CN113215257A - Nucleic acid composition, kit and detection method for detecting breast cancer related gene methylation - Google Patents
Nucleic acid composition, kit and detection method for detecting breast cancer related gene methylation Download PDFInfo
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Abstract
The invention discloses a nucleic acid composition, a kit and a detection method for detecting breast cancer related gene methylation, wherein the nucleic acid composition comprises: methylation specific primers and probes of target sites of RASSF1A gene; methylation specific primers and probes of the target site of the APC gene; methylation specific primers and probes of GSTP1 gene target sites; methylation specific primers and probes for target sites of the CDH1 gene; specific primers and probes for the ACTB reference gene; the kit prepared from the nucleic acid composition disclosed by the invention has the advantages of accurate and rapid detection result of breast cancer related gene methylation, high throughput, simplicity in operation, high accuracy and sensitivity and the like.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a nucleic acid composition, a kit and a detection method for detecting breast cancer related gene methylation.
Background
The number of patients diagnosed with cancer in 2020 is 1930 ten thousand, and 1000 ten thousand die of cancer. Currently, people worldwide 1/5 suffer from cancer throughout their lifetime. Breast cancer has become the most common cancer among women in a global population, accounting for 11.7% of newly developed cancer cases. The incidence of breast cancer worldwide has been on the rise since the end of the 70 s of the 20 th century. Worldwide, one woman is diagnosed with breast cancer every 26 seconds; worldwide, 200 ten thousand women suffer from breast diseases every year; almost every 1 minute a woman dies from breast cancer; every year, 50 million women die worldwide from breast cancer. Each year, about 30 ten thousand new cases of breast cancer occur in China. Breast tumors have a higher diagnostic rate in the united states, with early diagnosis and treatment greatly improving 5-year survival (89%).
X-ray mammography in healthy women has been a successful strategy to reduce breast cancer mortality over the past 50 years. Full Field Digital Mammography (FFDM), Digital Breast Tomography (DBT), Breast Computed Tomography (BCT), Automated Breast Ultrasound (ABUS), a fusion of FFDM and ABUS, a fusion of DBT and ABUS, Magnetic Resonance Imaging (MRI), optical imaging, radio wave imaging, tactile sensors, etc. have also been developed. All of these methods have the inevitable disadvantage that the imaging test results are subject to hysteresis and may cause radioactive damage to the subject. Therefore, the search for a new noninvasive and accurate method for early diagnosis and screening detection of gynecological tumors is a problem to be solved in clinic at present.
With the rapid development of the technology, tumor markers are developed into a new field of tumor diagnosis and treatment after image diagnosis and pathological diagnosis, and have a great influence on the diagnosis, monitoring and treatment of tumors. Tumor markers can be detected in body fluids or tissues and can reflect the existence, differentiation degree, prognosis estimation, treatment effect judgment and the like of tumors. At present, the development of genetic diagnosis technology is continuous, and the hope is brought to the early diagnosis of breast cancer, wherein one field is based on DNA methylation detection. Aberrant DNA methylation is a recognized epigenetic marker of breast cancer. Changes in methylation at an early stage of cancer progression may be clinically relevant for cancer detection and prognosis-based treatment decisions. DNA aberrant methylation occurs in almost all tumors, making it a reliable target for tumor diagnosis. The research finds that DNA abnormal methylation usually appears in the early stage of tumor, and is a potential index for early screening of tumor, disease risk prediction, clinical course monitoring and curative effect evaluation.
The CDH1(Cadherin 1) gene encodes a classical Cadherin of the Cadherin superfamily. This calcium-dependent intercellular adhesion protein consists of five extracellular cadherin repeats, a transmembrane domain and a highly conserved cytoplasmic tail. Loss of function of this gene is thought to promote cancer progression by increasing proliferation, invasion and/or metastasis. The extracellular domain of this protein mediates bacterial adhesion to mammalian cells, while the cytoplasmic domain is essential for internalization. Mutations in this gene have been associated with gastric cancer, breast cancer, colorectal cancer, thyroid cancer, ovarian cancer and meibomian periodontal syndrome.
RASSF1A (Ras Association Domain Family Member 1) gene encodes a protein similar to RAS effector protein, and is a potential tumor suppressor. The target gene regulated by RASSFIA relates to various contents such as gene transcription, signal transduction, cytoskeleton, cell cycle, cell adhesion, apoptosis and the like, and has wide biological action. RASSF1A is a novel tumor suppressor gene, and its role in the process of tumor development and development is particularly important. It is presently believed that inactivation of expression of the RASSF1A gene is primarily associated with aberrant hypermethylation, heterozygous deletions and chromosomal deletions of the promoter region. The frequency of inactivation of the RASSF1A gene in human tumors is very high. For example, the RASSF1A gene is inactivated by being methylated in 70% or more of small cell lung cancer, 91% of renal cell carcinoma, 62% of bladder cancer, 71% of thyroid cancer, 84% of nasopharyngeal carcinoma, 70% or more of prostate cancer, and the like.
The GSTP1(Glutathione S-Transferase Pi 1) gene encodes an enzyme that plays an important role in detoxification by catalyzing the binding of a number of hydrophobic and electrophilic compounds to reduced Glutathione. This GST family member is a polymorphic gene encoding active, functionally distinct GSTP1 variant proteins that are thought to play a role in xenobiotic metabolism and in susceptibility to cancer and other diseases. The GSTP1 gene polymorphism is related to breast cancer occurrence, and epigenetic markers including GSTP1 gene methylation are important indexes for detecting breast cancer.
The apc (adenomatosus Polyposis coli) gene encodes a tumor suppressor protein that promotes rapid degradation of CTNNB1 and is involved in Wnt signaling as a negative regulator, the activity of which is related to its phosphorylation state. It activates GEF activity of SPTA13 and ARHGEF 4. Plays a role in Hepatocyte Growth Factor (HGF) induced cell migration. It is also involved in other processes including cell migration, adhesion, transcriptional activation and apoptosis. Diseases associated with APC include Familial Adenomatous Polyposis (FAP), hereditary desmoid disease, colorectal cancer as well as partial gastric cancer, breast cancer.
