shown in SEQ ID NO: 55 and 56; the primer pair for PENK methylation level detection has the nucleotide sequence of SEQ ID NO: 59 and 60 or SEQ ID NO: 63 and 64; the primer pair for the detection of the methylation level of PITX2 has the sequence of SEQ ID NO: 67 and 68 or the sequences shown in SEQ ID NOs: 71 and 72; the primer pair used for the detection of the level of methylation of PTGS2 has the sequence of SEQ ID NO: 75 and 76 or SEQ ID NO: 79 and 80; the primer pair for RARB methylation level detection has the sequence of SEQ ID NO: 83 and 84, SEQ ID NO: 87 and 88 or the sequences shown in SEQ ID NOs: 91 and 92; and the primer pair for detecting the methylation level of RASSF1A has the nucleotide sequence of SEQ ID NO: 95 and 96, SEQ ID NO: 99 and 100 or SEQ ID NO: 103 and 104.

In a preferred embodiment, the kit may further comprise a blocking primer, wherein the blocking primer binds to a primer having the sequence of SEQ ID NO: 11 and 12 have the sequence shown in SEQ ID NO: 13, and (c) a sequence set forth in (c); and a polypeptide having the sequence of SEQ ID NO: 15 and 16 have the sequence shown in SEQ ID NO: 17; and a polypeptide having the sequence of SEQ ID NO: 19 and 20 have the sequence shown in SEQ ID NO: 21; and a polypeptide having the sequence of SEQ ID NO: 23 and 24 have the sequence shown in SEQ ID NO: 25; and a polypeptide having the sequence of SEQ ID NO: 27 and 28 have the sequence shown in SEQ ID NO: 29; and a polypeptide having the sequence of SEQ ID NO: 31 and 32 have the sequence shown in SEQ ID NO: 33; and a polypeptide having the sequence of SEQ ID NO: 35 and 36 have the sequence shown in SEQ ID NO: 37; and a polypeptide having the sequence of SEQ ID NO: 39 and 40 has the sequence shown in SEQ ID NO: 41; and a polypeptide having the sequence of SEQ ID NO: 43 and 44 has the sequence shown in SEQ ID NO: 45, and (c) a sequence shown as 45; and a polypeptide having the sequence of SEQ ID NO: 47 and 48 have the sequence shown in SEQ ID NO: 49; and a polypeptide having the sequence of SEQ ID NO: 51 and 52 have the sequence shown in SEQ ID NO: 53, or a sequence shown in SEQ ID NO; and a polypeptide having the sequence of SEQ ID NO: 55 and 56 has the sequence shown in SEQ ID NO: 57; and a polypeptide having the sequence of SEQ ID NO: 59 and 60 has the sequence shown in SEQ ID NO: 61; and a polypeptide having the sequence of SEQ ID NO: 63 and 64 has the sequence shown in SEQ ID NO: 65; and a polypeptide having the sequence of SEQ ID NO: 67 and 68 have the sequence shown in SEQ ID NO: 69; and a polypeptide having the sequence of SEQ ID NO: 71 and 72 has the sequence shown in SEQ ID NO: 73; and a polypeptide having the sequence of SEQ ID NO: 75 and 76 have the sequence shown in SEQ ID NO: 77; and a polypeptide having the sequence of SEQ ID NO: 79 and 80 have the sequence shown in SEQ ID NO: 81; and a polypeptide having the sequence of SEQ ID NO: 83 and 84 has the sequence shown in SEQ ID NO: 85; and a polypeptide having the sequence of SEQ ID NO: 87 and 88 has the sequence shown in SEQ ID NO: 89; and a polypeptide having the sequence of SEQ ID NO: 91 and 92 have the sequence shown in SEQ ID NO: 93; and a polypeptide having the sequence of SEQ ID NO: 95 and 96 has the sequence shown in SEQ ID NO: 97; and a polypeptide having the sequence of SEQ ID NO: 99 and 100 has the sequence shown in SEQ ID NO: 101; and to a polypeptide having the sequence of SEQ ID NO: 103 and 104 have the sequence shown in SEQ ID NO: 105; wherein the blocking primer has a 3' terminal modification that is resistant to extension amplification by a DNA polymerase.

In a preferred embodiment, the kit may further comprise a probe, wherein the probe binds to a polypeptide having the sequence of SEQ ID NO: 11 and 12 have the sequence shown in SEQ ID NO: 14, or a sequence shown in fig. 14; and a polypeptide having the sequence of SEQ ID NO: 15 and 16 have the sequence shown in SEQ ID NO: 18, or a sequence shown in seq id no; and a polypeptide having the sequence of SEQ ID NO: 19 and 20 have the sequence shown in SEQ ID NO: 22; and a polypeptide having the sequence of SEQ ID NO: 23 and 24 have the sequence shown in SEQ ID NO: 26; and a polypeptide having the sequence of SEQ ID NO: 27 and 28 have the sequence shown in SEQ ID NO: 30; and a polypeptide having the sequence of SEQ ID NO: 31 and 32 have the sequence shown in SEQ ID NO: 34; and a polypeptide having the sequence of SEQ ID NO: 35 and 36 have the sequence shown in SEQ ID NO: 38; and a polypeptide having the sequence of SEQ ID NO: 39 and 40 has the sequence shown in SEQ ID NO: 42; and a polypeptide having the sequence of SEQ ID NO: 43 and 44 has the sequence shown in SEQ ID NO: 46; and a polypeptide having the sequence of SEQ ID NO: 47 and 48 have the sequence shown in SEQ ID NO: 50; and a polypeptide having the sequence of SEQ ID NO: 51 and 52 have the sequence shown in SEQ ID NO: 54, or a sequence shown in SEQ ID NO; and a polypeptide having the sequence of SEQ ID NO: 55 and 56 has the sequence shown in SEQ ID NO: 58; and a polypeptide having the sequence of SEQ ID NO: 59 and 60 have the sequence shown in SEQ ID NO: 62; and a polypeptide having the sequence of SEQ ID NO: 63 and 64 have the sequence shown in SEQ ID NO: 66; and a polypeptide having the sequence of SEQ ID NO: 67 and 68 have the sequence shown in SEQ ID NO: 70; and a polypeptide having the sequence of SEQ ID NO: 71 and 72 has the sequence shown in SEQ ID NO: 74; and a polypeptide having the sequence of SEQ ID NO: 75 and 76 have the sequence shown in SEQ ID NO: 78, or a sequence shown in seq id no; and a polypeptide having the sequence of SEQ ID NO: 79 and 80 have the sequence shown in SEQ ID NO: 82; and a polypeptide having the sequence of SEQ ID NO: 83 and 84 has the sequence shown in SEQ ID NO: 86; and a polypeptide having the sequence of SEQ ID NO: 87 and 88 has the sequence shown in SEQ ID NO: 90; and a polypeptide having the sequence of SEQ ID NO: 91 and 92 have the sequence shown in SEQ ID NO: 94; and a polypeptide having the sequence of SEQ ID NO: 95 and 96 has the sequence shown in SEQ ID NO: 98, or a sequence shown in seq id no; and a polypeptide having the sequence of SEQ ID NO: 99 and 100 have the sequence shown in SEQ ID NO: 102; and to a polypeptide having the sequence of SEQ ID NO: 103 and 104 have the sequence shown in SEQ ID NO: 106, wherein the probe has a fluorescent group at one terminus and a fluorescence quencher group at the other terminus.

In a more preferred embodiment, the primer pair and the corresponding blocking primer and probe are included in the kit.

In some embodiments, the kit further comprises a nucleic acid having SEQ ID NO: 107 and 108 and a primer pair having the sequences shown in SEQ ID NOs: 109, and is used for performing PCR amplification reaction by using the ACTB gene or the segment thereof which is treated by the bisulfite and is used as the reference gene in the biological sample as a template.

In a preferred embodiment, the kit further comprises a DNA extraction reagent and a bisulfite reagent. Preferably, the bisulphite reagent comprises sodium bisulphite.

In a preferred embodiment, the kit further comprises instructions describing the method of use of the kit and the processing of the test results by logistic regression.

The prostate cancer status includes susceptibility to prostate cancer and presence, progression, subtype and/or stage of prostate cancer.

The biological sample is selected from the group consisting of blood, serum, plasma, urine, urethral flushing fluid, semen and circulating cells of the subject.

The method and the kit provided by the application provide a new way for predicting, diagnosing and evaluating the prostatic cancer, which is rapid, reliable and accurate.

Drawings

FIG. 1 shows Receiver Operating Characteristic (ROC) curves for methylation levels of 10 biomarker genes.

Fig. 2 shows the distribution of methylation levels (expressed as Ct values) of the 10 biomarker genes in different stages of prostate cancer, wherein fig. 2A shows the distribution of methylation levels of APC, CCND2, CDH1, GSTP1, MCAM and PENK, and fig. 2B shows the distribution of methylation levels of PITX2, PTGS2, RARB and RASSF 1A.

Fig. 3 shows the distribution of methylation levels (expressed as Ct values) of 10 marker genes in different prostate cancer subtypes, wherein fig. 3A shows the distribution of methylation levels of APC, CCND2, CDH1, GSTP1, MCAM and PENK, and fig. 3B shows the distribution of methylation levels of PITX2, PTGS2, RARB and RASSF 1A.

FIG. 4 shows Receiver Operating Characteristic (ROC) curves of a logistic regression model constructed using 10 marker genes;

FIG. 5 shows Receiver Operating Characteristic (ROC) curves for logistic regression models constructed using the 5 most characteristic marker genes.

Detailed Description

Unless otherwise defined, technical terms used in the present application have the meanings commonly understood by those skilled in the art to which the present invention belongs.

The present application relates in one aspect to a method of identifying a prostate cancer status in a subject, comprising the steps of: 1) collecting a biological sample from the subject; 2) detecting the methylation level of a biomarker gene in the biological sample, wherein the biomarker gene is selected from one or more of the following genes: APC (adenosine polysaccharides coli), CCND2(cyclin D2), CDH1(cadherin 1), GSTP1 (glutaminone S-transferase pi 1), MCAM (melanomycel addition module), PENK (propkephalin), PITX2(paired lipid hormone 2), PTGS2 (prostagladin-endoglycoxide synthase 2), RARB (retinoic acid receptor a) and RASSF1A (Ras association domain family member 1); and 3) comparing the methylation level detected in step 2) with a normal methylation level of the corresponding biomarker gene in the population to determine the prostate cancer status in the subject.

As used herein, the term "subject" refers to an individual (preferably a human) having or suspected of having a disease, or, in predicting a susceptibility, may also include healthy individuals. The term is often used interchangeably with "patient", "test subject", "treatment subject", and the like.

The term "population" as used herein generally refers to a healthy population. Where reference is made to a particular disease (such as prostate cancer), the "population" may include individuals who do not have that particular disease but who may have other diseases. In addition, only a part of individuals may be selected as a "population" according to the characteristics such as age, sex, health condition, menopause or not. "Normal methylation levels in a population" can be determined by testing a sufficient number of individuals, or can be found in the available clinical literature. In some cases, the normal level refers to no methylation.

The term "prostate cancer status" as used herein includes a subject's susceptibility to prostate cancer as well as the presence, progression, subtype and/or stage of prostate cancer. In some embodiments, the susceptibility of the subject to prostate cancer may be predicted from the methylation level of the biomarker gene in the subject. In other embodiments, the presence or absence of prostate cancer in a subject can be identified based on the methylation level of the biomarker gene in the subject; and identifying the subtype and/or stage of prostate cancer, if present. The prostate cancer subtypes may include adenocarcinoma, transitional cell carcinoma, squamous carcinoma, and the like. Stages of prostate cancer may include stages I (IA, IB or IC), II, III and IV. In some embodiments, the prostate cancer is stage I prostate cancer. In some embodiments, the prostate cancer is stage II prostate cancer. In some embodiments, the prostate cancer is stage III prostate cancer. In other embodiments, the prostate cancer is stage IV prostate cancer.

