CN118064593A - Prostate cancer biomarker, prostate cancer detection kit and application - Google Patents
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract
The application belongs to the field of molecular biology detection, and particularly relates to a prostate cancer biomarker, a prostate cancer detection kit and application. The kit for detecting the prostate cancer provided by the application can effectively distinguish a prostate cancer patient from a healthy subject by detecting the methylation level of the prostate cancer biomarker (the full length or partial region of the negative chain of the CHr7:150955661-150956061 of the KCNH2 gene and the full length or partial region of the positive chain of the CHr16:23835973-23836051 of the PRKCB gene) in a sample, and provides a new idea for noninvasive, high-sensitivity and high-specificity detection of the prostate cancer.
Description
Technical Field
The application belongs to the field of molecular biology detection, and particularly relates to a prostate cancer biomarker, a prostate cancer detection kit and application.
Background
Prostate cancer is a malignant tumor with a high incidence rate in older men, and with the aggravation of aging population and the change of life style and dietary structure, the incidence rate of Chinese prostate cancer has shown a remarkable rising trend in recent years, and the incidence rate of male tumors is the sixth.
Currently, clinical diagnosis of prostate cancer mainly depends on serum PSA, transrectal prostate ultrasound, and the like. The detection sensitivity and specificity of serum PSA are low, and the PSA detection is easy to generate false positive results to cause excessive diagnosis, so that the subjects bear unnecessary physiological, psychological and economic burdens.
The development of tumor molecular markers is a new field of tumor diagnosis and treatment after image diagnosis and pathological diagnosis, and has great influence on the diagnosis, monitoring and treatment of tumors. Screening of multi-site high-sensitivity and high-specificity DNA methylation biomarkers from prostate cancer urine can be used for identifying cancer patients, and is helpful for early detection of prostate cancer, so that survival rate and quality of life of patients are improved.
Disclosure of Invention
Aiming at the defects of the prior art, the application aims to provide a prostate cancer biomarker, a prostate cancer detection kit and application thereof, and aims to solve the technical problems of insufficient diagnosis means, low detection accuracy, detection sensitivity and specificity to be improved and the like of the prostate cancer in the prior art.
To achieve the above object, the present application provides a prostate cancer biomarker, which uses GRCh38.p14 as a reference genome and comprises the full length or partial region of the negative strand of Ch7: 150955661-150956061 of KCNH2 gene and the full length or partial region of the positive strand of Ch16: 23835973-23836051 of PRKCB gene.
The application also provides a prostate cancer detection kit comprising reagents for detecting the methylation level of the prostate cancer biomarker.
Preferably, the above reagents comprise a first pair of methylation primers for detecting the methylation level of the full length or partial region of the negative strand of Chr7:150955661-150956061, and a second pair of methylation primers for detecting the methylation level of the full length or partial region of the positive strand of Chr16: 23835973-23836051.
Further preferably, the above reagents further comprise a first pair of unmethylated primers for detecting the methylation level of the full length or partial region of the negative strand of Chur 7:150955661-150956061, and a second pair of unmethylated primers for detecting the methylation level of the full length or partial region of the positive strand of Chur 16: 23835973-23836051.
Preferably, the nucleotide sequence of the first methylation primer pair is shown as SEQ ID NO. 10-11, the nucleotide sequence of the first unmethylation primer pair is shown as SEQ ID NO. 12-13, the nucleotide sequence of the second methylation primer pair is shown as SEQ ID NO. 14-15, and the nucleotide sequence of the second unmethylation primer pair is shown as SEQ ID NO. 16-17.
Preferably, the reagent further includes a first detection probe corresponding to the first methylation primer, and a second detection probe corresponding to the second methylation primer.
Further preferably, the above reagents include a first methylation primer pair of SEQ ID NO. 10-11 and a first detection probe of SEQ ID NO.25, and a second methylation primer pair of SEQ ID NO. 14-15 and a second detection probe of SEQ ID NO. 26.
Preferably, the kit for detecting prostate cancer further comprises one or more of a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a detection primer pair of an internal reference gene, a detection probe of an internal reference gene, a positive control and a negative control.
Preferably, the first detection probe, the second detection probe, and the detection probe of the reference gene each have a fluorescent reporter group at the 5 'end and a fluorescent quenching group at the 3' end.
The application also provides application of the prostate cancer biomarker or the prostate cancer detection kit in preparation of a prostate cancer diagnosis product.