At present, the invention solves the problems that the nucleic acid composition and the kit which have accurate results, fast timeliness, high detection flux, good specificity and good sensitivity cannot be found in the early detection of breast cancer methylation, can indirectly provide effective information for the early diagnosis of human breast cancer, and provides a simple, convenient and easy method for the early detection and early treatment of breast cancer and the monitoring of treatment effect.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a nucleic acid composition, a kit and a detection method for detecting methylation of breast cancer related genes.
In order to achieve the above object, the present invention adopts the following technical solutions:
a nucleic acid composition for detecting breast cancer-associated gene methylation, comprising: methylation specific primers and probes of target sites of RASSF1A gene; methylation specific primers and probes of the target site of the APC gene; methylation specific primers and probes of GSTP1 gene target sites; methylation specific primers and probes for target sites of the CDH1 gene; specific primers and probes for the ACTB reference gene;
methylation specific primers and probes for target sites of RASSF1A gene include:
a forward primer: 5 '-CGGGGTTCGTTTGTGGTTTC-3' SEQ ID 01,
reverse primer: 5 '-CCGATTAAATCGTACTTCGC-3' SEQ ID 02,
detecting a probe: 5 'tag-TCGCGTTTGTAGCGTTTAAAGT-3' tag SEQ ID 03;
methylation specific primers and probes for the target site of the APC gene include:
a forward primer: 5 '-TAGGTATTGAGTTTTTTTTCGGTAA-3' SEQ ID 04,
reverse primer: 5 '-CAAAATCGACCGAACACCG-3' SEQ ID 05,
detecting a probe: 5 'tag-TTTCGGTTTGTTTCGTCGTTGTTCG-3' tag SEQ ID 06;
methylation specific primers and probes for the GSTP1 gene target site include:
a forward primer: 5 '-CGTTGGAGTTTTCGTCGTA-3' SEQ ID 07,
reverse primer: 5 '-GAAAACCCGACCTAATACTACGAAT-3' SEQ ID 08,
detecting a probe: 5 'tag-TCGTTATTATGAGTACGCGCGGTTCGC-3' tag SEQ ID 09;
methylation specific primers and probes for the target site of the CDH1 gene include:
a forward primer: 5 '-TATTTCGGATTTTTGATTTGCG-3' SEQ ID 10,
reverse primer: 5 '-TAAAAAAAAAAAACGATAACGACGA-3' SEQ ID 11,
detecting a probe: 5 'tag-ACGTATTCGGTCGTAAGTTTCGCGTT-3' tag SEQ ID 12;
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGGAGGAGTTTAGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTTCCGAAACGAAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACCACCCAAACACAATAACAAACACA-3' tag SEQ ID 15.
A kit for detecting breast cancer-associated gene methylation, comprising: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A genes, methylation specific primers and probes of target sites of APC genes, methylation specific primers and probes of target sites of GSTP1 genes, methylation specific primers and probes of target sites of CDH1 genes, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
methylation specific primers and probes for target sites of RASSF1A gene include:
a forward primer: 5 '-CGGGGTTCGTTTGTGGTTTC-3' SEQ ID 01,
reverse primer: 5 '-CCGATTAAATCGTACTTCGC-3' SEQ ID 02,
detecting a probe: 5 'tag-TCGCGTTTGTAGCGTTTAAAGT-3' tag SEQ ID 03;
methylation specific primers and probes for the target site of the APC gene include:
a forward primer: 5 '-TAGGTATTGAGTTTTTTTTCGGTAA-3' SEQ ID 04,
reverse primer: 5 '-CAAAATCGACCGAACACCG-3' SEQ ID 05,
detecting a probe: 5 'tag-TTTCGGTTTGTTTCGTCGTTGTTCG-3' tag SEQ ID 06;
methylation specific primers and probes for the GSTP1 gene target site include:
a forward primer: 5 '-CGTTGGAGTTTTCGTCGTA-3' SEQ ID 07,
reverse primer: 5 '-GAAAACCCGACCTAATACTACGAAT-3' SEQ ID 08,
detecting a probe: 5 'tag-TCGTTATTATGAGTACGCGCGGTTCGC-3' tag SEQ ID 09;
methylation specific primers and probes for the target site of the CDH1 gene include:
a forward primer: 5 '-TATTTCGGATTTTTGATTTGCG-3' SEQ ID 10,
reverse primer: 5 '-TAAAAAAAAAAAACGATAACGACGA-3' SEQ ID 11,
detecting a probe: 5 'tag-ACGTATTCGGTCGTAAGTTTCGCGTT-3' tag SEQ ID 12;
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGGAGGAGTTTAGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTTCCGAAACGAAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACCACCCAAACACAATAACAAACACA-3' tag SEQ ID 15.
In the kit for detecting methylation of breast cancer related genes, the final concentration of the components of the PCR reaction solution is as follows: 1 XPCR buffer solution, 0.4-0.6 MuM RASSF1A forward primer, 0.4-0.6 MuM RASSF1A reverse primer, 0.2-0.3 MuM RASSF1A detection probe, 0.4-0.6 MuM APC forward primer, 0.4-0.6 MuM APC reverse primer, 0.2-0.3 MuM APC detection probe, 0.4-0.6 MuM GSTP1 forward primer, 0.4-0.6 MuM GSTP1 reverse primer, 0.2-0.3 MuM GSTP1 detection probe, 0.4-0.6 MuM CDH1 forward primer, 0.4-0.6 MuM CDH1 reverse primer, 0.2-0.3 MuM CDH1 detection probe, 0.1-0.3 MuM ACTB forward primer, 0.1-0.1 MuM CDB 3 primer, 0.0.5 MuM Taq enzyme sample DNA detection probe, 0.05-0.05 MuM DNA polymerase chain reaction probe, 0.05-0.05 MuM DNA detection probe.
In the foregoing kit for detecting methylation of breast cancer-related genes, the positive quality control substance is genomic DNA of the ssci methylase-treated Hct116 cell line.
In the kit for detecting methylation of breast cancer related genes, the negative quality control product is genomic DNA of Hct116 cell line with genes DNMT1 and DNMT3b knocked out.