In the methods of the invention, treatment of the subject may also be scheduled based on the stage of the prostate cancer, e.g., including performing further tests on the subject, performing surgery, performing medication, and taking no further action. In other embodiments, the methods of the invention further comprise measuring the methylation level of one or more of the APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF1A genes, or fragments thereof, in the subject again after the subject is treated; and correlating the measurement to prostate cancer status to identify whether the treatment results in an alteration of prostate cancer status in the subject. In some embodiments, the correlating is performed by a software classification algorithm.

The detection of the methylation level in step 2) comprises extracting DNA from the biological sample and treating with bisulfite, followed by a PCR amplification reaction using methylation specific primer pairs. Wherein bisulfite treatment causes the deamination of unmethylated cytosine residues in the DNA duplex to uracil; whereas methylated cytosine residues remain unchanged. Thus, in a subsequent PCR amplification reaction, methylated cytosine residue sites on the template are paired as cytosine residues with guanine residues in the primers, while unmethylated cytosine residue sites are paired as uracil residues with adenine residues in the primers. The inventors designed multiple primer pairs for each biomarker gene to detect the methylation level of a target region within each biomarker gene, wherein the target regions are respectively selected from the group consisting of SEQ ID NOs: 1-10, and fragments of at least 15 bases in length that are contiguous in the sequence set forth; and the nucleic acid sequences of the primer pairs are respectively identical, complementary or hybridized to the target regions. The primer pairs provided herein utilize this methylation difference to detect the level of methylation of a target region within a biomarker gene. When the target region of the biomarker gene is unmethylated, the primer pair used is not able to pair-bind efficiently to the target region as a template (bisulfite treated) in a PCR amplification reaction and no (or very little) amplification product is produced; and when the target gene of the biomarker gene is methylated, the primer pair used can be effectively paired and combined with the target region (treated by bisulfite) as a template in a PCR amplification reaction, thereby generating an amplification product. Such differences in amplification reactions can be monitored in real time as the amplification reaction progresses, or can be judged by detecting the amplification products. The present inventors have screened multiple primer pairs (see below) for the biomarker genes over multiple trials, which, alone or in combination, can help identify the prostate cancer status in the subject.

The term "biomarker gene or fragment thereof" is often used herein in reference to detecting methylation levels because in a PCR amplification reaction, the primer pair used does not distinguish between the entire gene or a fragment thereof in the selection of the template, as long as the length of the template is not less than the length of the region to be amplified (in fact, in the course of DNA extraction and subsequent bisulfite treatment, it often results in fragmentation of the gene into fragments of different sizes).

In some preferred embodiments, the present invention measures marker gene methylation using the HeavyMethyl method, so a blocking primer is designed in addition to the ordinary Taqman primer. The blocking primers are designed on the nucleotide sequence to pair with a template sequence in the region amplified by the corresponding primer pair. In addition, the blocking primer introduces chemical modifications at the 3 ' -OH, such as C3 Spacer (C3 Spacer), C6 Spacer (C6 Spacer), inverted3 ' end (inverted3 ' end), 3 ' phosphate (3 ' P), etc., which render the DNA polymerase incapable of amplification. In an embodiment of the method of the invention, the nucleotide sequence of the blocking primer is designed to bind to the unmethylated template (sulfite-treated) but not to the methylated template (sulfite-treated). Thus, when methylation does not occur in the region corresponding to the blocking primer, it can prevent the corresponding amplification reaction from proceeding, thereby improving the specificity of the detection method of the present invention.

In a more preferred embodiment of the method of the invention, the method further comprises the use of a fluorescent probe to monitor and/or quantify the PCR amplification reaction in real time. The 5' end of the probe used can be a reporting fluorophore such as FAM, JOE, TET, HEX, Cy3, Texas Red, Rox or Cy 5; the quenching group at the 3' end is BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, or MGB.

Detection of the methylation level of the biomarker gene in the methods of the present invention includes detecting the presence or absence of methylation in the biomarker gene, and quantitatively and qualitatively detecting the methylation.

The biological sample is selected from fluid or tissue cells extracted from the subject, including blood, serum, plasma, urine, urethral flushing fluid, semen, circulating cells, and the like, preferably plasma, serum, and urine.

In the methods of the invention, the age of the subject may also be taken into account for predicting the prostate cancer status in said subject.

In some embodiments, the methods of the invention further comprise the step of providing a written or electronic report of the prediction of prostate cancer, and optionally, the report comprises a prediction of the presence or absence or likelihood of prostate cancer in the subject or of the risk of stratification of prostate cancer in the subject.

In some embodiments, the methods of the invention further comprise establishing a report of the relative level of biomarker gene methylation for a physician and transmitting such report by mail, fax, mailbox, or the like. In one embodiment, a data stream comprising a report of the methylation level of a biomarker gene is transmitted over the internet.

In some embodiments, a statistical method is used to construct a diagnostic model based on the methylation levels of the biomarker genes, the statistical method selected from the following methods: multiple linear regression, lookup tables, decision trees, support vector machines, Probit regression, logistic regression, cluster analysis, neighborhood analysis, genetic algorithms, bayesian and non-bayesian methods, and the like.

In other embodiments, predictive or diagnostic models based on biomarker gene methylation levels are provided. The model may be in the form of software code, computer readable format, or written instructions for assessing the relative methylation levels of biomarker genes.

With the method of the invention new and important additional information is available which assists the physician in grading the patient's risk of getting prostate cancer and planning the diagnostic steps to be taken next. The methods provided herein are similarly also useful for assessing the risk of prostate cancer in asymptomatic high risk patients, as well as for use as a screening tool for the general population. It is contemplated that the methods of the present invention may be used by clinicians as part of a comprehensive evaluation of other predictive and diagnostic indicators.

The methods of the invention can be used to assess the therapeutic efficacy of existing chemotherapeutic agents and candidate chemotherapeutic agents, as well as other types of cancer treatment modalities. For example, a biological sample can be taken from a subject before or after treatment or during treatment and the detection of the level of biomarker gene methylation is performed as described above, and the change in cancer status in the subject is identified by the detection result, thereby determining the efficacy of the treatment.

The methods of the invention can also be used to identify whether a subject is potentially developing cancer. Detecting the relative levels of biomarker gene methylation in biological samples taken from the subject over time, thereby interpreting changes in biomarker methylation levels that are characteristic of cancer as progressing toward the development of cancer.

The combination of the biomarker genes provides a sensitive, specific and accurate means for predicting the presence of or detecting prostate cancer in different stages of prostate cancer progression. The assessment of the methylation level in the biological sample can also be correlated with the presence of a pre-malignant or pre-clinical condition in the patient. Thus, the disclosed methods can be used to predict or detect the presence of prostate cancer, the stage of prostate cancer, the subtype of prostate cancer, the benign or malignant nature of prostate cancer, the metastatic potential of prostate cancer, the histological type of neoplasm associated with prostate cancer, the indolence or aggressiveness of cancer, and other prostate cancer characteristics associated with preventing, diagnosing, characterizing, and treating prostate cancer in a patient.

The methods of the invention are also useful for assessing the efficacy of a candidate drug for inhibiting prostate cancer, assessing the efficacy of a prostate cancer therapy, monitoring the progression of prostate cancer, selecting an agent or therapy for inhibiting prostate cancer, monitoring the treatment of a prostate cancer patient, monitoring the status of prostate cancer inhibition in a patient, and assessing the carcinogenic potential of a test compound by detecting the level of methylation of a biomarker gene in a test animal following exposure to the test compound.

The invention also provides kits for detecting prostate cancer status. In some embodiments, the kit can include a DNA extraction reagent and a bisulfite reagent. DNA extraction reagents may include lysis buffer, binding buffer, wash buffer, and elution buffer. Lysis buffers are typically composed of protein denaturants, detergents, pH buffers and nuclease inhibitors. The binding buffer is typically composed of a protein denaturant and a pH buffer. The washing buffer solution is divided into a washing buffer solution A and a washing buffer solution B: the washing buffer solution A consists of a protein denaturant, a nuclease inhibitor, a detergent, a pH buffer and ethanol; the washing buffer B consists of nuclease inhibitor, pH buffer and ethanol. The elution buffer typically consists of a nuclease inhibitor and a pH buffer. The protein denaturant is selected from one or more of guanidinium isothiocyanate, guanidinium hydrochloride and urea; the detergent is selected from one or more of Tween20, IGEPAL CA-630, Triton X-100, NP-40 and SDS; the pH buffering agent is selected from one or more of Tris, boric acid, phosphate, MES and HEPES; the nuclease inhibitor is one or more selected from EDTA, EGTA and DEPC. Bisulfite reagents include bisulfite buffers and protection buffers. Wherein the bisulfite is selected from one or more of sodium bisulfite, sodium sulfite, sodium bisulfite, ammonium bisulfite and ammonium sulfite; the protective buffer solution consists of an oxygen radical scavenger, and the oxygen radical scavenger is one or more selected from hydroquinone, vitamin E derivatives, gallic acid, Trolox, trihydroxybenzoic acid and trihydroxybenzoic acid derivatives.

The kit comprises a primer pair for methylation specific PCR amplification reaction of one or more of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes. These primer pairs detect methylation of at least one nucleotide sequence in the nucleotide sequence of the target region of the corresponding gene, respectively.

The kit of the present invention may further comprise blocking primers and probes used in combination with the above-described primer pairs (the blocking primers and probes are described above and below).

In certain embodiments, the kit can further comprise instructions for using the kit to extract DNA from a biological sample and treat the DNA with a bisulfite reagent. In other embodiments, the kit further comprises instructions for measuring the level of the biomarker in the subject using the reagents within the kit. In still other embodiments, the kit comprises instructions for using the kit to determine the status of prostate cancer in a subject.

The invention also provides a method for detecting the methylation level of the biomarker gene or the fragment thereof by using the kit, which comprises the following steps: extracting DNA in a biological sample by using a DNA extraction reagent, and treating the extracted DNA by using a bisulfite reagent; and detecting the methylation level of the biomarker gene by using the provided primer pair by using the treated DNA as a template.

The biomarker gene methylation level measurement method may be selected from one or more of the following methods: real-time fluorescence PCR, digital PCR, bisulfite sequencing, methylation specificity PCR, restriction enzyme analysis, high-resolution melting curve technology, gene chip technology and flight time mass spectrum.

The invention is further described below by way of examples.

Example 1: DNA extraction

The DNA extraction reagent consists of a lysis buffer, a binding buffer, a washing buffer and an elution buffer. The lysis buffer consists of protein denaturants, detergents, pH buffers and nuclease inhibitors. The binding buffer consists of a protein denaturant and a pH buffer. The washing buffer solution is divided into a washing buffer solution A and a washing buffer solution B, wherein the washing buffer solution A consists of a protein denaturant, a nuclease inhibitor, a detergent, a pH buffer agent and ethanol; the washing buffer B consists of nuclease inhibitor, pH buffer and ethanol. The elution buffer consists of a nuclease inhibitor and a pH buffer. Wherein the protein denaturant is: guanidine hydrochloride; the detergent is: tween 20; the pH buffer is: Tris-HCl; the nuclease inhibitor is: EDTA.