In general, compared with the prior art, the above technical solution conceived by the present application mainly has the following technical advantages:
(1) The kit for detecting the prostate cancer provided by the application can effectively distinguish a prostate cancer patient from a healthy subject by detecting the methylation level of the prostate cancer biomarker (the full length or partial region of the negative chain of the Chr7:150955661-150956061 of the KCNH2 gene and the full length or partial region of the positive chain of the Chr16:23835973-23836051 of the PRKCB gene) in a sample, improves the detection rate of the prostate cancer, has high detection accuracy, can effectively avoid the detection of false positive and false negative results, and provides the biomarker capable of detecting the prostate cancer with high sensitivity and high specificity.
(2) In a preferred embodiment, the prostate cancer detection kit provided by the application provides a new idea for noninvasive, high-sensitivity and high-specificity detection of prostate cancer by detecting the methylation level of the Chr7:150955661-150956061 negative strand of KCNH2 gene and the Chr16:23835973-23836051 positive strand of PRKCB gene, wherein the sensitivity of a urine sample for diagnosing prostate cancer is 86.6%, the specificity of a urine sample for detecting healthy subjects is 91.1%, and the specificity of a urine sample for detecting benign diseases of the urinary system is 93.0%.
Detailed Description
The present application will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
The term "subject" may be a mammal or a cell, tissue, organ or portion of a mammal. In the present application, mammal means any kind of mammal, preferably a human (including a human, a human subject or a human patient).
The term "diagnosis" refers to determining the health status of a subject, and encompasses detecting the presence or absence of a disease, responding to a therapeutic regimen, assessing risk of recurrence, assessing risk and extent of cancerous lesions, prognostic assays, and the like. In some cases, the term "diagnosis" refers to the use of a single factor in determining, validating or confirming a clinical state of a patient. In some embodiments, "detecting" prostate cancer refers to detecting the presence or absence of a disease, i.e., determining whether a subject has prostate cancer.
The term "sample" refers to any substance, including biological samples, that may contain a target area for analysis. In the present application, a "sample" or "biological sample" may be a sample obtained directly from a biological source or a sample that is processed. Samples or biological samples include, but are not limited to, body fluids (e.g., whole blood, plasma, serum, cerebral spinal fluid, synovial fluid, urine, sweat, semen, stool, sputum, tears, mucus, amniotic fluid, or the like), exudates, bone marrow samples, ascites, pelvic rinse, pleural fluid, spinal fluid, lymph fluid, eye fluid, nasal, laryngeal or genital swab extracts, cell suspensions of digestive tissue, or extracts of fecal matter, and tissue and organ samples from humans, animals (e.g., non-human mammals) and plants, and processed samples derived therefrom.
The term "methylation" is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. DNA methylation refers to covalent binding of a methyl group at the 5 th carbon position of cytosine of a genomic CpG dinucleotide under the action of a DNA methyltransferase. DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression.
The term "methylation level" refers to whether or not cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/proportion/percentage of methylation, representing both qualitative and quantitative concepts. In practical application, different detection indexes can be adopted to compare the DNA methylation level according to practical conditions. As in some cases, the comparison may be made based on Ct values detected by the sample; in some cases, the ratio of gene methylation in the sample, i.e., number of methylated molecules/(number of methylated molecules+number of unmethylated molecules). Times.100, can be calculated and then compared; in some cases, statistical analysis and integration are performed on each index to obtain a final determination index.
The term "biomarker" refers to a biochemical marker that can label changes in system, organ, tissue, cell and subcellular structure or function, or changes that may occur, such as proteins, DNA or RNA, etc., for a very broad range of uses. Can be used for disease diagnosis, disease stage judgment or for evaluating the safety and effectiveness of new drugs or new therapies in target populations. Screening biomarkers for disease screening and early diagnosis can greatly improve the clinical treatment effect of patients.
The term "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction, PCR) to amplify a sequence of interest based on a polynucleotide sequence corresponding to a gene of interest or a portion thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer depends on many factors, including temperature, source of primer, method used, and the like. For example, for diagnostic and prognostic applications, the oligonucleotide primers will typically contain at least 10, 15, 20, 25 or more nucleotides, but may also contain fewer nucleotides, depending on the complexity of the target sequence. In the present application, the term "primer" refers to a pair of primers capable of hybridizing to a double strand of a target DNA molecule or to regions of the target DNA molecule flanking the nucleotide sequence to be amplified. "primer pair" refers to a group of forward and reverse primers.
The term "TaqMan probe" refers to a stretch of oligonucleotide sequences comprising a5 'fluorescent reporter group and a 3' quencher group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce because of the presence of a quenching group near the fluorescent group. During amplification, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of the DNA polymerase (e.g., taq enzyme) digests the probe and the fluorescent group is far from the quenching group, its energy is not absorbed, i.e., a fluorescent signal is generated. The fluorescence signal is also identical to the target fragment with a synchronous exponential increase per PCR cycle. When the fluorescent groups carried by the detection probes are different, the TAQMAN QPCR reaction system can meet the requirement of simultaneously detecting a plurality of target genes.