A detection method for detecting methylation of a breast cancer-associated gene, comprising the steps of:
the method comprises the following steps: extracting DNA of a sample to be detected, and taking the DNA after conversion treatment as a genome DNA sample of a PCR template;
step two: carrying out PCR amplification on a genome DNA sample by using a kit for detecting methylation of breast cancer related genes;
the kit comprises: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A genes, methylation specific primers and probes of target sites of APC genes, methylation specific primers and probes of target sites of GSTP1 genes, methylation specific primers and probes of target sites of CDH1 genes, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
methylation specific primers and probes for target sites of RASSF1A gene include:
a forward primer: 5 '-CGGGGTTCGTTTGTGGTTTC-3' SEQ ID 01,
reverse primer: 5 '-CCGATTAAATCGTACTTCGC-3' SEQ ID 02,
detecting a probe: 5 'tag-TCGCGTTTGTAGCGTTTAAAGT-3' tag SEQ ID 03;
methylation specific primers and probes for the target site of the APC gene include:
a forward primer: 5 '-TAGGTATTGAGTTTTTTTTCGGTAA-3' SEQ ID 04,
reverse primer: 5 '-CAAAATCGACCGAACACCG-3' SEQ ID 05,
detecting a probe: 5 'tag-TTTCGGTTTGTTTCGTCGTTGTTCG-3' tag SEQ ID 06;
methylation specific primers and probes for the GSTP1 gene target site include:
a forward primer: 5 '-CGTTGGAGTTTTCGTCGTA-3' SEQ ID 07,
reverse primer: 5 '-GAAAACCCGACCTAATACTACGAAT-3' SEQ ID 08,
detecting a probe: 5 'tag-TCGTTATTATGAGTACGCGCGGTTCGC-3' tag SEQ ID 09;
methylation specific primers and probes for the target site of the CDH1 gene include:
a forward primer: 5 '-TATTTCGGATTTTTGATTTGCG-3' SEQ ID 10,
reverse primer: 5 '-TAAAAAAAAAAAACGATAACGACGA-3' SEQ ID 11,
detecting a probe: 5 'tag-ACGTATTCGGTCGTAAGTTTCGCGTT-3' tag SEQ ID 12;
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGGAGGAGTTTAGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTTCCGAAACGAAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACCACCCAAACACAATAACAAACACA-3' tag SEQ ID 15;
step three: and determining whether the sample to be detected is methylated or not according to the fluorescence Ct values of the RASSF1A, APC, GSTP1, CDH1 and ACTB gene amplification results.
In the aforementioned detection method for detecting methylation of breast cancer-related genes, in the first step, the reagent used in the conversion treatment is bisulfite or bisulfite.
In the second step of the detection method for detecting methylation of breast cancer-related genes, the reaction procedure of PCR amplification is: 95 ℃ for 10min,45cycles for 95 ℃ for 15sec, 60 ℃ for 30sec (fluorescence collection).
The invention has the advantages that:
the invention discovers that the nucleic acid composition of RASSF1A, APC, GSTP1, CDH1 specific forward primer, specific reverse primer and specific probe has synergistic effect on the sensitivity and specificity of detecting the methylation of breast cancer related genes;
the gene ACTB is used as an internal reference gene to control the quality of a sample, the situation that housekeeping genes are possibly methylated is considered, the position without CpG sites is selected when an internal reference primer and a probe are designed, and the sequence after bisulfite treatment is designed, so that the quality control of the housekeeping genes on the sample is ensured;
the invention designs the primer and the probe with high specificity, configures the kit with convenient use and reliable detection result, and combines the scientific and reasonable PCR reaction system, so that the invention has the characteristics of rapidness, high flux, sensitivity and good specificity, when the kit is used for screening the breast cancer, the sensitivity is 95 percent, the negative detectable rate (specificity) is 85 percent, the rapid and accurate measurement of the methylation degree of the breast cancer related gene is realized, the breast cancer can be indirectly and timely and effectively diagnosed and treated, the medical cost is reduced, the social resource is saved, and the life quality is improved.
Drawings
FIG. 1 is a graph showing the amplification curve of a methylation positive sample of RASSF1A gene according to the present invention;
FIG. 2 is a graph showing the amplification of a sample positive for methylation of the APC gene according to the present invention;
FIG. 3 is a graph showing the amplification of a methylation positive sample of GSTP1 gene according to the present invention;
FIG. 4 is a graph showing the amplification curve of a methylation positive sample of the CDH1 gene according to the present invention;
FIG. 5 is a graph of the amplification curve of a negative sample according to the present invention;
FIG. 6 is a graph showing no amplification of both the target gene and the reference gene according to the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
A detection method for detecting methylation of a breast cancer-associated gene, comprising the steps of:
the method comprises the following steps: extracting DNA of a sample to be detected, and taking the DNA after conversion treatment as a genome DNA sample of a PCR template;
the reagent adopted in the conversion treatment is bisulfite or bisulfite, and the sequence after the bisulfite treatment is adopted for design, so that the quality control of housekeeping genes on the sample can be ensured; the examples herein are not exhaustive, and any examples are applicable to the present invention as long as the sequences can be pre-processed.
Step two: carrying out PCR amplification on a genome DNA sample by using a kit for detecting methylation of breast cancer related genes; preferably, the reaction procedure for PCR amplification is: 95 ℃ for 10min,45cycles for 95 ℃ for 15sec, 60 ℃ for 30sec (fluorescence collection).
The kit comprises: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A genes, methylation specific primers and probes of target sites of APC genes, methylation specific primers and probes of target sites of GSTP1 genes, methylation specific primers and probes of target sites of CDH1 genes, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
preferably, the positive quality control product is genomic DNA of Hct116 cell line treated with SssI methylase, C in all CG sequences of the genome can be methylated at C5, the negative quality control product is genomic DNA of Hct116 cell line with genes DNMT1 and DNMT3b knocked out, and the template-free control product does not contain human genomic DNA.