In this example, a plasma sample of a prostate cancer patient is taken as an example, and plasma DNA is extracted. The extraction method comprises the following steps:

(1) adding lysis buffer solution with the same volume into 1mL of blood plasma, adding proteinase K and Carrier RNA to make the final concentrations of the proteinase K and the Carrier RNA respectively 100mg/L and 1 mu g/mL, shaking and uniformly mixing, and incubating at 55 ℃ for 30 min;

(2) 100. mu.L of magnetic beads (purchased from Life technologies, cat. No.: 37002D) were added and incubated for 1 hour with shaking;

(3) adsorbing the magnetic beads by a magnetic separator, and discarding the supernatant solution;

(4) adding 1mL of cleaning buffer solution A for resuspending the magnetic beads, and shaking and cleaning for 1 min;

(5) adsorbing the magnetic beads by a magnetic separator, and discarding the supernatant;

(6) adding 1mL of cleaning buffer B for resuspending the magnetic beads, and shaking and cleaning for 1 min;

(7) adsorbing the magnetic beads by a magnetic separator, and discarding the supernatant solution;

(8) centrifuging at 10000rpm for 1min, adsorbing magnetic beads with a magnetic separator, and removing residual supernatant;

(9) opening the cover of the centrifugal tube filled with the magnetic beads, placing the centrifugal tube on a metal bath at the temperature of 55 ℃, and airing for 10 min;

(10) adding 100 μ L elution buffer solution to resuspend the magnetic beads, placing on 65 deg.C metal bath, and shaking for elution for 10 min;

(11) adsorbing the magnetic beads by using a magnetic separator, taking out a buffer solution containing the target DNA, quantifying the DNA, and marking;

(12) the eluted DNA is stored in a refrigerator at 4 ℃ for standby or stored in a refrigerator at-20 ℃ for long-term storage.

Example 2: bisulfite treatment of DNA

Bisulfite treating DNA is by using a bisulfite reagent consisting of a bisulfite buffer and a protective buffer; the bisulfite buffer solution is a mixed liquid of sodium bisulfite and water; the protective buffer solution is a mixed liquid of oxygen radical scavenger hydroquinone and water.

In this embodiment, the DNA extracted in example 1 is used as a treatment target, and the bisulfite is used to treat the DNA, which includes the following steps:

(1) preparing a bisulfite buffer: weighing 1g of sodium bisulfite powder, and adding water to prepare 3M buffer solution;

(2) preparing a protection buffer solution: weighing 1g of hydroquinone reagent, and adding water to prepare 0.5M protective buffer solution;

(3) mixing 100 mu L of DNA solution, 200 mu L of bisulfite buffer solution and 50 mu L of protective solution, and shaking and mixing uniformly;

(4) and (3) heat treatment: 5min at 95 ℃, 60min at 80 ℃ and 10min at 4 ℃;

(5) adding 1mL of DNA binding buffer solution into the DNA solution treated by the bisulfite, adding 50 mu l of magnetic beads, and oscillating and incubating for 1 h;

(6) adsorbing the magnetic beads by a magnetic separator, and discarding the supernatant solution;

(7) adding 0.5mL of cleaning buffer solution A for resuspending the magnetic beads, and shaking and cleaning for 1 min;

(8) adsorbing the magnetic beads by a magnetic separator, and discarding the supernatant;

(9) adding 0.5mL of cleaning buffer B for resuspending the magnetic beads, and shaking and cleaning for 1 min;

(10) adsorbing the magnetic beads by a magnetic separator, and discarding the supernatant solution;

(11) centrifuging at 10000rpm for 1min, adsorbing magnetic beads with a magnetic separator, and removing residual supernatant;

(12) placing the centrifugal tube filled with the magnetic beads on a metal bath at 55 ℃, uncovering and airing for 10 min;

(13) adding 50 μ L elution buffer solution to resuspend the magnetic beads, placing on 65 deg.C metal bath, and shaking for elution for 10 min;

(14) and adsorbing the magnetic beads by using a magnetic separator, taking out the buffer solution containing the target DNA, quantifying the DNA, and marking.

Example 3: real-time fluorescence PCR detection of DNA methylation and primer group verification

This example measures methylation levels of biomarker genes, exemplified by real-time fluorescent PCR. The detection genes are APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes, and the reference gene is ACTB. This example used bisulfite treated DNA from example 2 as a template for real-time fluorescent PCR amplification. And 3 multi-hole detection is carried out on the DNA sample to be detected, the negative quality control product, the positive quality control product and the template-free control. The negative quality control product and the positive quality control product are respectively prepared as follows: adding 400 mu L of human leukocyte DNA with the concentration of 10 ng/mu L into TE buffer solution containing 1% BSA, mixing uniformly, and fixing the volume of the solution to 200mL to obtain a negative quality control substance with the concentration of 0.02 ng/mu L; 384. mu.L of human leukocyte DNA with the concentration of 10 ng/. mu.L and 16. mu.L of Hela cell DNA with the concentration of 10 ng/. mu.L are added into TE buffer solution containing 1% BSA, mixed evenly and metered to 200mL to obtain a positive quality control substance with the concentration of 0.02 ng/. mu.L, wherein the content of the positive DNA is 4%.

For the APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes, many sets of primers and probe combinations can be designed, and the performance of each set of probe and primer combination may be different, so that the verification through experiments is needed.

Thus, we designed various primers and probes for the APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes, which are identical to, complementary to or hybridized to SEQ ID NO: 1-10 or a complement thereof; the methylated and unmethylated nucleic acid sequences were then used as templates to verify the effectiveness of the primers and probes. The following optimal primer groups and primer groups of the reference gene ACTB are screened out according to the real-time fluorescent PCR amplification result:

APC primer set 1

Primer 1: 5'-TCGCGAGGGTATATTTTCGAGG-3' SEQ ID NO 11

Primer 2: 5'-ATCCGCATCCAACGAATTACAC-3' SEQ ID NO 12

Blocking primers: SEQ ID NO 13:5 '-TTGAGGGGTATGGGGTTAGGGTTAGG-C3-3'

And (3) probe: SEQ ID NO 14:5 '-HEX-ACACAAAACCCCGCCCAACCG-BHQ 1-3'

APC primer set 2

Primer 1: 5'-GGCGTTTTTTATTTTCGTCGGG-3' SEQ ID NO 15

Primer 2: 5'-TACCCCATTTCCGAATCCGAC-3' SEQ ID NO 16

Blocking primers: SEQ ID NO 17:5 '-GTTTTTTATTTTTGTTGGGAGTTTGTTGATTG-C3-3'

And (3) probe: SEQ ID NO 18:5 '-HEX-TACGCCCACACCCAACCAATCG-BHQ 1-3'

CCND2 primer set 1

Primer 1: SEQ ID NO 195 '-TACGGTTTATTTAGTTCGCGTTTTAT-3'

Primer 2: SEQ ID NO 20: 5'-CCAACGCCCTAATATACTAACCAAACT-3'

Blocking primers: SEQ ID NO 21:5 '-TTTAGTTTGTGTTTTATTTTGTTTTGTTGGTTTTT-C3-3'

And (3) probe: SEQ ID NO 22:5 '-Texas Red-AAACCCGAACAAAAACGCGAAAAAC-BHQ 2-3'

CCND2 primer set 2

Primer 1: 5'-TGCGTTAGAGTACGTGTTAGGGTC-3' SEQ ID NO 23

Primer 2: 5'-CGAATAAAAAATTAAATCCGACTCC-3' SEQ ID NO 24

Blocking primers: SEQ ID NO 25:5 '-GAGTATGTGTTAGGGTTGATTGTGTTGGT-C3-3'

And (3) probe: SEQ ID NO 26:5 '-Texas Red-AACCGACTACGATAAAATCGCCGCC-BHQ 2-3'

CCND2 primer set 3

Primer 1: SEQ ID NO 27: 5'-GTTGTATCGGTGTGGTTACGTTTAGC-3'

Primer 2: 5'-TCTAAAAACTCTTCGAACGCCGT-3' SEQ ID NO 28

Blocking primers: SEQ ID NO 29:5 '-ATTGGTGTGGTTATGTTTAGTGTAGATATTTTGG-C3-3'

And (3) probe: SEQ ID NO 30:5 '-Texas Red-TCGCCCCTACATCTACTAACAAACCGC-BHQ 2-3'

CDH1 primer set 1

Primer 1: SEQ ID NO 31: 5'-AGTGGAATTAGAATCGTGTA-3'

Primer 2: 5'-CCACAACCAATCAACAAC-3' SEQ ID NO 32

Blocking primers: SEQ ID NO 33:5 '-GGAATTAGAATTGTGTAGGTTTTATAAT-C3-3'

And (3) probe: SEQ ID NO 34:5 '-Texas Red-CGAAACTAACGACCCGCCCA-BHQ 2-3'

CDH1 primer set 2

Primer 1: SEQ ID NO 35: 5'-TCGGAATTGTAAAGTATTTGTG-3'

Primer 2: 5'-GTCCCTCGCAAATCAAAA-3' SEQ ID NO 36

Blocking primers: SEQ ID NO 37:5 '-TGGAATTGTAAAGTATTTGTGAGTTTGT-C3-3'

And (3) probe: SEQ ID NO 38 5 '-Texas Red-CTACAACAACAACAACAACGCCGAA-BHQ 2-3'

GSTP1 primer set 1

Primer 1: 5'-TTTCGGTTAGTTGCGCGG-3' SEQ ID NO 39: 5'-TTTCGGTTAGTTGCGCGG-3'

Primer 2: SEQ ID NO 40: 5'-CTAAAAAATCCCGCGAACTCC-3'

Blocking primers: SEQ ID NO 41:5 '-TTGTGTGGTGATTTTGGGGATTTTAG-C3-3'

And (3) probe: SEQ ID NO 42:5 '-JOE-ACCGCAAAAAAACGCCCTAAAATC-BHQ 1-3'

GSTP1 primer set 2

Primer 1: 5'-TCGTTGGAGTTTCGTCGTCGT-3' SEQ ID NO 43

Primer 2: 5'-AACCCCCGTCCCGAATCT-3' SEQ ID NO 44

Blocking primers: SEQ ID NO 455 '-GGAGTTTTGTTGTTGTAGTTTTTGTTATTAGTGAG-C3-3'

And (3) probe: SEQ ID NO 46:5 '-JOE-ACGCGAACCGCGCGTACTCACT-BHQ 1-3'

MCAM primer set 1

Primer 1: 5'-GACGTAACGTTTCGATTTC-3' SEQ ID NO 47

Primer 2: 5'-CTTCCCAAACTAATCTACG-3' SEQ ID NO 48

Blocking primers: SEQ ID NO 49:5 '-ATGTTTTGATTTTGTTGTGTTGTTGGTTTTG-C3-3'

And (3) probe: SEQ ID NO 50:5 '-FAM-ACTCGCCGCCTACTACTACTATCC-BHQ 1-3'

MCAM primer set 2

Primer 1: SEQ ID NO 51: 5'-TTAGGGGGGCGGGGGAG-3'

Primer 2: SEQ ID NO 52: 5'-CCTCGACCAATCAAACCCCG-3'

Blocking primers: SEQ ID NO 535 '-GTGGGGGAGGGGGTTTGGAGTGTTT-C3-3'

And (3) probe: SEQ ID NO 54:5 '-FAM-CCGACTCTAACCTACCTCCGACA-BHQ 1-3'

MCAM primer set 3

Primer 1: 5'-GGAGTAGTAGCGGATTTG-3' SEQ ID NO 55

Primer 2: 5'-CCAACACTAAAACTAAAACAATATC-3' SEQ ID NO 56

Blocking primers: SEQ ID NO 575 '-GTAGTGGATTTGGTTTTTGGTGTTTTTAAGG-C3-3'

And (3) probe: SEQ ID NO 58:5 '-FAM-ACCGACGACAACGACAATAATACAATC-BHQ 1-3'

