CN108048570A - For primer pair, kit and the method for colorectal cancer related gene Septin9 DNA methylation assays - 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The present invention relates to a kind of primer pairs for colorectal cancer related gene Septin9 DNA methylation assays.The primer pair includes Septin9 forward primers, reverse primer and detection probe, β Actin forward primers, reverse primer and detection probe.The present invention relates to a kind of kits for colorectal cancer related gene Septin9 DNA methylation assays.The kit includes the PCR reaction solution containing above-mentioned primer and probe, including forward primer, reverse primer, detection probe, 10 × PCR buffer, dNTP, nuclease-free water and Ex Taq enzymes.The invention further relates to a kind of methods for colorectal cancer related gene Septin9 DNA methylation assays.Kit and its detection method provided by the invention have the advantages that testing result is accurate, detection flux is high, specificity is good, sensitivity is good, detection time is quick, easy to use and can effectively meet clinical requirement.
Description
Technical field
The present invention relates to vitro diagnostic techniques fields, particularly, are related to a kind of for colorectal cancer related gene Septin9
Primer pair, kit and the method for DNA methylation assay.
Background technology
Septin9 genes are a kind of potential tumor suppressor genes of discovered in recent years, are to adjust on human chromosomal 2q23
Control one of gene of cell normal growth.Its albumen encoded is located at kytoplasm, is a kind of albumen of high glycosylation, is that p53 is situated between
The downstream signal for the cell cycle regulating led can make abnormal cells arrest in G2 phases, prevention cell Proliferation.Septin9 starts
Sub- abnormal methylation can inhibit to transcribe, and G2/M checkpoint regulations is caused to get muddled, cause abnormal cell proliferation.Septin9
Gene methylation and its missing of expression are related to kinds cancer or tumor cell line, including colorectal cancer, gallbladder cancer, leaching
Bar knurl, colorectal cancer, adenocarcinoma of esophagus, breast cancer and leukaemia, wherein Septin9 gene promoter methylations are in colorectal cancer patients
Middle accounting rate is more than 70%.In addition, the APC, SHP1, E-cadherin, ER, the Septin9 that are found in colorectal cancer patients blood plasma,
In the gene that 7 kinds of high-frequencies such as SEMA3B, and 3OST2 methylate, only Septin9 genes are in the blood of non-colorectal cancer patients
It is to methylate in conspicuousness low frequency in slurry, this illustrates that Septin9 can be as the tumor markers of colorectal cancer early stage.It is another
Aspect, enhancing the expression of Septin9mRNA and albumen can make tumor suppressor gene recapture effect by using demethylating agent, do
The generation for being likely to reduced Septin9 abnormal methylations is disturbed down, and after use in conjunction histon deacetylase (HDAC) inhibitor, is inhibited
The effect of growth of tumour cell is more notable, this provides experimental basis for the treatment of tumour, has important meaning to tumor patient prognosis
Justice.
Colorectal cancer is a kind of malignant tumour of serious threat human health, and incidence is only second to lung cancer and breast cancer, position
Occupy malignant tumour the 3rd.WHO statistics show 5 years survival rates of colorectal cancer patients in north america up to 61%, and I
State only has 32%.Research thinks that 5 years survival rates have apparent correlation with the severity of disease during diagnosis, and progressive stage is suffered from
5 years survival rates of person are less than 10%, and 5 years survival rates of the colorectal cancer patients early diagnosed are then up to 92%.From 2008
To between 2013, colorectal cancer incidence rate and the death rate are in ascendant trend year by year.It is worth noting that in recent years in the world
The morbidity crowd of interior colorectal cancer has the trend of rejuvenation, and China young people (<30 years old) incidence of colorectal cancer is up to
15%, so as to greatly compromise the life security and health of China people.Therefore, it is early to find, early diagnose, early treatment pair
It is of great significance in improving treatment of colorectal cancer effect.
DNA methylation refers to that under the action of dnmt rna the methyl (- CH3) of S-adenosylmethionine is covalently tied
It closes on 5 carbon atoms of cytimidine (C) base of DNA molecular, forms 5-methylcytosine (5mC), and do not change DNA's
Sequence.Numerous researchs show that the occurrence and development of SEPT9 gene promoter methylations and colorectal cancer are closely related, it is knot
The special molecular labeling of the carcinoma of the rectum.Therefore, the methylation state of colorectal cancer SEPT9 gene promoters is detected, to colorectal cancer
Diagnosis, treatment, Index for diagnosis etc. be of great significance.