As an example, the final concentrations of the components of the PCR reaction solution were: 1 XPCR buffer, 0.4-0.6 MuM RASSF1A forward primer, 0.4-0.6 MuM RASSF1A reverse primer, 0.2-0.3 MuM RASSF1A detection probe, 0.4-0.6 MuM APC forward primer, 0.4-0.6 MuM APC reverse primer, 0.2-0.3 MuM APC detection probe, 0.4-0.6 MuM GSTP1 forward primer, 0.4-0.6 MuM GSTP1 reverse primer, 0.2-0.3 MuM GSTP1 detection probe, 0.4-0.6 MuM CDH1 forward primer, 0.4-0.6 MuM CDH1 reverse primer, 0.2-0.3 MuM CDH1 detection probe, 0.1-0.3 MuM ACTB forward primer, 0.1-0.1 MuM CDB 3 MuM CDTP 3 primer, 0.0.5 MuM Taq 2-0.05 MuM DNA polymerase chain reaction probe, 0.05-0.05 mM DNA; it should be noted that: the reagent formulation of the reaction solution is not limited, and is provided as a preferred embodiment, and other reaction solution formulations capable of being matched with the amplification of the sample DNA are also applicable to the present invention.
Methylation specific primers and probes for target sites of RASSF1A gene include:
a forward primer: 5 '-CGGGGTTCGTTTGTGGTTTC-3' SEQ ID 01,
reverse primer: 5 '-CCGATTAAATCGTACTTCGC-3' SEQ ID 02,
detecting a probe: 5 'tag-TCGCGTTTGTAGCGTTTAAAGT-3' tag SEQ ID 03, preferably, the 5 'tag is FAM and the 3' tag is BHQ 1;
methylation specific primers and probes for the target site of the APC gene include:
a forward primer: 5 '-TAGGTATTGAGTTTTTTTTCGGTAA-3' SEQ ID 04,
reverse primer: 5 '-CAAAATCGACCGAACACCG-3' SEQ ID 05,
detecting a probe: 5 'tag-TTTCGGTTTGTTTCGTCGTTGTTCG-3' tag SEQ ID 06, preferably, the 5 'tag is ROX and the 3' tag is BHQ 2;
methylation specific primers and probes for the GSTP1 gene target site include:
a forward primer: 5 '-CGTTGGAGTTTTCGTCGTA-3' SEQ ID 07,
reverse primer: 5 '-GAAAACCCGACCTAATACTACGAAT-3' SEQ ID 08,
detecting a probe: 5 'tag-TCGTTATTATGAGTACGCGCGGTTCGC-3' tag SEQ ID 09, preferably, the 5 'tag is TAMRA and the 3' tag is BHQ 2;
methylation specific primers and probes for the target site of the CDH1 gene include:
a forward primer: 5 '-TATTTCGGATTTTTGATTTGCG-3' SEQ ID 10,
reverse primer: 5 '-TAAAAAAAAAAAACGATAACGACGA-3' SEQ ID 11,
detecting a probe: 5 'tag-ACGTATTCGGTCGTAAGTTTCGCGTT-3' tag SEQ ID 12, preferably, the 5 'tag is VIC and the 3' tag is BHQ 1;
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGGAGGAGTTTAGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTTCCGAAACGAAACG-3' SEQ ID 14,
detecting a probe: 5 'CY 5-ACCACCACCCAAACACAATAACAAACACA-3' marker SEQ ID15, preferably, the 5 'marker is CY5 and the 3' marker is BHQ 2;
remarking: the purity of the primers should be PAGE or HPLC grade.
BHQ1 and BHQ2 are only preferred as quenching marker groups, and the selection of fluorescent markers is also preferred, so long as the fluorescent markers can be used for marking products.
Step three: and determining whether the sample to be detected is methylated or not according to the fluorescence Ct values of the RASSF1A, APC, GSTP1, CDH1 and ACTB gene amplification results.
Here, it is to be emphasized that: the detection method of the present invention is not limited, and any detection method using the nucleic acid composition of the present invention is not specifically exemplified herein, as long as it is within the scope of the present invention.
The technical effects of the invention are verified through experiments as follows:
first, a kit for an experiment was prepared using the following procedure.
Firstly, materials: a kit for detecting methylation of a breast cancer-associated gene, comprising: methylation specific primers for target sites of RASSF1A gene; a hydrolysis probe specific for the RASSF1A gene; methylation specific primers of the target site of the APC gene; a hydrolysis probe specific for the APC gene; methylation specific primers for the GSTP1 gene target site; a GSTP1 gene specific hydrolysis probe; methylation specific primers for the target site of the CDH1 gene; a CDH1 gene specific hydrolysis probe; primers for the ACTB reference gene; hydrolysis probes for ACTB reference genes;
methylation specific primers and probes for target sites of RASSF1A gene include:
a forward primer: 5 '-CGGGGTTCGTTTTGTGGTTTC-3' (SEQ ID NO.1)
Reverse primer: 5 '-CCGATTAAATCCGTACTTCGC-3' (SEQ ID NO.2)
Detecting a probe: 5 'FAM-TCGCGTTTGTTAGCGTTTAAAGT-3' BHQ1(SEQ ID NO.3)
Methylation specific primers and probes for the target site of the APC gene include:
a forward primer: 5 '-TAGGTATTGACGTTTTTTTTCGGTAA-3' (SEQ ID NO.4)
Reverse primer: 5 '-CAAAATCGAACCGAACACCG-3' (SEQ ID NO.5)
Detecting a probe: 5 'ROX-TTTCGGTTTTGTTTCGTCGTTGTTCG-3' BHQ2(SEQ ID NO.6)
Methylation specific primers and probes for the GSTP1 gene target site include:
a forward primer: 5 '-CGTTGGAGTTTCGTCGTCGTA-3' (SEQ ID NO.7)
Reverse primer: 5 '-GAAAACCCGAACCTAATACTACGAAT-3' (SEQ ID NO.8)
Detecting a probe: 5 'TAMRA-TCGTTATTAGTGAGTACGCGCGGTTCGC-3' BHQ2(SEQ ID NO.9)