PENK primer set 1

Primer 1: 5'-ACGGTGAGGTTTTACGTT-3' SEQ ID NO 59

Primer 2: SEQ ID NO 60: 5'-CCAAACCCGCTCAACAA-3'

Blocking primers: SEQ ID NO 61:5 '-TGAGGTTTTATGTTTGTTAGTATATTTGGGTTTG-C3-3'

And (3) probe: SEQ ID NO 62:5 '-HEX-ACGAACGTCGAAAAAAAACGAACCCG-BHQ 1-3'

PENK primer set 2

Primer 1: 5'-TCGTTTTTTCGTTTTTGGTTTGC-3' SEQ ID NO 63

Primer 2: 5'-AACCCGCAAAAATACTCCTTTCT-3' SEQ ID NO 64

Blocking primers: SEQ ID NO 655 '-TTGTTTTTGGTTTGTGGTGTTTTTTTTGG-C3-3'

And (3) probe: SEQ ID NO 66 5 '-HEX-TCCGCGACCCAAAACAAAATTCC-BHQ 1-3'

PITX2 primer set 1

Primer 1: SEQ ID NO 67: 5'-TTCGTGTGGGGAGTGACGTGAC-3'

Primer 2: 5'-TCGTTTCTCGTCGCCCCTACT-3' SEQ ID NO 68

Blocking primers: SEQ ID NO 695 '-GTGGGGAGTGATGTGATGTTAGTAGAGATTTTATT-C3-3'

And (3) probe: SEQ ID NO 70:5 '-HEX-ACCGCCCGCGCGCCACTATAC-BHQ 1-3'

PITX2 primer group 2

Primer 1: SEQ ID NO 71: 5'-CGGAGTCGGGAGAGCGAAAG-3'

Primer 2: SEQ ID NO 72: 5'-TAAAACTACCGAATTAACGCAAATTACT-3'

Blocking primers: SEQ ID NO 73: 5 '-TTGGGAGAGTGAAAGGAGAGGGGATT-C3-3'

And (3) probe: SEQ ID NO 74: 5 '-HEX-TCCTCGATTAACTCCTAAATACCCCGCC-BHQ 1-3'

PTGS2 primer set 1

Primer 1: SEQ ID NO 75: 5'-GTTTTCGGAAGCGTTCGGGT-3'

Primer 2: SEQ ID NO 76: 5'-TACGTAAACCCGATAAAAACAAAATT-3'

Blocking primers: SEQ ID NO 77:5 '-TGGAAGTGTTTGGGTAAAGATTGTGAAG-C3-3'

And (3) probe: SEQ ID NO 78 5 '-Texas Red-CCCAAACGCACAAATTTCCGCC-BHQ 2-3'

PTGS2 primer set 2

Primer 1: SEQ ID NO 79: 5'-GCGGAAAGAAATAGTTATTTCGTTAT-3'

Primer 2: SEQ ID NO 80: 5'-TATATAACTAAACGCCAAAACCGC-3'

Blocking primers: SEQ ID NO 815 '-GAAATAGTTATTTTGTTATATGGGTTTGGTTTTTAG-C3-3'

And (3) probe: SEQ ID NO 82 5 '-Texas Red-TCCTAAACGTACTCCTAACGCTCACTA-BHQ 2-3'

RARB primer set 1

Primer 1: SEQ ID NO 83: 5'-GCGTATAGAGGAATTTAAAGTGTGG-3'

Primer 2: SEQ ID NO 84: 5'-ACGCCTTTTTATTTACGACGACTTAAC-3'

Blocking primers: SEQ ID NO 85: 5 '-TTATTTACAACAACTTAACTTAAAAAACAATATTCCACC-C3-3'

And (3) probe: SEQ ID NO 86: 5 '-FAM-TATTCCGCCTACGCCCGCTCG-BHQ 1-3'

RARB primer set 2

Primer 1: SEQ ID NO 87: 5'-GAATTTTTTTATGCGAGTTGTTTGAGG-3'

Primer 2: SEQ ID NO 88: 5'-TTCCGAATACGTTCCGAATCCTACC-3'

Blocking primers: SEQ ID NO 89: 5 '-TTATGTGAGTTGTTTGAGGATTGGGATGTTGAG-C3-3'

And (3) probe: SEQ ID NO 90: 5 '-FAM-AACAAACCCTACTCGAATCGCTCGCG-BHQ 1-3'

RARB primer set 3

Primer 1: SEQ ID NO 91: 5'-TGGGAATTTTTCGTTTCGGTT-3'

Primer 2: SEQ ID NO 92: 5'-ACACGTAAACTATTAATCTTTTTCCCAAC-3'

Blocking primers: SEQ ID NO 93: 5 '-CATAAACTATTAATCTTTTTCCCAACCCCAAATC-C3-3'

And (3) probe: SEQ ID NO 94: 5 '-FAM-TCATTTACCATTTTCCAAACTTACTCGACC-BHQ 1-3'

RASSF1A primer set 1

Primer 1: 5'-AAGGAAGGGTAAGGCG-3' SEQ ID NO 95: 5'-AAGGAAGGGTAAGGCG-3'

Primer 2: SEQ ID NO 96: 5' -CGAACGAAACCACAAAAC-3

Blocking primers: SEQ ID NO 97:5 '-GTGGGGGGGGTTTTGTGAG-C3-3'

And (3) probe: SEQ ID NO 98:5 '-FAM-AACCCCGACTTCAACGCCTC-BHQ 1-3'

RASSF1A primer set 2

Primer 1: SEQ ID NO 99: 5'-TTGTTAGCGTTTAAAGTTAGC-3'

Primer 2: SEQ ID NO 100: 5' -TAAAATTACACGCGATACCC-3

Blocking primers: SEQ ID NO 101:5 '-AGTGTTTAAAGTTAGTGAAGTATGGGTT-C3-3'

And (3) probe: SEQ ID NO 102:5 '-FAM-ACACGCTCCAACCGAATACGACC-BHQ 1-3'

RASSF1A primer set 3

Primer 1: 5'-TCGTTTTTAGTTCGCGG-3' SEQ ID NO 103

Primer 2: SEQ ID NO 104: 5' -TTAACTACCCCTTCCGCT-3

Blocking primers: SEQ ID NO 105:5 '-TAGTTTGTGGGGTTTGTTATGTATATGT-C3-3'

And (3) probe: SEQ ID NO 106:5 '-FAM-ACAAACCTTTACGCACGACGCCC-BHQ 1-3'

Primer group for reference gene ACTB

Primer 1: SEQ ID NO 107: 5'-GTGATGGAGGAGGTTTAGTAAGT-3'

Primer 2: 5'-CCAATAAAACCTACTCCTCCCTT-3' SEQ ID NO 108: 5'-CCAATAAAACCTACTCCTCCCTT-3'

And (3) probe: SEQ ID NO 109:5 '-Cy 5-ACCACCACCCAACACACAATAACAAACACA-BHQ 3-3'

Multiple groups of primers and probes can distinguish methylated templates from unmethylated templates, and can be used as primers and probes for detecting methylation of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes. Although the effect of different primers and probe combinations is slightly different, the above primers and probes are suitable for methylation detection of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes, respectively. Table 1 below shows the results of detection of the synthesized methylated and unmethylated templates (bisulfite treated) of the above genes using the respective primer and probe combinations. Clearly, each primer and probe combination designed is highly specific for methylated templates.

Table 1 shows the results (Ct, mean) of the primer sets designed for the methylated and unmethylated templates

In addition, we used different cancer patients and healthy human DNA as templates to further verify the effectiveness of the primer and probe combination. Plasma samples of 5 prostate cancers, 3 liver cancers and 5 healthy persons were subjected to DNA extraction in example 1, followed by bisulfite treatment of the DNA template in example 2. And (3) carrying out a real-time fluorescence PCR experiment by using the combination of the primers and the probes. The Ct values of the marker genes of the cancer sample and the healthy human sample were measured, respectively, and the results are shown in tables 2 to 4 below, wherein the results of the detection of the primer sets APC, CCND2, CDH1 and GSTP1 are shown in table 2, the results of the detection of the primer sets MCAM, PENK, PITX2 and PTGS2 are shown in table 3, and the results of the detection of the primer sets PTGS2, RARB and RASSF1A are shown in table 4.

TABLE 2 detection of methylation level of a given gene in individuals with known prostate cancer status (including healthy individuals) for each primer set

Abbreviation Pc for prostate cancer; heca: liver cancer; con health

TABLE 3 detection of methylation level of a given gene in individuals with known prostate cancer status (including healthy individuals) for each primer set

	MCAM-1	MCAM-2	MCAM-3	PENK-1	PENK-2	PITX2-1	PITX2-2	PTGS2-1
									PC 1	34.75	36.04	36.24	34.08	36.26	30.96	32.92	35.14
PC 2	33.31	35.81	35.61	3546	36.28	30.8	33.58	34.68
									PC 3	33.77	35.52	36.08	34.37	35.05	31.33	33.89	33.3
PC 4	33.73	35.73	36.26	35.36	35.64	32.36	32.91	33.39
									PC 5	34.72	34.09	35.81	35.14	36.57	31.71	32.26	34.9
HeCa 1	No Ct	42.79	No Ct	No Ct	No Ct	No Ct	41.23	No Ct
									HeCa 2	42.37	No Ct	43.18	No Ct	44.26	No Ct	No Ct	No Ct
HeCa
	3	44.21	No Ct	No Ct	No Ct	No Ct	43.82	No Ct	No Ct
Con 1										No Ct	No Ct	No Ct	No Ct	No Ct	No Ct	No Ct	No Ct
	Con 2	No Ct	No Ct	No Ct	No Ct	42.19	No Ct	No Ct	43.65
Con 3										44.82	No Ct	42.69	No Ct	No Ct	No Ct	No Ct	No Ct
	Con 4	No Ct	43.98	No Ct	43.28	No Ct	No Ct	No Ct	No Ct
Con 5										No Ct	No Ct	No Ct	No Ct	No Ct	No Ct	No Ct	No Ct

Abbreviations: PC: prostate cancer; and (3) HeCa: liver cancer; con: health care

TABLE 4 detection of methylation level of a given gene in individuals with known prostate cancer status (including healthy individuals) for each primer set

	PTGS2-2	RARB-1	RARB-2	RARB-3	RASSF1A-1	RASSF1A-2	RASSF1A-3
								PC 1	36.15	31.15	36.33	34.52	33.26	37.56	35.3
PC 2	37.9	30.56	36.46	35.34	36.39	37.07	37.22
								PC 3	37.5	32.11	36.31	33.9	34.74	36.37	37.11
PC 4	36.85	30.83	34.81	33.87	36.17	36.77	35.68
								PC 5	34.36	30.6	34.85	36.3	35.08	37.67	37.79
HeCa 1	No Ct	No Ct	No Ct	No Ct	No Ct	No Ct	No Ct
								HeCa 2	No Ct	42.36	No Ct	No Ct	No Ct	41.98	No Ct
HeCa
	3	43.29	No Ct	43.85	No Ct	No Ct	No Ct	No Ct
Con 1									No Ct	No Ct	No Ct	43.62	No Ct	No Ct	No Ct
	Con 2	No Ct	No Ct	No Ct	No Ct	No Ct	No Ct	44.29
Con 3									42.18	No Ct	44.81	No Ct	No Ct	No Ct	No Ct
	Con 4	No Ct	No Ct	No Ct	No Ct	No Ct	42.83	No Ct
Con 5									No Ct	44.23	No Ct	No Ct	No Ct	No Ct	No Ct

As can be seen from the Ct values of the methylation detection of the APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes of the cancer patient samples and the healthy human samples, the above primer and probe combinations have high specificity amplification on the methylated DNA of the prostate cancer, and other cancers and healthy humans have no amplification or have amplification Ct values larger than 40. Although the amplified Ct values of the prostate cancer samples of the primer and probe combinations of different groups are slightly different, the amplified Ct values are obviously different from those of other cancer and healthy human samples, so that the primer combinations are all suitable for detecting the prostate cancer.