The term "methylation-specific PCR" is one of the most sensitive experimental techniques currently studied for methylation, and a minimum of about 50pg of DNA methylation can be found. After the single-stranded DNA is treated by a methylation conversion reagent, all unmethylated cytosines are deaminated to uracil, and methylated cytosines in CpG sites are kept unchanged, two pairs of primers aiming at methylated and unmethylated sequences are respectively designed, and the methylated and unmethylated DNA sequences can be distinguished by PCR amplification.
The term "methylation specific fluorescent quantitative PCR (qMSP)" is an experimental technique combining fluorescent quantitative PCR technology and methylation specific PCR technology. In the technology, proper primer pairs are designed based on sequence differences of DNA in different methylation states after the DNA is converted by a methylation conversion reagent, so that methylated sequences and unmethylated sequences are distinguished, but qMSP final detection indexes are fluorescent signals, so that fluorescent probes or fluorescent dyes are required to be added in addition to methylation detection primers in a qMSP reaction system. Compared with the traditional methylation specific PCR technology, qMSP has higher sensitivity and specificity for detecting the DNA methylation level, is more suitable for detecting trace amount of DNA fragments which are mixed in the DNA of patients in early cancer, does not need gel electrophoresis detection, and is simpler and more convenient to operate. In the present disclosure, methylation primer pairs are added when performing real-time quantitative methylation-specific PCR, and if the Ct value meets the requirement (e.g., ct.ltoreq.45 in urine samples), it indicates that the target sequence is methylated.
The application provides a prostate cancer biomarker, which takes GRCh38.p14 as a reference genome and comprises the full length or partial region of the negative strand of Ch7: 150955661-150956061 of KCNH2 gene and the full length or partial region of the positive strand of Ch16: 23835973-23836051 of PRKCB gene.
The application also provides a prostate cancer detection kit comprising reagents for detecting the methylation level of the prostate cancer biomarker.
In some embodiments, the above reagents include a first methylation primer pair for detecting the methylation level of a full length or partial region of the negative strand of Chr7:150955661-150956061, and a second methylation primer pair for detecting the methylation level of a full length or partial region of the positive strand of Chr16: 23835973-23836051.
It will be appreciated that in detecting the methylation levels of the Chr7:150955661-150956061 negative strand and the Chr16:23835973-23836051 positive strand, the full length region of the region may be detected, and also a portion of the region may be detected.
In some embodiments, the above reagents include a first methylation primer pair for detecting the methylation level of a full length or partial region of the negative strand of Chr7:150955661-150956061, and a second methylation primer pair for detecting the methylation level of a full length or partial region of the positive strand of Chr16: 23835973-23836051.
In a preferred embodiment, the nucleotide sequence of the first methylation primer pair is shown as SEQ ID NO. 10-11, and the nucleotide sequence of the second unmethylated primer pair is shown as SEQ ID NO. 14-15.
In some embodiments, the above reagents further comprise a first pair of unmethylated primers for detecting the methylation level of the full length or partial region of the negative strand of Chr7:150955661-150956061, and a second pair of unmethylated primers for detecting the methylation level of the full length or partial region of the positive strand of Chr16: 23835973-23836051.
In a preferred embodiment, the above-mentioned primer set includes a first methylation primer pair of SEQ ID NOS.10-11 and a first unmethylated primer pair of SEQ ID NOS.12-13, and a second methylation primer pair of SEQ ID NOS.14-15 and a second unmethylated primer pair of SEQ ID NOS.16-17.
It is also within the scope of the present application if a primer pair has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.) or more sequence identity with the nucleotide sequence indicated by the first methylated primer pair, the first unmethylated primer pair, the second methylated primer pair, the second unmethylated primer pair, and the primer pair also has a certain prostate cancer diagnostic function (e.g., a specificity or sensitivity is comparable to, slightly reduced, slightly increased, or substantially increased, etc.) as compared to the primer pair of the present application.
In some embodiments, the reagent further comprises a first detection probe corresponding to the first methylation primer and a second detection probe corresponding to the second methylation primer.
In a preferred embodiment, the reagents include a first methylation primer pair of SEQ ID NO. 10-11 and a first detection probe of SEQ ID NO.25, and a second methylation primer pair of SEQ ID NO. 14-15 and a second detection probe of SEQ ID NO. 26.
In some embodiments, the kit further comprises one or more of a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a detection primer pair for an internal reference gene, a detection probe for an internal reference gene, a positive control, and a negative control.