Methylation specific primers and probes for the target site of the CDH1 gene include:
a forward primer: 5 '-TATTTCGGATTTTTTGATTTGCG-3' (SEQ ID NO.10)
Reverse primer: 5 '-TAAAAAAAAAAAAACGATAACGACGA-3' (SEQ ID NO.11)
Detecting a probe: 5 'VIC-ACGTATTCGGGTCGTAAGTTTCGCGTT-3' BHQ1(SEQ ID NO.12)
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGGAGGAGGTTTAGTAAGTT-3' (SEQ ID NO.13)
Reverse primer: 5 '-GCACTCTTCCGAAAACGAAACG-3' (SEQ ID NO.14)
Detecting a probe: 5 'CY 5-ACCACCACCCAACACACAATAACAAACACA-3' BHQ2(SEQ ID NO.15)
Selecting a positive quality control product and a negative quality control product:
positive quality control product and negative quality control product. The positive quality control product is the genome DNA of the Hct116 cell line treated by SssI methylase, and can ensure that C in all CpG sequences in the genome is methylated at the position of C5; the negative quality control product is the genome DNA of the Hct116 cell line with genes DNMT1 and DNMT3b knocked out;
thirdly, PCR reaction solution composition:
the kit comprises a PCR reaction solution containing the specific primer and the probe, wherein the PCR reaction solution comprises:
RASSF1A forward primer: 5' -CGGGGTTCGTTTTGTGGTTTC-3
RASSF1A reverse primer: 5' -CCGATTAAATCCGTACTTCGC-3
RASSF1A detection probe: 5 'FAM-TCGCGTTTGTTAGCGTTTAAAGT-3' BHQ1
APC forward primer: 5' -TAGGTATTGACGTTTTTTTTCGGTAA-3
APC reverse primer: 5' -CAAAATCGAACCGAACACCG-3
APC detection probe: 5 'ROX-TTTCGGTTTTGTTTCGTCGTTGTTCG-3' BHQ2
GSTP1 forward primer: 5' -CGTTGGAGTTTCGTCGTCGTA-3
GSTP1 reverse primer: 5' -GAAAACCCGAACCTAATACTACGAAT-3
GSTP1 detection probe: 5 'TAMRA-TCGTTATTAGTGAGTACGCGCGGTTCGC-3' BHQ2
CDH1 forward primer: 5' -TATTTCGGATTTTTTGATTTGCG-3
CDH1 reverse primer: 5' -TAAAAAAAAAAAAACGATAACGACGA-3
CDH1 detection probe: 5 'VIC-ACGTATTCGGGTCGTAAGTTTCGCGTT-3' BHQ1
ACTB forward primer: 5' -GTGATGGAGGAGGTTTAGTAAGTT-3
ACTB reverse primer: 5' -GCACTCTTCCGAAAACGAAACG-3
ACTB detection probe: 5 'CY 5-ACCACCACCCAACACACAATAACAAACACA-3' BHQ2
2X PCR buffer solution, Taq enzyme, dNTP and nuclease-free water. Were purchased from biotechnology, inc.
The final concentration of the PCR reaction solution components was:
1 XPCR buffer, 0.6. mu.M (. mu.mol/L) RASSF1A forward primer, 0.6. mu.M (. mu.mol/L) RASSF1A reverse primer, 0.3. mu.M (. mu.mol/L) RASSF1A detection probe, 0.6. mu.M (. mu.mol/L) APC forward primer, 0.6. mu.M (. mu.mol/L) APC reverse primer, 0.3. mu.M (. mu.mol/L) APC detection probe, 0.6. mu.M (. mu.mol/L) GSTP1 forward primer, 0.6. mu.M (. mu.mol/L) GSTP1 reverse primer, 0.3. mu.M (. mu.mol/L) GSTP1 detection probe, 0.6. mu.M (. mu.mol/L) CDH1 forward primer, 0.6. mu.M (. mu.mol/L) CDH1 reverse primer, 0.3. mu.M (. mu.mol/L) GSTP 1L forward primer, 0.M ACTB 2. mu.mol/L detection probe, 0.M ACTB detection probe, 0.25mM (mmol/L) dNTP;
(II) detecting the methylation of the breast cancer related gene by using the kit sample prepared by the method:
first, technical principle
A pair of specific primers and probes are designed through promoter regions of breast cancer related genes and reference genes in a human genome. Then, the primers and the probes are used for amplifying the DNA of the sample converted by the bisulfite, whether the sample to be detected is methylated or not is determined according to the Ct value of the relative fluorescence value of the PCR amplification result of the related genes, and the risk of the breast cancer is indirectly determined according to the methylation.
Second, detection method
The method comprises the following steps: extracting DNA of a sample to be detected, carrying out bisulfite conversion treatment on the DNA, and carrying out qPCR amplification by using the converted DNA as a PCR template.
Wherein, the reagent used for the Conversion treatment is bisulfite or bisulfite, and other auxiliary reagents (corresponding reagents are purchased from epiect Fast Bis μ lfite Conversion Kit of QIAGEN, Germany);
step two: providing the kit for detecting methylation of breast cancer related genes, and carrying out PCR amplification on a template;
step three: determining whether the sample to be detected is methylated or not according to the relative fluorescence value Ct of the PCR amplification result of the related gene;
the specific detection method comprises the following steps:
one) plasma separation
1. 10ml of peripheral blood was drawn using a free DNA sampling tube and plasma separation was performed the first time after arrival at the laboratory.
2. Centrifuging at 1600rpm for 20min at 4 deg.C, and packaging the plasma in sterile centrifuge tubes.
3. The plasma after the primary separation was centrifuged at 16,000rpm for 10min at 4 ℃ to remove residual cells. After centrifugation, the plasma was dispensed into sterile centrifuge tubes.