Example four: sensitivity and specificity of kit for detecting plasma of prostate cancer patients and benign disease patients

All samples were collected from the general naval hospital of the liberated military of Chinese people, using 211 samples from patients pathologically determined for prostate cancer and 225 samples from patients pathologically determined for benign conditions (see Table 5). Prostate cancer samples include all stages and common subtypes of disease. Prostate cancer patients are diagnosed by imaging and pathological diagnosis, the sample stage is based on the international TNM stage standard, and the sample subtype is determined according to tissue biopsy and immunohistochemistry methods. Benign samples include the common types of benign disorders seen throughout the study population. A complete clinical pathology report was obtained after surgery, including patient age, race, stage, subtype and coding collection site for each sample.

Table 5 prostate cancer stage and other characteristics of the sample subjects collected.

DNA was extracted by the DNA extraction method of example 1, the DNA template was treated with bisulfite by the method of example 2, and real-time fluorescence PCR experiments were performed using the primer and probe combinations (primer set 1 for each biomarker gene) provided in example 3 to detect the APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes and the reference gene ACTB, and finally the Ct values of the respective genes in samples of healthy and prostate cancer patients were obtained. The methylation level of each gene can be reflected by this Ct value, as described in example 3 above.

Descriptive Statistics, Receiver Operating Characteristics (ROC) curves and graphical presentation of plasma biomarker levels were performed using commercially available software packages (IBM SPSS Statistics 24 and MedCalc11.4.2.0, available from IBM and MedCalc, respectively). Statistical differences were determined using the nonparametric Kruskal-Wallis test (ANOVA) followed by Dunn's multiple post-comparison test. For all statistical comparisons, a p-value <0.05 was considered statistically significant.

The methylation levels of the above 10 marker genes were detected in plasma from 211 patients pathologically determined prostate cancer and 225 individuals with benign prostate disease using a real-time fluorescent PCR assay. To help determine the ability of these biomarker genes to distinguish symptomatic similar cancers from benign prostate disease, all samples were obtained from the same clinical population. All samples were collected prior to any intervention and prior to the known disease state. The disease state is then determined by pathological examination of ex vivo tissues. Plasma was collected using a single sample collection protocol and compliance was monitored. This ensures sample quality and eliminates the possibility of any collection, processing, and biological bias in the sample collection. Normal healthy samples were not used in this study because they are generally more easily distinguished than benign conditions. These samples show that the average patient age among individuals with prostate cancer (63 years) is higher than those with benign conditions (50 years) and all increase with the staged progression of disease manifestations (table 5). Overall, the distribution of prostate cancer subtypes is similar to that seen in all prostate cancer cases in the human population, with a higher proportion of adenocarcinomas (90.5%) than other prostate cancers). The benign controls in the study represented common prostate disorders including prostatitis and prostatic hyperplasia, among others.

For the methylation level detection data for each biomarker, using medcalc11.4.2.0 software, a 95% confidence interval was selected, an ROC curve was generated and its area under the curve (AUC) value was calculated. The methylation levels AUC for the 10 biomarker genes in the prostate cancer sample were all greater than 0.700(p value < 0.05) and AUC between 0.731 and 0.888, relative to benign prostate disease (see FIG. 1 and Table 6).

TABLE 610 Area Under Curve (AUC) of Receiver Operating Characteristics (ROC) Curve analysis of marker genes

To determine whether certain biomarker genes have greater discrimination between different stages (especially early stages) of cancer, the discrimination in stage I and II (the most important stages of marker detection) samples for 10 biomarker genes (fig. 2) was compared. For phase I samples, both PITX2 and RARB had high discrimination (p value <0.001), then arranged in descending order as CDH1, CCND2, APC and PENK (p value 0.001 to 0.01), then GSTP1 and MCAM (p value 0.01 to 0.05). There were no significant differences between stage I cancer and benign disorders for PTGS2 and RASSF1A (p-value > 0.05). For phase II samples, both PITX2 and RARB again had very high discrimination (< 0.001 p value), followed by CDH1, APC, CCND2 and PENK (0.001 to 0.01 p value), followed by GSTP1 and MCAM (0.01 to 0.05 p value). PTGS2 and RASSF1A did not differ significantly (p value > 0.05).

The methylation levels of the above biomarker genes were also evaluated for statistically significant differences between samples from benign conditions and various subtypes of prostate cancer (figure 3). For adenocarcinoma, CDH1, PITX2 and RARB had high discrimination (p value <0.001), and then were arranged in descending order as CCND2, GSTP1, APC, PENK, RASSF1A, MCAM and PTGS2(p value 0.001 to 0.05). CCND2 and RARB have high discrimination for transitional cell carcinoma (p value <0.001), and then are arranged in descending order as PITX2, APC, CDH1, PENK, GSTP1, MCAM, RASSF1A and PTGS2(p value 0.001 to 0.05). For squamous carcinoma, PITX2 and RARB have high discrimination (p value <0.001), and then arranged in descending order as CDH1, APC, CCND2, MCAM, GSTP1, PENK, PTGS2 and RASSF1A (p value 0.001 to 0.05).

Detecting the methylation level of a single biomarker gene is superior to detecting the methylation level of multiple biomarker genes in terms of simplicity of operation and reduced cost. However, it is clear that the methylation level of a single biomarker gene may not provide information on the inherent diversity of complex diseases, so it is often also necessary to construct diagnostic models that employ multiple markers. The multi-marker diagnostic model needs to be performed by using a statistical analysis method, and a methylated gene marker diagnostic model is constructed by taking a logistic regression model as an example to detect the prostate cancer.

The training of the logistic regression model is performed as follows: samples were divided into cases and controls and then regression coefficients were optimized using IBM SPSS Statistics 24 software. There is one regression coefficient for each marker, plus one bias parameter, to maximize the likelihood that the logistic regression model will be used to train the data.

After training, the set of regression coefficients defines a logistic regression model. One skilled in the art can readily use this type of diagnostic model to predict the likelihood of any new sample being identified as a case or control by placing the methylation levels of the biomarkers into a logistic regression equation.

The 10 marker genes were all greater than 0.700 in methylation level AUC, and we subsequently combined the 10 marker genes using logistic regression to yield an AUC of 0.948 (standard error: 0.014; 95% CI: 0.907-0.974; p value <0.0001) (FIG. 4). To make the monitoring analysis method simpler, 5 marker genes with larger AUC values (APC, CCND2, CDH1, PITX2, and RARB) were combined and a logistic regression model was established. The AUC value obtained was 0.933 (standard error: 0.016; 95% CI: 0.889-0.964; p value <0.0001) (FIG. 5). The two models were further compared by determining the sensitivity of the model at a fixed value of specificity and the specificity of the model at a fixed value of sensitivity (tables 7 and 8). For example, one can choose a method that has a sum of sensitivity and specificity greater than about 155% when the method has a sensitivity greater than about 95%; or when the specificity of the method is greater than about 95%, the sum of the sensitivity and specificity is greater than about 160%. Generally, 10 markers are slightly more sensitive and specific than the logistic regression model of 5 markers, and 5 marker combinations are also better choices based on the procedure of the operational analysis and cost considerations.

TABLE 75 sensitivity of the logistic regression model for the most characteristic marker genes and 10 marker genes at the important specificity threshold

TABLE 85 specificity of the logistic regression model for the most characteristic marker genes and 10 marker genes at the important sensitivity threshold

It is noted that the methylation level measurements provided in this example were obtained using primer set 1 for each biomarker gene (e.g., APC primer set 1 for the APC gene; CCND2 primer set 1 for the CCND2 gene, and so on), but similar measurements were obtained using additional primer sets provided herein (data not shown).

According to the technical scheme provided by the invention, the methylation level of one or more genes or fragments thereof in the APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF1A genes is jointly detected, so that the sensitivity and specificity of prostate cancer detection are improved, and the correctness and reliability of the detection result are ensured. In addition, the primers provided by the kit can be used for rapidly and conveniently judging whether the sample is positive or not and judging the risk value by detecting the methylation of the biomarker genes in the sample and analyzing by using a logistic regression equation.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; those of ordinary skill in the art will understand that: modifications can be made to the technical solutions described in the foregoing embodiments, or some or all of the technical features can be equivalently replaced; the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention, and the technical solutions are all covered in the scope of the present specification.

SEQUENCE LISTING

<110> Beijing Aikelen medical science and technology Co., Ltd

<120> method and kit for identifying prostate cancer status

<130> 21115CI

<160> 109

<170> PatentIn version 3.5

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cagctgtgta atccgctgga tgcggaccag ggcgctcccc attcccgtcg ggagcccgcc 300

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<210> 8

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<213> Homo sapiens

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tctatcccag tactccgact tctctccgaa tagagaagct acgtgacttg ggaaagagct 720

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<210> 9

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gcgtaaaggg agagaagttg gtgctcaacg tgagccagga gcagcgtccc ggctcctccc 180