In some embodiments, the DNA extraction reagents described above include, but are not limited to, lysis buffers, binding buffers, wash buffers, and elution buffers.
In some embodiments, the methylation conversion reagent described above is used to deaminate unmethylated cytosines in DNA to uracil, while methylated cytosines remain unchanged, and can distinguish between methylated and unmethylated cytosines in, for example, cpG dinucleotide sequences, and the like. The methylation converting reagent of the present application is not particularly limited, and reagents reported in the conventional art for achieving cytosine to uracil conversion may be exemplified by, but not limited to, hydrazine salts, bisulfites (e.g., sodium bisulfate, potassium bisulfate, ammonium bisulfate) and bisulfites (e.g., sodium bisulfate, potassium bisulfate, calcium bisulfate, magnesium bisulfate, aluminum bisulfate, cesium bisulfate, ammonium bisulfate, etc.).
In some embodiments, the amplification reagents described above include one or more of amplification buffers, dNTPs, DNA polymerase, and Mg 2+.
In some embodiments, the reference gene may be, but is not limited to ACTB. In some specific embodiments of the application, the nucleotide sequence of the detection primer pair of the reference gene ACTB is shown as SEQ ID NO. 22-23, and the nucleotide sequence of the detection probe is shown as SEQ ID NO.24. It will be appreciated that in other embodiments, other genes may be selected as reference genes, and in this case, the detection primer pair and the detection probe of the reference gene may be designed correspondingly.
In some embodiments, the first detection probe, the second detection probe, and the detection probe of the reference gene are TaqMan probes, and the 5 'end thereof comprises a fluorescent reporter group, and the 3' end thereof comprises a fluorescence quenching group. The fluorescent reporter groups are independently selected from any one of FAM, ROX, VIC, HEX, NED, TET, JOE, CY and CY 5; the fluorescence quenching groups are independently selected from any one of TAMRA, MGB, BHQ, BHQ, BHQ2 and BHQ 3. It should be noted that examples of the fluorescent reporter group and the fluorescent quenching group of each detection probe each independently include, but are not limited to, those listed above.
In some embodiments, the positive control is a biomarker comprising methylation, which is used to monitor the detection performance of a primer pair or primer probe combination in a kit. The negative reference refers to a biomarker that does not contain methylation therein and is used to monitor whether the assay is contaminated.
In some embodiments, the above-described prostate cancer biomarkers can be detected by methods well known in the art, including, but not limited to, methylation-specific PCR (qMSP), sequencing by ligation (BSP), methylation-sensitive random primer polymerase chain reaction (MS AP-PCR), methylation-sensitive single nucleotide primer extension (MS-SNuPE), methylation-sensitive DNA restriction enzyme analysis, restriction enzyme-based sequencing, restriction enzyme-based microarray analysis, joint bisulfite restriction analysis (COBRA), methylation CpG island amplification (MCA), methylation CpG island amplification and microarray (MCAM), hpaII small fragment enrichment by ligation-mediated PCR (HELP), bisulfite microarray analysis, methylation-specific pyrosequencing, HELP-Seq), TET-assisted pyridine borane sequencing (TAPS), methylation DNA immunoprecipitation sequencing (dip-Seq), gal hydrolysis and ligation adapter-dependent PCR (GLAD-PCR), methylation DNA immunoprecipitation-microarray analysis (mep-microarray analysis), and magneto-sensitive dye-blot analysis using a magneto-sensitive dye-based magneto-resistive array.
The kit provided by the application can be used for diagnosing the prostate cancer. Such diagnostics include, but are not limited to, prostate cancer risk assessment, prognosis, disease recognition, diagnosis of the stage of the disorder, and screening of therapeutic targets. Optionally, the diagnosing further comprises predicting prostate cancer, i.e. when the methylation level of the prostate cancer biomarker in the individual is in an elevated state for a certain period of time, it is possible to develop prostate cancer, and the prostate cancer biomarker can be detected to play a role in prediction.
The application also provides application of the prostate cancer biomarker or the prostate cancer detection kit in preparation of a prostate cancer diagnosis product.
In some embodiments, the prostate cancer diagnostic product described above includes at least one of a primer, a probe, a kit, a chip, a sequencing library, a membrane strip, and a protein array. Alternatively, the product may be in the form of a lyophilized powder, solution, suspension, emulsion, or the like.
The invention also provides a method for monitoring whether a subject suffers from prostate cancer or whether a prostate cancer patient recurs, which comprises the following steps: extracting DNA of a urine sample of a subject, transforming the DNA by using a DNA transforming reagent, purifying the transformed DNA, performing qPCR amplification by using the kit, detecting methylation levels of a Chr7:150955661-150956061 negative strand of a KCNH2 gene and a Chr16:23835973-23836051 positive strand of a PRKCB gene by a qMSP method, and further judging whether the urine sample to be detected is negative or positive for prostate cancer.