II) extracting plasma DNA:
the DNA extraction kit was purchased from Jivan Biotechnology (Beijing) Ltd:
1. 2ml of plasma samples were taken and the corresponding amounts of reagents were added as shown in Table 1 according to the following system:
TABLE 1
| Volume of plasma | Proteinase K | Buffer GHP | FineMag Particles K |
| 2ml | 200μl | 3.0ml | 30μl |
2. After the sample and the reagent are fully mixed, the mixture is incubated at room temperature for 20min, and the mixture is inverted and mixed for 10sec every 3-5min, so that the magnetic beads and the nucleic acids are fully combined. After incubation, removing liquid drops on the inner wall of the tube wall by brief centrifugation;
3. placing the centrifugal tube in a magnetic force and standing for 2min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube;
4. adding 1ml buffer RBP, shaking and mixing for 1min to make the magnetic beads fully suspended, and centrifuging briefly to remove the dropping liquid on the inner wall;
5. placing the centrifugal tube in a magnetic force and standing for 1min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube;
6. adding 1ml 80% ethanol (ready for use), shaking and mixing for 1min each time to suspend the magnetic beads thoroughly, and centrifuging briefly to remove the inner wall dripping liquid.
7. Placing the centrifugal tube on magnetic force and standing for 1min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube.
8. Repeat step 6-7 once, total 2 ethanol elution.
9. Placing the centrifugal tube on magnetic force, and air drying at room temperature for 5-10 min.
10. And (3) taking down the centrifugal tube from the magnetic force, adding 45 mu l of buffer solution EB, shaking and uniformly mixing to suspend the magnetic beads, incubating for 5min at 56 ℃, slightly shaking the centrifugal tube every 2min during the incubation to fully absorb and remove the nucleic acid, and then centrifuging for a short time to remove the liquid on the tube wall and the tube cover.
11. The centrifuge tube was placed on a magnetic rack and allowed to stand for 2min, and when the magnetic beads were completely adsorbed, the DNA solution was carefully transferred to a 1.5ml collection tube and stored under appropriate conditions.
12. Taking 1 mul of DNA eluent to carry out the concentration determination of the Qubit HS, recording the data result, and storing the rest samples in a refrigerator at the temperature of-20 ℃ if the experiment is not continued.
Third) DNA transformation procedure:
1. the extracted DNA was formulated into a bisulfite conversion system according to the following formulation as shown in Table 2;
TABLE 2
| Reagent composition | Reaction volume (μ l) |
| Extracted DNA | 40 |
| Sulfite treatment | 85 |
| DNA protect buffer | 15 |
| Total of | 140 |
2. After the transformation system is configured, placing the obtained product in a PCR instrument for reaction according to the following reaction conditions, wherein the reaction program is shown in Table 3;
TABLE 3
3. DNA purification after bisulfite conversion:
4. the PCR tube was transiently detached and the bisulfite converted product was transferred to a 1.5ml EP tube.
5. Add 310. mu.l of buffer BL reagent to each sample
6. Add 250. mu.l of absolute ethanol to each sample, vortex the solution for 15 seconds, and centrifuge briefly
7. Transferring the mixture to a spin column, and centrifuging at 14000rpm for 1min
8. The filtrate was discarded, 500. mu.l of buffer BW was added, and the mixture was centrifuged at 14000rpm for 1min
9. Discarding the filtrate, adding 500. mu.l buffer BD, incubating for 15min, and centrifuging at 14000rpm for 1min
10. The filtrate was discarded, 500. mu.l of buffer BW was added, and the mixture was centrifuged at 14000rpm for 1min
11. Repeat step 10
12. Discarding the filtrate, adding 250 μ l of anhydrous ethanol, centrifuging at 14000rpm for 1min
13. Discarding the filtrate, centrifuging at 14000rpm for 1min
14. Drying at room temperature for 3-5min to remove residual ethanol;
15. add 15. mu.l buffer EB and incubate and elute at room temperature for 1min, 14000rpm centrifuge for 1min, collect DNA and store at-20 ℃.
Four) qPCR reaction configuration
1. The qPCR instrument was an ABI 7500 model real-time fluorescent quantitative PCR system (available from life technology, Inc.) of USA, and the reaction system was 20 μ l
2. qPCR reaction system configuration and conditions, as shown in table 4 below:
TABLE 4
| Composition (I) | Reaction volume (μ l) |
| DNA after bisulfite conversion | 2.0 |
| Primers | 5.6 |
| Probe | 1.4 |
| RNAse-free Water | 1.0 |
| 2X PCR Mix | 10.0 |
| Total of | 20 |
3. PCR reaction sample addition layout:
3 parallel detections are carried out on the DNA sample to be detected, the positive quality control product, the negative quality control product and the template-free control, and the 96-hole sample adding layout of the PCR instrument is shown in the following table 5. In Table 5, PC represents a Positive Control (Positive Control), NC represents a Negative Control (Negative Control), NTC represents a No-Template Control (No Template Control), and S represents a test sample (sample)
TABLE 5
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | PC | PC | PC | S7 | S7 | S7 | ||||||
| B | NC | NC | NC | S8 | S8 | S8 | ||||||
| C | S1 | S1 | S1 | S9 | S9 | S9 | ||||||
| D | S2 | S2 | S2 | S10 | S10 | S10 | ||||||
| E | S3 | S3 | S3 | S.. | S.. | S.. | ||||||
| F | S4 | S4 | S4 | NTC | NTC | NTC | ||||||
| G | S5 | S5 | S5 | |||||||||
| H | S6 | S6 | S6 |
4. The PCR reaction procedure is as in table 6:
TABLE 6
5. Analysis of detection results
1) Running amplification curve analysis without template control (NTC), wherein no amplification curve is required, which indicates that the experiment is pollution-free and can continue to analyze;
2) the internal reference genes (ACTB) of the quality control product should have amplification signals and present an S-shaped amplification curve, and the Ct values of the internal reference should accord with the results of the following table; the target gene of the negative quality control product rises due to no amplification curve, the Ct value of the gene of the positive quality control product is in accordance with the following table 7, and when the quality control product detection meets the following conditions, the experiment is proved to be effective, and the analysis can be continued;
TABLE 7
3) The internal reference genes of the sample have amplification signals and are in an S-shaped amplification curve, and the PCR detection result of the sample is interpreted according to the following table 8;
TABLE 8
Referring to fig. 1-6, fig. 1 is a graph of amplification of methylation positive samples of RASSF1A gene, fig. 2 is a graph of amplification of methylation positive samples of APC gene, fig. 3 is a graph of amplification of methylation positive samples of GSTP1 gene, fig. 4 is a graph of amplification of methylation positive samples of CDH1 gene, and fig. 5 is a graph of amplification of negative samples, it can be seen that the relationship between amplification curves of breast cancer-related methylation genes and ACTB internal reference genes in different methylation samples is shown, and if there is no amplification curve in both target genes and internal reference genes, fig. 6 shows that the sample is unqualified and needs repeated detection or resampling.