ctgctcattt taaaagcact tcttgtattg tttttaaggt gagaaatagg aaagaaaacg 240

ccggcttgtg cgctcgctgc ctgcctctct ggctgtctgc ttttgcaggg ctgctgggag 300

tttttaagct ctgtgagaat cctgggagtt ggtgatgtca gactagttgg gtcatttgaa 360

ggttagcagc ccgggtaggg ttcaccgaaa gttcactcgc atatattagg caattcaatc 420

tttcattctg tgtgacagaa gtagtaggaa gtgagctgtt cagaggcagg agggtctatt 480

ctttgccaaa ggggggacca gaattccccc atgcgagctg tttgaggact gggatgccga 540

gaacgcgagc gatccgagca gggtttgtct gggcaccgtc ggggtaggat ccggaacgca 600

ttcggaaggc tttttgcaag catttacttg gaaggagaac ttgggatctt tctgggaacc 660

ccccgccccg gctggattgg ccgagcaagc ctggaaaatg gtaaatgatc atttggatca 720

attacaggct tttagctggc ttgtctgtca taattcatga ttcggggctg ggaaaaagac 780

caacagccta cgtgccaaaa aaggggcaga gtttgatgga gttgggtg 828

<210> 10

<211> 684

<212> DNA

<213> Homo sapiens

<400> 10

atctccgcgt ggtgctttgc ggtcgccgtc gttgtggccg tccggggtgg ggtgtgagga 60

ggggacgaag gagggaagga agggcaaggc ggggggggct ctgcgagagc gcgcccagcc 120

ccgccttcgg gccccacagt ccctgcaccc aggtttccat tgcgcggctc tcctcagctc 180

cttcccgccg cccagtctgg atcctggggg aggcgctgaa gtcggggccc gccctgtggc 240

cccgcccggc ccgcgcttgc tagcgcccaa agccagcgaa gcacgggccc aaccgggcca 300

tgtcggggga gcctgagctc attgagctgc gggagctggc acccgctggg cgcgctggga 360

agggccgcac ccggctggag cgtgccaacg cgctgcgcat cgcgcggggc accgcgtgca 420

accccacacg gcagctggtc cctggccgtg gccaccgctt ccagcccgcg gggcccgcca 480

cgcacacgtg gtgcgacctc tgtggcgact tcatctgggg cgtcgtgcgc aaaggcctgc 540

agtgcgcgcg tgagtagtgg ccccgcgcgc ctacgagagc ggaaggggca gccaaggggc 600

agcgcagtcg ccgcgggtca agtcgcggca gagggggtcg gcggggacag ctcccgagga 660

ctaggtccgt tactttcgcc ccat 684

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 11

tcgcgagggt atattttcga gg 22

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 12

atccgcatcc aacgaattac ac 22

<210> 13

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 13

ttgaggggta tggggttagg gttagg 26

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 14

acacaaaacc ccgcccaacc g 21

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 15

ggcgtttttt attttcgtcg gg 22

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 16

taccccattt ccgaatccga c 21

<210> 17

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 17

gttttttatt tttgttggga gtttgttgat tg 32

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 18

tacgcccaca cccaaccaat cg 22

<210> 19

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 19

tacggtttat ttagttcgcg ttttat 26

<210> 20

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 20

ccaacgccct aatatactaa ccaaact 27

<210> 21

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 21

tttagtttgt gttttatttt gttttgttgg ttttt 35

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 22

aaacccgaac aaaaacgcga aaaac 25

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 23

tgcgttagag tacgtgttag ggtc 24

<210> 24

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 24

cgaataaaaa attaaatccg actcc 25

<210> 25

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 25

gagtatgtgt tagggttgat tgtgttggt 29

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 26

aaccgactac gataaaatcg ccgcc 25

<210> 27

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 27

gttgtatcgg tgtggttacg tttagc 26

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 28

tctaaaaact cttcgaacgc cgt 23

<210> 29

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 29

attggtgtgg ttatgtttag tgtagatatt ttgg 34

<210> 30

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 30

tcgcccctac atctactaac aaaccgc 27

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 31

agtggaatta gaatcgtgta 20

<210> 32

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 32

ccacaaccaa tcaacaac 18

<210> 33

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 33

ggaattagaa ttgtgtaggt tttataat 28

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 34

cgaaactaac gacccgccca 20

<210> 35

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 35

tcggaattgt aaagtatttg tg 22

<210> 36

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 36

gtccctcgca aatcaaaa 18

<210> 37

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 37

tggaattgta aagtatttgt gagtttgt 28

<210> 38

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 38

ctacaacaac aacaacaacg ccgaa 25

<210> 39

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 39

tttcggttag ttgcgcgg 18

<210> 40

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 40

ctaaaaaatc ccgcgaactc c 21

<210> 41

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 41

ttgtgtggtg attttgggga ttttag 26

<210> 42

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 42

accgcaaaaa aacgccctaa aatc 24

<210> 43

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 43

tcgttggagt ttcgtcgtcg t 21

<210> 44

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 44

aacccccgtc ccgaatct 18

<210> 45

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 45

ggagttttgt tgttgtagtt tttgttatta gtgag 35

<210> 46

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 46

acgcgaaccg cgcgtactca ct 22

<210> 47

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 47

gacgtaacgt ttcgatttc 19

<210> 48

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 48

cttcccaaac taatctacg 19

<210> 49

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 49

atgttttgat tttgttgtgt tgttggtttt g 31

<210> 50

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 50

actcgccgcc tactactact atcc 24

<210> 51

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 51

ttaggggggc gggggag 17

<210> 52

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 52

cctcgaccaa tcaaaccccg 20

<210> 53

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 53

gtgggggagg gggtttggag tgttt 25

<210> 54

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 54

ccgactctaa cctacctccg aca 23

<210> 55

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 55

ggagtagtag cggatttg 18

<210> 56

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 56

ccaacactaa aactaaaaca atatc 25

<210> 57

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 57

gtagtggatt tggtttttgg tgtttttaag g 31

<210> 58

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 58

accgacgaca acgacaataa tacaatc 27

<210> 59

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 59

acggtgaggt tttacgtt 18

<210> 60

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 60

ccaaacccgc tcaacaa 17

<210> 61

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 61

tgaggtttta tgtttgttag tatatttggg tttg 34

<210> 62

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 62

acgaacgtcg aaaaaaaacg aacccg 26

<210> 63

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 63

tcgttttttc gtttttggtt tgc 23

<210> 64

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 64

aacccgcaaa aatactcctt tct 23

<210> 65

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 65

ttgtttttgg tttgtggtgt tttttttgg 29

<210> 66

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 66

tccgcgaccc aaaacaaaat tcc 23

<210> 67

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 67

ttcgtgtggg gagtgacgtg ac 22

<210> 68

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 68

tcgtttctcg tcgcccctac t 21

<210> 69

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 69

gtggggagtg atgtgatgtt agtagagatt ttatt 35

<210> 70

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 70

accgcccgcg cgccactata c 21

<210> 71

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 71

cggagtcggg agagcgaaag 20

<210> 72

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 72

taaaactacc gaattaacgc aaattact 28

<210> 73

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 73

ttgggagagt gaaaggagag gggatt 26

<210> 74

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 74

tcctcgatta actcctaaat accccgcc 28

<210> 75

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 75

gttttcggaa gcgttcgggt 20

<210> 76

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 76

tacgtaaacc cgataaaaac aaaatt 26

<210> 77

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 77

tggaagtgtt tgggtaaaga ttgtgaag 28

<210> 78

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 78

cccaaacgca caaatttccg cc 22

<210> 79

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 79

gcggaaagaa atagttattt cgttat 26

<210> 80

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 80

tatataacta aacgccaaaa ccgc 24

<210> 81

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 81

gaaatagtta ttttgttata tgggtttggt ttttag 36

<210> 82

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 82

tcctaaacgt actcctaacg ctcacta 27

<210> 83

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 83

gcgtatagag gaatttaaag tgtgg 25

<210> 84

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 84

acgccttttt atttacgacg acttaac 27

<210> 85

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 85

ttatttacaa caacttaact taaaaaacaa tattccacc 39

<210> 86

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 86

tattccgcct acgcccgctc g 21

<210> 87

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 87

gaattttttt atgcgagttg tttgagg 27

<210> 88

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 88

ttccgaatac gttccgaatc ctacc 25

<210> 89

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 89

ttatgtgagt tgtttgagga ttgggatgtt gag 33

<210> 90

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 90

aacaaaccct actcgaatcg ctcgcg 26

<210> 91

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 91

tgggaatttt tcgtttcggt t 21

<210> 92

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 92

acacgtaaac tattaatctt tttcccaac 29

<210> 93

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 93

cataaactat taatcttttt cccaacccca aatc 34

<210> 94

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 94

tcatttacca ttttccaaac ttactcgacc 30

<210> 95

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 95

aaggaagggt aaggcg 16

<210> 96

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 96

cgaacgaaac cacaaaac 18

<210> 97

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 97

gtgggggggg ttttgtgag 19

<210> 98

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 98

aaccccgact tcaacgcctc 20

<210> 99

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 99

ttgttagcgt ttaaagttag c 21

<210> 100

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 100

taaaattaca cgcgataccc 20

<210> 101

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 101

agtgtttaaa gttagtgaag tatgggtt 28

<210> 102

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 102

acacgctcca accgaatacg acc 23

<210> 103

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 103

tcgtttttag ttcgcgg 17

<210> 104

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 104

ttaactaccc cttccgct 18

<210> 105

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> blocking primer

<400> 105

tagtttgtgg ggtttgttat gtatatgt 28

<210> 106

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 106

acaaaccttt acgcacgacg ccc 23

<210> 107

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 107

gtgatggagg aggtttagta agt 23

<210> 108

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 108

ccaataaaac ctactcctcc ctt 23

<210> 109

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Probe

<400> 109

accaccaccc aacacacaat aacaaacaca 30

Claims

1. A method of identifying a prostate cancer status in a subject, comprising the steps of:

1) detecting the methylation level of a biomarker gene in a biological sample from the subject, wherein the biomarker gene is selected from one or more of the following genes: APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF 1A; and

2) comparing the methylation level detected in step 1) with a normal methylation level of a corresponding biomarker gene in a population to determine the prostate cancer status in the subject.

2. The method of claim 1, further comprising performing step 1) again after the subject receives medical treatment and comparing the methylation level measurements obtained in two times to determine a change in prostate cancer status in the subject.

3. The method of claim 1 or 2, wherein the prostate cancer status comprises prostate cancer susceptibility and presence, progression, subtype and/or stage of prostate cancer.

4. The method of claim 1 or 2, wherein step 1) comprises extracting DNA from the biological sample and treating with bisulfite, such that unmethylated cytosine residues in the DNA are deaminated while methylated cytosine residues remain unchanged.

5. The method of claim 4, wherein the bisulfite salt is sodium bisulfite.

6. The method of claim 1 or 2, wherein the biomarker genes in step 1) are selected from 2 or more than 2 of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF 1A.

7. The method of claim 6, wherein the biomarker genes in step 1) are selected from 5 or more than 5 of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF 1A.

8. The method of claim 7, wherein the biomarker genes in step 1) are APC, CCND2, CDH1, PITX2, and RARB.

9. The method of claim 1 or 2, wherein the prostate cancer status is stage I or stage II prostate cancer and the biomarker gene is PITX2 and/or RARB.

10. The method of claim 1 or 2, wherein the prostate cancer status is adenocarcinoma and the biomarker genes are CDH1, PITX2, and/or RARB.

11. The method of claim 1 or 2, wherein the prostate cancer state is transitional cell carcinoma and the biomarker gene is CCND2 and/or RARB.

12. The method of claim 1 or 2, wherein the prostate cancer state is squamous carcinoma and the biomarker gene is PITX2 and/or RARB.

13. The method of claim 1 or 2, wherein step 1) comprises detecting the methylation level of a target region within the biomarker gene, the target region being a nucleotide sequence of at least 15 bases in length within the biomarker gene, or a complement thereof, respectively.

14. The method of claim 1 or 2, wherein in step 1) the method is performed in a batch process

Detection of methylation level of APC gene comprises using a nucleic acid having SEQ ID NO: 11 and 12 or a primer pair having the sequences shown in SEQ ID NOs: 15 and 16, and performing PCR amplification reaction by using the APC gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation level of CCND2 gene includes using a dna having SEQ ID NO: 19 and 20, a primer pair having the sequences shown in SEQ ID NOs: 23 and 24, or a primer pair having the sequences shown in SEQ ID NOs: 27 and 28, and carrying out PCR amplification reaction by using the CCND2 gene after bisulfite treatment of the biological sample or a fragment thereof as a template;

detection of the methylation level of the CDH1 gene includes using a dna having the sequence of SEQ ID NO: 31 and 32 or a primer pair having the sequences shown in SEQ ID NOs: 35 and 36, and carrying out PCR amplification reaction by using the CDH1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation levels of the GSTP1 gene includes using a nucleic acid having the sequence of SEQ ID NO: 39 and 40 or a primer pair having the sequences shown in SEQ ID NOs: 43 and 44, and carrying out PCR amplification reaction by using the GSTP1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of the methylation level of the MCAM gene involves the use of a nucleic acid sequence having SEQ ID NO: 47 and 48, a primer pair having the sequences shown in SEQ ID NOs: 51 and 52, or a primer pair having the sequences shown in SEQ ID NOs: 55 and 56, and carrying out PCR amplification reaction by taking the MCAM gene or the segment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation levels of PENK genes involves the use of a dna having the sequence of SEQ ID NO: 59 and 60 or a primer pair having the sequences shown in SEQ ID NOs: 63 and 64, and carrying out PCR amplification reaction by using the hydrosulfite-treated PENK gene or the fragment thereof in the biological sample as a template;

detection of methylation levels of PITX2 gene includes using a probe having the sequence of SEQ ID NO: 67 and 68 or a primer pair having the sequences shown in SEQ ID NOs: 71 and 72, and carrying out PCR amplification reaction by using the PITX2 gene or the fragment thereof after bisulfite treatment in the biological sample as a template;

detection of methylation levels of the PTGS2 gene included the use of a dna having the sequence of SEQ ID NO: 75 and 76 or a primer pair having the sequences shown in SEQ ID NOs: 79 and 80, and carrying out PCR amplification reaction by using the PTGS2 gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation levels of the RARB gene involves the use of a dna having the sequence of SEQ ID NO: 83 and 84, a primer pair having the sequences shown in SEQ ID NOs: 87 and 88, or a primer pair having the sequences shown in SEQ ID NOs: 91 and 92, and performing PCR amplification reaction by using the RARB gene or the fragment thereof treated by the bisulfite in the biological sample as a template; and

detection of the methylation level of the RASSF1A gene includes the use of a nucleic acid having the sequence of SEQ ID NO: 95 and 96, a primer pair having the sequences shown in SEQ ID NOs: 99 and 100, or a primer pair having the sequences shown in SEQ ID NOs: 103 and 104, and carrying out PCR amplification reaction by using the RASSF1A gene or the fragment thereof after bisulfite treatment in the biological sample as a template.