The following describes the above technical scheme in detail with reference to specific embodiments. It should be understood that materials of the same or similar type, model, quality, nature, or function as the reagents and instruments used in the examples described below may be used in the practice of the application. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 collection and processing of training set samples
The inventor collects 55 cases of cancer tissue samples clinically diagnosed with prostate cancer by himself, and 55 cases of paracancerous normal tissue samples are used as training sets. All tissue samples were formalin-fixed and paraffin-embedded samples, the collection process of all samples had been approved by the ethics committee, and all volunteers had signed informed consent prior to sample collection.
1) Extraction of DNA samples
For paraffin-embedded Tissue samples, QIAAMP DNA FFPE Tissue Kit (56404) was used to extract genomic DNA from the Tissue samples, see Kit instructions for specific procedures.
2) Bisulphite conversion and purification
The nucleic acid conversion reagent is prepared by respectively carrying out bisulphite conversion on the extracted DNA by using a nucleic acid purification reagent (Ehan apparatus 20200843) of Wuhan Ai Misen life technologies, and then purifying the converted DNA for subsequent experiments, wherein specific experimental operations are described in a kit instruction. After the conversion was completed, 30. Mu.L of purified water was used for elution and stored at-20℃for further use.
Example 2 prostate cancer biomarker methylation region screening based on training set samples
With GRCh38.p14 as the reference genome, the present application provides an isolated polynucleotide molecule SEQ ID NO.1 and SEQ ID NO.4 combination as a prostate cancer biomarker, SEQ ID NO.1 being located at the target region of KCNH2 (physical position being the Chun 7:150955661-150956061 minus strand) and SEQ ID NO.4 being located at the target region of PRKCB (physical position being the Chun 16:23835973-23836051 plus strand). After the above polynucleotide molecule is subjected to a methylation conversion reagent, the "C" in the DNA double strand is converted into the "U", and the "U" is converted into the "T" by the subsequent PCR, so that the "C" of the methylated DNA cannot be converted. Thus, the polynucleotide molecules obtained after bisulphite treatment of SEQ ID NO.1 include SEQ ID NO.2 (fully methylated sequence) and SEQ ID NO.3 (unmethylated sequence); the polynucleotide molecules obtained by bisulphite treatment of SEQ ID NO.4 include SEQ ID NO.5 (fully methylated sequence) and SEQ ID NO.6 (unmethylated sequence); the polynucleotide molecules obtained after bisulphite treatment of SEQ ID No.7 include SEQ ID No.8 (fully methylated sequence) and SEQ ID No.9 (unmethylated sequence), and specific information on the above polynucleotide molecules is shown in Table 1.
The bisulfite treated polynucleotide molecules design a methylation primer pair and a non-methylation primer pair specific for a prostate cancer biomarker, wherein the methylation primer pair does not amplify a non-methylated template and the non-methylation primer pair does not amplify a methylated template. According to the design principle of the primers, a first primer pair is designed by taking polynucleotide sequences SEQ ID NO.2 and SEQ ID NO.3 as templates, wherein the first primer pair comprises a first methylation primer pair shown by SEQ ID NO.10 and SEQ ID NO.11 and a first unmethylation primer pair shown by SEQ ID NO.12 and SEQ ID NO.13, and the position of an amplicon detected by the first primer pair is the negative strand of Chr7:150955776-15095917 (region 1). Designing a second primer pair by taking the polynucleotide sequences SEQ ID NO.5 and SEQ ID NO.6 as templates, wherein the second primer pair comprises a second methylation primer pair shown by SEQ ID NO.14 and SEQ ID NO.15 and a second unmethylation primer pair shown by SEQ ID NO.16 and SEQ ID NO.17, and the position of an amplicon detected by the second primer pair is the positive strand of chr16:23835973-23836051 (region 2). A third primer pair is designed by taking the polynucleotide sequences SEQ ID NO.8 and SEQ ID NO.9 as templates, and comprises a third methylation primer pair shown in SEQ ID NO.18 and SEQ ID NO.19 and a third unmethylation primer pair shown in SEQ ID NO.20 and SEQ ID NO.21, wherein the position of an amplicon detected by the third primer pair is the positive strand of chr16:23835814-23835971 (region 3).
Specific information of the first primer pair, the second primer pair and the third primer pair is shown in Table 2.