(III) the sensitivity and specificity detection of breast cancer and normal human plasma is carried out by using the kit sample:
plasma samples from 20 normal persons and 20 breast cancer patients were used as the subjects to be monitored. Free DNA was extracted from each sample. The extraction of DNA can be carried out by any standard means known in the art, and specifically, in this case, the sample DNA used is extracted by using a specific extraction protocol according to the detection system of example 2.
The DNA sample is then pretreated to convert the 5' unmethylated cytosine to uracil. In this experimental case, the pretreatment was achieved by bisulfite reagent treatment. The modification of bisulfite DNA was pretreated by conversion using the detection system in example 2.
Then, the pretreated DNA samples of 20 normal persons and 20 cases of breast cancer were added to the test system of the above case 2 to test multiplex RASSF1A, APC, GSTP1, CDH1 and reference gene ACTB. Real-time PCR was performed on bisulfite converted DNA.
Wherein the PCR conditions used in the experimental cases were performed according to the qPCR conditions in case 2.
Finally, tables 9 and 10 below show the results of using the multiplex assay of the present invention to test 20 normal human and 20 breast cancer patient samples. As can be seen from tables 9 and 10, the positive detection rate and the negative detection rate of this protocol were high.
TABLE 9 results of plasma sample detection of breast cancer patients using the kit of the present invention
| Sample positive determination method | Number of samples to be tested | Number of positive samples | Positive rate |
| At least one target positive | 20 | 19 | 95％ |
TABLE 10 results of the measurement of a plasma sample of a normal human by using the kit of the present invention
| Sample positive determination method | Number of samples to be tested | Number of negative samples | Negative rate |
| At least one target positive | 20 | 17 | 85％ |
According to the detection results in the table, when the kit is used for breast cancer screening, the positive detection rate (sensitivity) is 95%, and the negative detection rate (specificity) is 85%.
From the above verification experiments, it can be seen that: the nucleic acid composition of the RASSF1A, the APC, the GSTP1 and the CDH1 specific forward primer, the specific reverse primer and the specific probe has a synergistic effect on the sensitivity and specificity of detecting the methylation of the breast cancer related genes, and the detection of the breast cancer methylation by combining the RASSF1A, the APC, the GSTP1 and the CDH1 specific forward primer, the specific reverse primer and the specific probe is an innovative discovery, and can obtain results through non-mechanized experiments.
The invention is not aimed at diagnosing diseases, and the obtained detection can not directly judge whether the diseases exist, indirectly provides effective information for early diagnosis of human breast cancer, and provides a simple, convenient and easy method for early detection and treatment of human breast cancer.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
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Claims (8)
1. A nucleic acid composition for detecting methylation of a breast cancer-associated gene, comprising: methylation specific primers and probes of target sites of RASSF1A gene; methylation specific primers and probes of the target site of the APC gene; methylation specific primers and probes of GSTP1 gene target sites; methylation of target site of CDH1 Gene
Specific primers and probes; specific primers and probes for the ACTB reference gene;
the methylation specific primers and probes of the RASSF1A gene target site comprise:
a forward primer: 5 '-CGGGGTTCGTTTGTGGTTTC-3' SEQ ID 01,
reverse primer: 5 '-CCGATTAAATCGTACTTCGC-3' SEQ ID 02,
detecting a probe: 5 'tag-TCGCGTTTGTAGCGTTTAAAGT-3' tag SEQ ID 03;
the methylation specific primers and probes of the target site of the APC gene comprise:
a forward primer: 5 '-TAGGTATTGAGTTTTTTTTCGGTAA-3' SEQ ID 04,
reverse primer: 5 '-CAAAATCGACCGAACACCG-3' SEQ ID 05,
detecting a probe: 5 'tag-TTTCGGTTTGTTTCGTCGTTGTTCG-3' tag SEQ ID 06;
the methylation specific primers and probes of the GSTP1 gene target site comprise:
a forward primer: 5 '-CGTTGGAGTTTTCGTCGTA-3' SEQ ID 07,
reverse primer: 5 '-GAAAACCCGACCTAATACTACGAAT-3' SEQ ID 08,
detecting a probe: 5 'tag-TCGTTATTATGAGTACGCGCGGTTCGC-3' tag SEQ ID 09;
the methylation specific primers and probes of the CDH1 gene target site comprise:
a forward primer: 5 '-TATTTCGGATTTTTGATTTGCG-3' SEQ ID 10,
reverse primer: 5 '-TAAAAAAAAAAAACGATAACGACGA-3' SEQ ID 11,
detecting a probe: 5 'tag-ACGTATTCGGTCGTAAGTTTCGCGTT-3' tag SEQ ID 12;
the ACTB reference gene specific primers and probes comprise:
a forward primer: 5 '-GTGATGGAGGAGTTTAGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTTCCGAAACGAAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACCACCCAAACACAATAACAAACACA-3' tag SEQ ID 15.