15. The method of claim 14, wherein in step 1) the method further comprises

Detection of methylation level of APC gene comprises using a nucleic acid having SEQ ID NO: 11 and 12 and a primer pair having the sequences shown in SEQ ID NOs: 13, or a blocking primer having the sequence shown in SEQ ID NO: 15 and 16 and a primer pair having the sequences shown in SEQ ID NOs: 17, performing PCR amplification reaction by using the APC gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation level of CCND2 gene includes using a dna having SEQ ID NO: 19 and 20 and a primer pair having the sequences shown in SEQ ID NOs: 21 using a blocking primer having the sequence shown in SEQ ID NO: 23 and 24 and a primer pair having the sequences shown in SEQ ID NOs: 25, or a blocking primer having the sequence shown in SEQ ID NO: 27 and 28 and a primer pair having the sequences shown in SEQ ID NOs: 29, and carrying out PCR amplification reaction by using the CCND2 gene treated by bisulfite in the biological sample or a fragment thereof as a template;

detection of the methylation level of the CDH1 gene includes using a dna having the sequence of SEQ ID NO: 31 and 32 and a primer pair having the sequences shown in SEQ ID NOs: 33, or a blocking primer having the sequence shown in SEQ ID NO: 35 and 36 and a primer pair having the sequences shown in SEQ ID NOs: 37, and carrying out PCR amplification reaction by using the CDH1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation levels of the GSTP1 gene includes using a nucleic acid having the sequence of SEQ ID NO: 39 and 40 and a primer pair having the sequences shown in SEQ ID NOs: 41, or a blocking primer having the sequence shown in SEQ ID NO: 43 and 44 and a primer pair having the sequences shown in SEQ ID NOs: 45, and carrying out PCR amplification reaction by using the GSTP1 gene treated by bisulfite in the biological sample or a fragment thereof as a template;

detection of the methylation level of the MCAM gene involves the use of a nucleic acid sequence having SEQ ID NO: 47 and 48 and a primer pair having the sequences shown in SEQ ID NOs: 49 using a blocking primer having the sequence shown in SEQ ID NO: 51 and 52 and a primer pair having the sequences shown in SEQ ID NOs: 53, or a blocking primer having the sequence shown in SEQ ID NO: 55 and 56 and a primer pair having the sequences shown in SEQ ID NOs: 57, performing PCR amplification reaction by using the MCAM gene treated by the bisulfite in the biological sample or the fragment thereof as a template;

detection of methylation levels of PENK genes involves the use of a dna having the sequence of SEQ ID NO: 59 and 60 and a primer pair having the sequences shown in SEQ ID NOs: 61, or a blocking primer having the sequence shown in SEQ ID NO: 63 and 64 and a primer pair having the sequences shown in SEQ ID NOs: 65, performing PCR amplification reaction by using the PENK gene treated by the bisulfite in the biological sample or the fragment thereof as a template;

detection of methylation levels of PITX2 gene includes using a probe having the sequence of SEQ ID NO: 67 and 68 and a primer pair having the sequences shown in SEQ ID NOs: 69, or using a blocking primer having the sequence shown in SEQ ID NO: 71 and 72 and a primer pair having the sequences shown in SEQ ID NOs: 73, and carrying out PCR amplification reaction by using the PITX2 gene or the fragment thereof after bisulfite treatment in the biological sample as a template;

detection of methylation levels of the PTGS2 gene included the use of a dna having the sequence of SEQ ID NO: 75 and 76 and a primer pair having the sequences shown in SEQ ID NOs: 77, or a blocking primer having the sequence shown in SEQ ID NO: 79 and 80 and a primer pair having the sequences shown in SEQ ID NOs: 81, and carrying out PCR amplification reaction by using the PTGS2 gene or the segment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation levels of the RARB gene involves the use of a dna having the sequence of SEQ ID NO: 83 and 84 and a primer pair having the sequences shown in SEQ ID NOs: 85, using a blocking primer having the sequence shown in SEQ ID NO: 87 and 88 and a primer pair having the sequence shown in SEQ ID NO: 89, or a blocking primer having the sequence shown in SEQ ID NO: 91 and 92 and a primer pair having the sequences shown in SEQ ID NOs: 93, and carrying out PCR amplification reaction by using the RARB gene or the fragment thereof treated by the bisulfite in the biological sample as a template; and

detection of the methylation level of the RASSF1A gene includes the use of a nucleic acid having the sequence of SEQ ID NO: 95 and 96 and a primer pair having the sequences shown in SEQ ID NOs: 97 using a blocking primer having the sequence shown in SEQ ID NO: 99 and 100 and a primer pair having the sequences shown in SEQ ID NOs: 101, or a blocking primer having the sequence shown in SEQ ID NO: 103 and 104 and a primer pair having the sequences shown in SEQ ID NOs: 105, performing PCR amplification reaction by using the RASSF1A gene or the fragment thereof after bisulfite treatment in the biological sample as a template,

wherein the blocking primer has a 3' terminal modification that is resistant to extension amplification by a DNA polymerase.

16. The method of claim 15, wherein in step 1) the method further comprises

Detection of methylation level of APC gene comprises using a nucleic acid having SEQ ID NO: 11 and 12, a primer pair having the sequences shown in SEQ ID NOs: 13 and a blocking primer having the sequence shown in SEQ ID NO: 14, or a probe having a sequence shown in SEQ ID NO: 15 and 16, a primer pair having the sequences shown in SEQ ID NOs: 17 and a blocking primer having the sequence shown in SEQ ID NO: 18, performing PCR amplification reaction by using the APC gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation level of CCND2 gene includes using a dna having SEQ ID NO: 19 and 20, a primer pair having the sequences shown in SEQ ID NOs: 21 and a blocking primer having the sequence shown in SEQ ID NO: 22, using a probe having the sequence shown in SEQ ID NO: 23 and 24, a primer pair having the sequences shown in SEQ ID NOs: 25 and a blocking primer having the sequence shown in SEQ ID NO: 26, or a probe having the sequence shown in SEQ ID NO: 27 and 28, a primer pair having the sequences shown in SEQ ID NOs: 29 and a blocking primer having the sequence shown in SEQ ID NO: 30, and performing PCR amplification reaction by using the CCND2 gene treated by bisulfite in the biological sample or a fragment thereof as a template;

detection of the methylation level of the CDH1 gene includes using a dna having the sequence of SEQ ID NO: 31 and 32, a primer pair having the sequences shown in SEQ ID NOs: 33 and a blocking primer having the sequence shown in SEQ ID NO: 34, or a probe having the sequence shown in SEQ ID NO: 35 and 36, a primer pair having the sequences shown in SEQ ID NOs: 37 and a blocking primer having the sequence shown in SEQ ID NO: 38, and carrying out PCR amplification reaction by using the CDH1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation levels of the GSTP1 gene includes using a nucleic acid having the sequence of SEQ ID NO: 39 and 40, a primer pair having the sequences shown in SEQ ID NOs: 41 and a blocking primer having the sequence shown in SEQ ID NO: 42, or a probe having the sequence shown in SEQ ID NO: 43 and 44, a primer pair having the sequences shown in SEQ ID NOs: 45 and a blocking primer having the sequence shown in SEQ ID NO: 46, and carrying out PCR amplification reaction by using the GSTP1 gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of the methylation level of the MCAM gene involves the use of a nucleic acid sequence having SEQ ID NO: 47 and 48, a primer pair having the sequences shown in SEQ ID NOs: 49 and a blocking primer having the sequence shown in SEQ ID NO: 50, using a probe having the sequence shown in SEQ ID NO: 51 and 52, a primer pair having the sequences shown in SEQ ID NOs: 53 and a blocking primer having the sequence shown in SEQ ID NO: 54, or a probe having the sequence shown in SEQ ID NO: 55 and 56, a primer pair having the sequences shown in SEQ ID NOs: 57 and a blocking primer having the sequence shown in SEQ ID NO: 58, and performing PCR amplification reaction by using the MCAM gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation levels of PENK genes involves the use of a dna having the sequence of SEQ ID NO: 59 and 60, a primer pair having the sequences shown in SEQ ID NOs: 61 and a blocking primer having the sequence shown in SEQ ID NO: 62, or a probe having the sequence shown in SEQ ID NO: 63 and 64, a primer pair having the sequences shown in SEQ ID NOs: 65 and a blocking primer having the sequence shown in SEQ ID NO: 66, and carrying out PCR amplification reaction by using the hydrosulfite-treated PENK gene or the fragment thereof in the biological sample as a template;

detection of methylation levels of PITX2 gene includes using a probe having the sequence of SEQ ID NO: 67 and 68, a primer pair having the sequences shown in SEQ ID NOs: 69 and a blocking primer having the sequence shown in SEQ ID NO: 70, or using a probe having the sequence shown in SEQ ID NO: 71 and 72, a primer pair having the sequences shown in SEQ ID NOs: 73 and a blocking primer having the sequence shown in SEQ ID NO: 74, and carrying out PCR amplification reaction by using the PITX2 gene or the fragment thereof after bisulfite treatment in the biological sample as a template;

detection of methylation levels of the PTGS2 gene included the use of a dna having the sequence of SEQ ID NO: 75 and 76, a primer pair having the sequences shown in SEQ ID NOs: 77 and a blocking primer having the sequence shown in SEQ ID NO: 78, or a probe having the sequence shown in SEQ ID NO: 79 and 80, a primer pair having the sequences shown in SEQ ID NO: 81 and a blocking primer having the sequence shown in SEQ ID NO: 82, and carrying out PCR amplification reaction by using the PTGS2 gene or the fragment thereof treated by the bisulfite in the biological sample as a template;

detection of methylation levels of the RARB gene involves the use of a dna having the sequence of SEQ ID NO: 83 and 84, a primer pair having the sequences shown in SEQ ID NOs: 85 and a blocking primer having the sequence shown in SEQ ID NO: 86, using a probe having the sequence shown in SEQ ID NO: 87 and 88, a primer pair having the sequences shown in SEQ ID NOs: 89 and a blocking primer having the sequence shown in SEQ ID NO: 90, or using a probe having the sequence shown in SEQ ID NO: 91 and 92, a primer pair having the sequences shown in SEQ ID NOs: 93 and a blocking primer having the sequence shown in SEQ ID NO: 94, and carrying out PCR amplification reaction by using the RARB gene or the fragment thereof treated by the bisulfite in the biological sample as a template; and

detection of the methylation level of the RASSF1A gene includes the use of a nucleic acid having the sequence of SEQ ID NO: 95 and 96, a primer pair having the sequences shown in SEQ ID NOs: 97 and a blocking primer having the sequence shown in SEQ ID NO: 98, using a probe having the sequence shown in SEQ ID NO: 99 and 100, a primer pair having the sequences shown in SEQ ID NOs: 101 and a blocking primer having the sequence shown in SEQ ID NO: 102, or a probe having the sequence shown in SEQ ID NO: 103 and 104, a primer pair having the sequences shown in SEQ ID NOs: 105 and a blocking primer having the sequence shown in SEQ ID NO: 106, performing PCR amplification reaction by using the RASSF1A gene or the fragment thereof after bisulfite treatment in the biological sample as a template,

wherein the probe has a fluorescent group at one end and a fluorescence quenching group at the other end.