TABLE 1 target region of KCNH2 Gene and target region of PRKCB Gene
TABLE 2 nucleotide sequences of the first, second, third primer pairs
Taking the tissue sample DNA converted by the bisulfite as a template, adding SYBR Green PCR Mix, a methylation primer pair and a non-methylation primer pair of a biomarker, and an upstream primer (SEQ ID NO.22: 5'-AAGGTGGTTGGGTGGTTGTTTTG-3') and a downstream primer (SEQ ID NO.23: 5'-AATAACACCCCCACCCTGC-3') of an internal reference gene ACTB. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4. The PCR amplified products were sent to Sanger sequencing, and the sequencing peak pattern was analyzed based on the methylation status of individual CpG sites within the target region of the target gene as determined by Sanger.
TABLE 3 SYBR Green PCR reaction system
| Component (A) | Dosage (mu L) |
| SYBR Green PCR Mix | 17.5 |
| Methylation (unmethylation) upstream primer pair (10. Mu.M) | 0.5 |
| Methylation (unmethylation) downstream primer pair (10. Mu.M) | 0.5 |
| Upstream primer of ACTB (10. Mu.M) | 0.5 |
| Downstream primer of ACTB (10. Mu.M) | 0.5 |
| Template DNA | 4 |
| Ultrapure water | Supplement to 25 |
Table 4 SYBR Green PCR reaction procedure
Analysis of results: methylation of cytosine in a CpG nucleotide is classified as methylated and unmethylated, where methylation is classified as fully methylated and partially methylated, and a CpG dinucleotide site is considered partially methylated if sequencing of cytosine at that site reveals both a C and a T at the cytosine position. If more than 95% of the C's in CpG dinucleotide sites in an amplicon are methylated, the sample is considered fully methylated in this region. Comparing the sequencing result of the amplified product with the pathological result according to the above standard, and calculating the sensitivity and specificity of the target region according to the methylation state of CpG sites of the amplified product. Sensitivity = proportion of samples positive for pathology results that detected methylation positive; specificity = proportion of methylation negative samples with negative pathological results. The sensitivity and the specificity of the target area on the training set sample are shown in Table 5, the sensitivity of the detected tissue sample is more than or equal to 80%, and the area with the specificity of the detected tissue sample is more than or equal to 80% is selected for the next verification.
TABLE 5 sensitivity and specificity of target regions of KCNH2 Gene and PRKCB Gene on training set samples
As shown in Table 5, the detection sensitivities of the region 1 and the region 2 on the tissue sample are 87.3% and 89.1%, respectively, the detection specificities are 81.8% and 83.6%, respectively, and the screening conditions that the sensitivity of the detected tissue sample is not less than 80% and the specificity of the detected tissue sample is not less than 80% are satisfied, so that the next verification can be performed. The detection specificity of region 3 on the tissue sample was 78.2%, and the above screening conditions were not satisfied.
Example 3 analysis of the Performance of zone 1, zone 2 to detect urine samples (test sets) based on qMSP method
The differential methylation sites screened in example 2 were evaluated in 27 urine samples from prostate cancer and 82 urine samples from healthy subjects using the qMSP method. The volume of each urine sample is larger than 10mL, the collection process of all urine samples is approved by the ethics committee, all volunteers sign the informed consent, and all urine samples are anonymized.
Specific operations using the Wuhan Ai Misen life technologies Co., ltd nucleic acid extraction kit (Ehan mechanical arm 20210740) are described in the kit instruction. The genomic DNA of each extracted sample was subjected to bisulfite conversion using a nucleic acid conversion kit (Kagaku Co., ltd.) as a nucleic acid purification reagent (Ehan apparatus 20200843), and after completion of the conversion, 30. Mu.L of purified water was used for elution.
QMSP reaction: designing a primer pair and a detection probe for methylation specific fluorescence quantitative PCR (qMSP) by taking DNA sequences of the region 1 and the region 2 after bisulfite conversion as templates respectively, wherein the primer pair is required to have good amplification specificity, namely, the primer pair only amplifies the DNA templates subjected to methylation, but does not amplify the DNA templates not subjected to methylation, and simultaneously does not cause other nonspecific amplification; in addition, the primer set is required to have a good amplification efficiency, that is, an amplification efficiency of 90% to 110% for each target region. The nucleotide sequences of the primer pair and the detection probe satisfying the above requirements are shown in Table 6, wherein the probes used are TaqMan probes. The qPCR reaction based on the TaqMan probes can realize simultaneous detection of a plurality of target genes in one reaction system, and at the moment, only the fluorescent groups carried by the 5' -end of each target gene specific detection probe are required to be different. In this example, each qPCR reaction system needs to detect 2 target genes, namely a target region (biomarker) and an internal reference gene ACTB, wherein the fluorescent group at the 5 'end of the detection probe (SEQ ID NO.24: GGAGTG GTTTTTGGGTTTG) of the ACTB is FAM, and the fluorescent quenching group MGB at the 3' end. The detection probe of region 1 (SEQ ID NO.25: CGCCCAAACTCCTACAA), the 5 '-terminal fluorescent group being ROX and the 3' -terminal fluorescent quenching group MGB. The 5 '-end fluorescent group of the detection probe (SEQ ID NO.26: GAACTACCGCGAACCT) of the region 2 is VIC, and the 3' -end is a fluorescence quenching group MGB. And (3) artificially synthesizing the primer and the probe for standby.