2. A kit for detecting methylation of a breast cancer-associated gene, comprising: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A genes, methylation specific primers and probes of target sites of APC genes, methylation specific primers and probes of target sites of GSTP1 genes, methylation specific primers and probes of target sites of CDH1 genes, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
the methylation specific primers and probes of the RASSF1A gene target site comprise:
a forward primer: 5 '-CGGGGTTCGTTTGTGGTTTC-3' SEQ ID 01,
reverse primer: 5 '-CCGATTAAATCGTACTTCGC-3' SEQ ID 02,
detecting a probe: 5 'tag TCGCGTTTGTAGCGTTTAAAGT-3' tag SEQ ID 03;
the methylation specific primers and probes of the target site of the APC gene comprise:
a forward primer: 5 '-TAGGTATTGAGTTTTTTTTCGGTAA-3' SEQ ID 04,
reverse primer: 5 '-CAAAATCGACCGAACACCG-3' SEQ ID 05,
detecting a probe: 5 'tag-TTTCGGTTTGTTTCGTCGTTGTTCG-3' tag SEQ ID 06;
the methylation specific primers and probes of the GSTP1 gene target site comprise:
a forward primer: 5 '-CGTTGGAGTTTTCGTCGTA-3' SEQ ID 07,
reverse primer: 5 '-GAAAACCCGACCTAATACTACGAAT-3' SEQ ID 08,
detecting a probe: 5 'tag-TCGTTATTATGAGTACGCGCGGTTCGC-3' tag SEQ ID 09;
the methylation specific primers and probes of the CDH1 gene target site comprise:
a forward primer: 5 '-TATTTCGGATTTTTGATTTGCG-3' SEQ ID 10,
reverse primer: 5 '-TAAAAAAAAAAAACGATAACGACGA-3' SEQ ID 11,
detecting a probe: 5 'tag-ACGTATTCGGTCGTAAGTTTCGCGTT-3' tag SEQ ID 12;
the ACTB reference gene specific primers and probes comprise:
a forward primer: 5 '-GTGATGGAGGAGTTTAGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTTCCGAAACGAAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACCACCCAAACACAATAACAAACACA-3' tag SEQ ID 15.
3. The kit for detecting methylation of breast cancer-related genes according to claim 2, wherein the PCR reaction solution comprises the following components in final concentration: 1 XPCR buffer solution, 0.4-0.6 MuM RASSF1A forward primer, 0.4-0.6 MuM RASSF1A reverse primer, 0.2-0.3 MuM RASSF1A detection probe, 0.4-0.6 MuM APC forward primer, 0.4-0.6 MuM APC reverse primer, 0.2-0.3 MuM APC detection probe, 0.4-0.6 MuM GSTP1 forward primer, 0.4-0.6 MuM GSTP1 reverse primer, 0.2-0.3 MuM GSTP1 detection probe, 0.4-0.6 MuM CDH1 forward primer, 0.4-0.6 MuM CDH1 reverse primer, 0.2-0.3 MuM CDH1 detection probe, 0.1-0.3 MuM MACH forward primer, 0.1-0.3 MuM MACH 3 primer, 0.5 MuM Taq DNA polymerase chain reaction probe, 0.05-0.05 MuM DNA polymerase chain terminator, 0.05-0.05 mM DNA.
4. The kit of claim 2, wherein the positive quality control product is genomic DNA of Hct116 cell line treated with SssI methylase.
5. The kit of claim 2, wherein the negative quality control product is genomic DNA of Hct116 cell line after knocking out genes DNMT1 and DNMT3 b.
6. A detection method for detecting methylation of a breast cancer-associated gene, comprising the steps of:
the method comprises the following steps: extracting DNA of a sample to be detected, and taking the DNA after conversion treatment as a genome DNA sample of a PCR template;
step two: carrying out PCR amplification on a genome DNA sample by using a kit for detecting methylation of breast cancer related genes;
the kit comprises: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A genes, methylation specific primers and probes of target sites of APC genes, methylation specific primers and probes of target sites of GSTP1 genes, methylation specific primers and probes of target sites of CDH1 genes, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
the methylation specific primers and probes of the RASSF1A gene target site comprise:
a forward primer: 5 '-CGGGGTTCGTTTGTGGTTTC-3' SEQ ID 01,
reverse primer: 5 '-CCGATTAAATCGTACTTCGC-3' SEQ ID 02,
detecting a probe: 5 'tag-TCGCGTTTGTAGCGTTTAAAGT-3' tag SEQ ID 03;
the methylation specific primers and probes of the target site of the APC gene comprise:
a forward primer: 5 '-TAGGTATTGAGTTTTTTTTCGGTAA-3' SEQ ID 04,
reverse primer: 5 '-CAAAATCGACCGAACACCG-3' SEQ ID 05,
detecting a probe: 5 'tag-TTTCGGTTTGTTTCGTCGTTGTTCG-3' tag SEQ ID 06;
the methylation specific primers and probes of the GSTP1 gene target site comprise:
a forward primer: 5 '-CGTTGGAGTTTTCGTCGTA-3' SEQ ID 07,
reverse primer: 5 '-GAAAACCCGACCTAATACTACGAAT-3' SEQ ID 08,
detecting a probe: 5 'tag-TCGTTATTATGAGTACGCGCGGTTCGC-3' tag SEQ ID 09;
the methylation specific primers and probes of the CDH1 gene target site comprise:
a forward primer: 5 '-TATTTCGGATTTTTGATTTGCG-3' SEQ ID 10,
reverse primer: 5 '-TAAAAAAAAAAAACGATAACGACGA-3' SEQ ID 11,
detecting a probe: 5 'tag-ACGTATTCGGTCGTAAGTTTCGCGTT-3' tag SEQ ID 12;
the ACTB reference gene specific primers and probes comprise:
a forward primer: 5 '-GTGATGGAGGAGTTTAGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTTCCGAAACGAAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACCACCCAAACACAATAACAAACACA-3' tag SEQ ID 15;
step three: and determining whether the sample to be detected is methylated or not according to the fluorescence Ct values of the RASSF1A, APC, GSTP1, CDH1 and ACTB gene amplification results.
7. The method according to claim 6, wherein in the first step, the reagent used in the conversion treatment is bisulfite or bisulfite.
8. The method according to claim 6, wherein in the second step, the PCR amplification reaction procedure is as follows: 95 ℃ for 10min,45cycles for 95 ℃ for 15sec, 60 ℃ for 30sec (fluorescence collection).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064788A (en) * | 2022-08-01 | 2023-05-05 | 山东大学 | Multiplex gene methylation detection fluorescent quantitative PCR kit for early screening of breast cancer |
| CN116064788B (en) * | 2022-08-01 | 2023-09-08 | 山东大学 | Multiplex gene methylation detection fluorescent quantitative PCR kit for early screening of breast cancer |
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