17. The method of claim 1 or 2, wherein step 1) further comprises using a peptide having the sequence of SEQ ID NO: 107 and 108 and a primer pair having the sequences shown in SEQ ID NOs: 109, and carrying out PCR amplification reaction by using the ACTB gene or the segment thereof which is treated by the bisulfite and is used as the reference gene in the biological sample as a template.

18. The method of claim 1 or 2, wherein step 2) comprises determining prostate cancer status in the subject based on logistic regression from the methylation levels of the biomarker genes.

19. The method of claim 1 or 2, wherein the biological sample is selected from the group consisting of blood, serum, plasma, urine, urethral flushing fluid, semen, and circulating cells of the subject.

20. A kit for identifying a prostate cancer state in a subject, comprising a primer pair for detecting a methylation level of a biomarker gene in a biological sample from the subject, wherein the primer pair is used for performing a PCR amplification reaction with bisulfite-treated the biomarker gene or a fragment thereof as a template; the biomarker genes are selected from one or more of the following genes: APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB and RASSF 1A.

21. The kit of claim 20, wherein the biomarker genes are selected from 2 or more of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF 1A.

22. The kit of claim 20, wherein the biomarker genes are selected from 5 or more of APC, CCND2, CDH1, GSTP1, MCAM, PENK, PITX2, PTGS2, RARB, and RASSF 1A.

23. The kit of claim 20, wherein the biomarker genes are APC, CCND2, CDH1, PITX2, and RARB.

24. The kit of claim 20, wherein the prostate cancer status is stage I or stage II prostate cancer and the biomarker gene is PITX2 and/or RARB.

25. The kit of claim 20, wherein the prostate cancer state is adenocarcinoma and the biomarker genes are CDH1, PITX2, and/or RARB.

26. The kit of claim 20, wherein the prostate cancer state is transitional cell carcinoma and the biomarker gene is CCND2 and/or RARB.

27. The kit of claim 20, wherein the prostate cancer state is squamous carcinoma and the biomarker gene is PITX2 and/or RARB.

28. The kit of claim 20, wherein

The primer pair for APC methylation level detection has the sequence of SEQ ID NO: 11 and 12 or SEQ ID NO: 15 and 16;

the primer pair for detecting the methylation level of CCND2 has the sequence shown in SEQ ID NO: 19 and 20, SEQ ID NO: 23 and 24 or SEQ ID NO: 27 and 28;

the primer pair for detection of the level of methylation of CDH1 has the sequence of SEQ ID NO: 31 and 32 or SEQ ID NO: 35 and 36;

the primer pair for detecting the methylation level of GSTP1 has the nucleotide sequence shown in SEQ ID NO: 39 and 40 or SEQ ID NO: 43 and 44;

the primer pair for detecting the MCAM methylation level has the sequence of SEQ ID NO: 47 and 48, SEQ ID NO: 51 and 52 or the sequences shown in SEQ ID NO: 55 and 56;

the primer pair for PENK methylation level detection has the nucleotide sequence of SEQ ID NO: 59 and 60 or SEQ ID NO: 63 and 64;

the primer pair for the detection of the methylation level of PITX2 has the sequence of SEQ ID NO: 67 and 68 or the sequences shown in SEQ ID NOs: 71 and 72;

the primer pair used for the detection of the level of methylation of PTGS2 has the sequence of SEQ ID NO: 75 and 76 or SEQ ID NO: 79 and 80;

the primer pair for RARB methylation level detection has the sequence of SEQ ID NO: 83 and 84, SEQ ID NO: 87 and 88 or the sequences shown in SEQ ID NOs: 91 and 92; and

the primer pair used for the detection of the methylation level of RASSF1A has the sequence of SEQ ID NO: 95 and 96, SEQ ID NO: 99 and 100 or SEQ ID NO: 103 and 104.

29. The kit of claim 28, further comprising a blocking primer, wherein

And a polypeptide having the sequence of SEQ ID NO: 11 and 12 have the sequence shown in SEQ ID NO: 13, and (c) a sequence set forth in (c);

and a polypeptide having the sequence of SEQ ID NO: 15 and 16 have the sequence shown in SEQ ID NO: 17;

and a polypeptide having the sequence of SEQ ID NO: 19 and 20 have the sequence shown in SEQ ID NO: 21;

and a polypeptide having the sequence of SEQ ID NO: 23 and 24 have the sequence shown in SEQ ID NO: 25;

and a polypeptide having the sequence of SEQ ID NO: 27 and 28 have the sequence shown in SEQ ID NO: 29;

and a polypeptide having the sequence of SEQ ID NO: 31 and 32 have the sequence shown in SEQ ID NO: 33;

and a polypeptide having the sequence of SEQ ID NO: 35 and 36 have the sequence shown in SEQ ID NO: 37;

and a polypeptide having the sequence of SEQ ID NO: 39 and 40 has the sequence shown in SEQ ID NO: 41;

and a polypeptide having the sequence of SEQ ID NO: 43 and 44 has the sequence shown in SEQ ID NO: 45, and (c) a sequence shown as 45;

and a polypeptide having the sequence of SEQ ID NO: 47 and 48 have the sequence shown in SEQ ID NO: 49;

and a polypeptide having the sequence of SEQ ID NO: 51 and 52 have the sequence shown in SEQ ID NO: 53, or a sequence shown in SEQ ID NO;

and a polypeptide having the sequence of SEQ ID NO: 55 and 56 has the sequence shown in SEQ ID NO: 57;

and a polypeptide having the sequence of SEQ ID NO: 59 and 60 has the sequence shown in SEQ ID NO: 61;

and a polypeptide having the sequence of SEQ ID NO: 63 and 64 has the sequence shown in SEQ ID NO: 65;

and a polypeptide having the sequence of SEQ ID NO: 67 and 68 have the sequence shown in SEQ ID NO: 69;

and a polypeptide having the sequence of SEQ ID NO: 71 and 72 has the sequence shown in SEQ ID NO: 73;

and a polypeptide having the sequence of SEQ ID NO: 75 and 76 have the sequence shown in SEQ ID NO: 77;

and a polypeptide having the sequence of SEQ ID NO: 79 and 80 have the sequence shown in SEQ ID NO: 81;

and a polypeptide having the sequence of SEQ ID NO: 83 and 84 has the sequence shown in SEQ ID NO: 85;

and a polypeptide having the sequence of SEQ ID NO: 87 and 88 has the sequence shown in SEQ ID NO: 89;

and a polypeptide having the sequence of SEQ ID NO: 91 and 92 have the sequence shown in SEQ ID NO: 93;

and a polypeptide having the sequence of SEQ ID NO: 95 and 96 has the sequence shown in SEQ ID NO: 97;

and a polypeptide having the sequence of SEQ ID NO: 99 and 100 has the sequence shown in SEQ ID NO: 101; and

and a polypeptide having the sequence of SEQ ID NO: 103 and 104 have the sequence shown in SEQ ID NO: 105;

30. The kit of claim 28 or 29, further comprising a probe, wherein

And a polypeptide having the sequence of SEQ ID NO: 11 and 12 have the sequence shown in SEQ ID NO: 14, or a sequence shown in fig. 14;

and a polypeptide having the sequence of SEQ ID NO: 15 and 16 have the sequence shown in SEQ ID NO: 18, or a sequence shown in seq id no;

and a polypeptide having the sequence of SEQ ID NO: 19 and 20 have the sequence shown in SEQ ID NO: 22;

and a polypeptide having the sequence of SEQ ID NO: 23 and 24 have the sequence shown in SEQ ID NO: 26;

and a polypeptide having the sequence of SEQ ID NO: 27 and 28 have the sequence shown in SEQ ID NO: 30;

and a polypeptide having the sequence of SEQ ID NO: 31 and 32 have the sequence shown in SEQ ID NO: 34;

and a polypeptide having the sequence of SEQ ID NO: 35 and 36 have the sequence shown in SEQ ID NO: 38;

and a polypeptide having the sequence of SEQ ID NO: 39 and 40 has the sequence shown in SEQ ID NO: 42;

and a polypeptide having the sequence of SEQ ID NO: 43 and 44 has the sequence shown in SEQ ID NO: 46;

and a polypeptide having the sequence of SEQ ID NO: 47 and 48 have the sequence shown in SEQ ID NO: 50;

and a polypeptide having the sequence of SEQ ID NO: 51 and 52 have the sequence shown in SEQ ID NO: 54, or a sequence shown in SEQ ID NO;

and a polypeptide having the sequence of SEQ ID NO: 55 and 56 has the sequence shown in SEQ ID NO: 58;

and a polypeptide having the sequence of SEQ ID NO: 59 and 60 have the sequence shown in SEQ ID NO: 62;

and a polypeptide having the sequence of SEQ ID NO: 63 and 64 have the sequence shown in SEQ ID NO: 66;

and a polypeptide having the sequence of SEQ ID NO: 67 and 68 have the sequence shown in SEQ ID NO: 70;

and a polypeptide having the sequence of SEQ ID NO: 71 and 72 has the sequence shown in SEQ ID NO: 74;

and a polypeptide having the sequence of SEQ ID NO: 75 and 76 have the sequence shown in SEQ ID NO: 78, or a sequence shown in seq id no;

and a polypeptide having the sequence of SEQ ID NO: 79 and 80 have the sequence shown in SEQ ID NO: 82;

and a polypeptide having the sequence of SEQ ID NO: 83 and 84 has the sequence shown in SEQ ID NO: 86;

and a polypeptide having the sequence of SEQ ID NO: 87 and 88 has the sequence shown in SEQ ID NO: 90;

and a polypeptide having the sequence of SEQ ID NO: 91 and 92 have the sequence shown in SEQ ID NO: 94;

and a polypeptide having the sequence of SEQ ID NO: 95 and 96 has the sequence shown in SEQ ID NO: 98, or a sequence shown in seq id no;

and a polypeptide having the sequence of SEQ ID NO: 99 and 100 have the sequence shown in SEQ ID NO: 102; and

and a polypeptide having the sequence of SEQ ID NO: 103 and 104 have the sequence shown in SEQ ID NO: 106 of the sequence shown in the figure 106,

31. The kit of claim 20, further comprising a polypeptide having the sequence of SEQ ID NO: 107 and 108 and a primer pair having the sequences shown in SEQ ID NOs: 109, and is used for performing PCR amplification reaction by using the ACTB gene or the segment thereof which is treated by the bisulfite and is used as the reference gene in the biological sample as a template.

32. The kit of claim 20, further comprising a DNA extraction reagent and a bisulfite reagent.

33. The kit of claim 32 wherein the bisulfite reagent comprises sodium bisulfite.

34. The kit of claim 20, wherein the prostate cancer status comprises a susceptibility to prostate cancer and the presence, progression, subtype and/or stage of prostate cancer.

35. The kit of claim 20, wherein the biological sample is selected from the group consisting of blood, serum, plasma, urine, urethral flushing fluid, semen, and circulating cells of the subject.

36. The kit of claim 20, further comprising instructions describing the method of use of the kit and processing the test results by logistic regression.