Primer probe combinations for use in the qMSP method of Table 6
The qMSP reaction was performed using the DNA of the urine sample converted by the bisulfite as a template and using the primer probe combination provided in Table 6, and the amplification system and the amplification procedure are shown in tables 7 and 8. In qPCR reaction of each sample, the amount of the reference gene ACTB needs to be detected for monitoring the sample quality and interpretation of the results. In each PCR reaction plate, besides the experimental hole for detecting the sample to be detected, a positive control hole and a negative control hole are also required to be arranged at the same time. The template of the positive control well is prepared by mixing 10 3 copies/. Mu.L of plasmid containing the converted ACTB sequence and 10 3 copies/. Mu.L of plasmid containing the target region sequence (the sequence which is completely methylated and converted by hydrogen sulfite) in equal volume, and other components are the same as the experimental tube; the template of the negative control well was TE buffer and the other components were the same as the experimental tube.
After qPCR reaction is finished, a baseline is adjusted (a signal value corresponding to the 3 rd to 15 th cycles is generally set as a baseline level), a threshold value is set, the threshold value is required to be positioned in an exponential amplification period, a straight line crossing the threshold value and being parallel to an X axis is called a threshold line, and the cycle number corresponding to the intersection point of the threshold line and an amplification curve is a Ct value. Analyzing the result of qPCR reaction, requiring ① negative control tube without amplification; ② The positive control PCR tube has obvious index increasing period, and the Ct value of the target gene of the positive control PCR tube is between 26 and 30; ③ The Ct value of the reference gene ACTB of the sample to be detected is less than or equal to 33. If the positive control, the negative control and the reference gene all meet the requirements, the detection result of the sample to be detected can be analyzed and the result can be interpreted, otherwise, the detection must be carried out again when the experiment is invalid.
TABLE 7 PCR reaction system
| Component (A) | Specification of specification | Volume (mu L) |
| Platinum IIPCR buffer | 5× | 5 |
| dNTPs | 2.5MM each | 3 |
| Methylation upstream primer for each region | 10μM | 0.5 |
| Methylation downstream primer for each region | 10μM | 0.5 |
| Detection probes for each zone | 10μM | 0.5 |
| Upstream primer of ACTB gene | 10μM | 0.5 |
| Downstream primer of ACTB gene | 10μM | 0.5 |
| Detection probe of ACTB gene | 10μM | 0.5 |
| DNA polymerase | / | 0.5 |
| DNA of sample to be tested | / | 5 |
| Purified water | / | Supplement to 25 |
Table 8 PCR reaction procedure
4) Interpretation of results
If the positive judgment value of the urine sample is 45 and the Ct value of amplification by using a certain pair of methylation detection primer pair and detection probe is less than or equal to 45, the sample is considered to be methylation positive in the amplification region, and the sample is a prostate cancer positive sample; if Ct value > 45 amplified using a certain pair of methylation detection primer and probe, the sample is considered to be methylation negative in this amplified region, and the sample is a prostate cancer negative sample. The detection results are shown in Table 9.
Table 9qMSP method detects the performance of region 1, region 2 diagnostic test set urine samples
As can be seen from Table 9, for a few test set urine samples, the sensitivity of detecting prostate cancer urine samples in region 1 and region 2 is greater than 77%, and the specificity of detecting healthy subject urine samples is greater than or equal to 92.7%, so that false negative samples can be effectively prevented from being detected.
In view of the difference in sensitivity of the first primer probe combination and the second primer probe combination for detecting the prostate cancer urine sample, if the combination detection may have a synergistic effect, the sensitivity of the prostate cancer detection is improved. When the prostate cancer is diagnosed in a combined way, the judgment standard of the positive sample is as follows: if the Ct value of amplified region 1 is less than or equal to 45, region 1 is considered methylation positive, and if the Ct value of amplified region 1 is > 45, region 1 is considered methylation negative, and region 2 is determined as above. If at least one region in the region 1 or the region 2 in a certain sample to be detected is methylation positive, the sample is a positive prostate cancer sample; if the region 1 and the region 2 in a certain sample to be tested are methylation negative, the sample is a prostate cancer negative sample. The area combination test results are shown in Table 10.
TABLE 10 Performance of first primer probe combinations and second primer probe combinations in combination diagnosis of urine samples from subjects
In Table 10, the sensitivity of the first primer probe combination and the second primer probe combination for diagnosing prostate cancer plasma sample was 85.2%, which is superior to the sensitivity of single primer probe combination for detecting prostate cancer (see Table 9). The specificity of the first primer probe combination and the second primer probe combination for jointly detecting the urine sample of the healthy person is 91.1 percent, the specificity for detecting the benign disease of the urinary system is 91.5 percent, which is slightly lower than that of the single primer probe combination, but still at a higher level.
Example 4 Performance of prostate cancer biomarker to detect clinical urine samples
In order to realize noninvasive detection, further verify and improve the sensitivity of the kit for diagnosing prostate cancer urine samples, the embodiment prescribes blinding 82 prostate cancer urine samples diagnosed by pathological detection, 248 healthy subject urine samples and 43 urine samples of subjects with benign diseases of the urinary system (including prostatic hyperplasia, urinary tract infection, glandular cystitis, kidney stones, hydronephrosis and the like) according to an in-vitro diagnostic reagent clinical test scheme, then a test operator performs a test, performs interpretation according to an interpretation standard, and compares a detection result with a pathological result (gold standard), thereby verifying the clinical effectiveness of the biomarker. The specific detection process is the same as in example 3, and the detection results are shown in Table 11.
Table 11 performance of prostate cancer biomarkers to detect clinical urine samples
As can be seen from table 11, prostate cancer patients can be effectively distinguished from control groups (including healthy subjects, subjects with benign urological disease) by detecting the methylation levels of region 1 and region 2 in the urine sample. Specifically, the sensitivity of the detection of the urine sample of the prostate cancer reaches 86.6 percent, the specificity of the urine sample of the healthy subject reaches 91.1 percent, and the specificity of the urine sample of the benign disease of the urinary system reaches 93.0 percent.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the application and is not intended to limit the application, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the application are intended to be included within the scope of the application.
Claims (10)
1. A prostate cancer biomarker, characterized in that grch38.p14 is used as a reference genome, said prostate cancer biomarker comprising the full length or partial region of the chr7:150955661-150956061 negative strand of the KCNH2 gene and the full length or partial region of the chr16:23835973-23836051 positive strand of the PRKCB gene.
2. A prostate cancer detection kit comprising reagents for detecting the methylation level of the prostate cancer biomarker of claim 1.
3. The kit of claim 2, wherein the reagents comprise a first methylation primer pair for detecting the methylation level of the full length or partial region of the negative strand of chr7:150955661-150956061 and a second methylation primer pair for detecting the methylation level of the full length or partial region of the positive strand of chr16: 23835973-23836051.
4. The kit of claim 3, wherein the reagents further comprise a first pair of unmethylated primers for detecting the methylation level of the full length or partial region of the negative strand of Chr7:150955661-150956061 and a second pair of unmethylated primers for detecting the methylation level of the full length or partial region of the positive strand of Chr16: 23835973-23836051.
5. The kit for detecting prostate cancer according to claim 4, wherein the nucleotide sequence of the first methylation primer pair is shown in SEQ ID NO. 10-11, the nucleotide sequence of the first unmethylation primer pair is shown in SEQ ID NO. 12-13, the nucleotide sequence of the second methylation primer pair is shown in SEQ ID NO. 14-15, and the nucleotide sequence of the second unmethylation primer pair is shown in SEQ ID NO. 16-17.
6. The kit of claim 3, wherein the reagent further comprises a first detection probe corresponding to the first methylation primer and a second detection probe corresponding to the second methylation primer.
7. The kit of claim 6, wherein the reagents comprise a first methylation primer pair of SEQ ID NO. 10-11 and a first detection probe of SEQ ID NO.25, and a second methylation primer pair of SEQ ID NO. 14-15 and a second detection probe of SEQ ID NO. 26.
8. The kit for detecting prostate cancer according to any one of claims 3to 7, further comprising one or more of a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a detection primer pair of an internal reference gene, a detection probe of an internal reference gene, a positive control, and a negative control.
9. The kit of claim 8, wherein the first detection probe, the second detection probe, and the detection probe of the reference gene each have a fluorescent reporter group at their 5 'end and a fluorescent quenching group at their 3' end.
10. Use of a prostate cancer biomarker according to claim 1 or a prostate cancer detection kit according to any of claims 2 to 9 in the manufacture of a prostate cancer diagnostic product.
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