At present, traditional methylation detecting method includes：Methylation status of PTEN promoter and bisulfite sequencing.However
These methods are low and the shortcomings that sensibility is not high there are cumbersome, accuracy, limit its extensively should in clinical labororatory
With.
The present invention examines patient's plasma free nucleic acid Septin9 gene methylations using fluorescent real time PCR technology
It surveys, after carrying out bisulfite processing to sample, nucleic acid is expanded by specific primer and probe, to reach fast
The purpose of speed detection Septin9 gene methylations, it is final to provide effective information indirectly for Human colorectal carcinoma early diagnosis, it is tied for people
The carcinoma of the rectum early finds that early treatment provides a kind of easy-to-use method.
The content of the invention
In order to solve above-mentioned traditional methylation detecting method, there are the technologies that cumbersome, accuracy are low and sensibility is not high
Problem, the present invention provide that a kind of easy to operate, accuracy is high and sensibility is high and is used for colorectal cancer based on Fluorescence PCR assay
Primer pair, kit and its method of related gene Septin9 DNA methylation assays.
The present invention provides a kind of primer pair for colorectal cancer related gene Septin9 DNA methylation assays, including pin
The specific primer and probe of promoter region and β-Actin reference genes to Septin9 genes, it is as follows：
Septin9 forward primers：5'-AAATAATCCCATCCAACTA-3'(SEQ ID NO.1),
Septin9 reverse primers：5'-GATTYGTTGTTTATTAGTTATTATGT-3'(SEQ ID NO.2),
Septin9 detection probes：5'FAM-TTAACCGCGAAATCCGAC-3'MGB(SEQ ID NO.3)；
β-Actin forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3'(SEQ ID NO.4),
β-Actin reverse primers：5'-CCAAACTATAACCTCTACAACCTTCAAAA-3'(SEQ ID NO.5),
β-Actin detection probes：5'VIC-CCCATTAACTAAACACAACCT-3'MGB(SEQ ID NO.6).
Wherein, Y is to annex base, i.e. Y=C/T.
The present invention also provides a kind of kit for colorectal cancer related gene Septin9 DNA methylation assays, including
PCR reaction solution containing such as above-mentioned primer and probe, the PCR reaction solution include：Septin9 forward primers, Septin9 are reversed
Primer, Septin9 detection probes, β-Actin forward primers, β-Actin reverse primers, β-Actin detection probes, 10 × PCR
Buffer, dNTP and nuclease-free water.
In a kind of preferred embodiment of the kit provided by the invention, the kit further includes Ex Taq enzymes.
In a kind of preferred embodiment of the kit provided by the invention, the ingredient final concentration of the PCR reaction solution
For：1 × PCR buffer, 0.4 μM of Septin9 forward primer, 0.4 μM of Septin9 reverse primer, 0.4 μM of Septin9 inspection
Probing pin, 0.4 μM of β-Actin forward primer, 0.4 μM of β-Actin reverse primer, 0.4 μM of β-Actin detection probe, 0.25mM
dNTP。
In a kind of preferred embodiment of the kit provided by the invention, the kit further includes positive reference substance
And negative controls, the positive reference substance methylate standard items DNA for Septin9, the negative controls methylate to be non-
Standard items DNA.
The wherein described standard items DNA that methylates is handled for normal human peripheral blood's genomic DNA through I methylases of Sss, can be made
C in all CG sequences of genome methylates on C5 positions；The non-standard items DNA that methylates is normal human peripheral blood's gene
Group DNA.
The present invention also provides a kind of method for colorectal cancer related gene Septin9 DNA methylation assays, including such as
Lower step：
Step 1：The DNA of sample extracting to be detected is taken, conversion processing is carried out to it, moulds of the DNA after conversion as PCR
Plate；
Step 2：The above-mentioned kit for colorectal cancer related gene Septin9 DNA methylation assays is provided, to the mould
Plate carries out PCR amplification；
Step 3：It is to be detected to determine according to the relative fluorescence CT values of Septin9 and β-Actin gene PCR amplifications
The methyl rate of sample, i.e., Septin9/ β in the ratio divided by positive reference substance of Septin9/ β-Actin in sample to be detected-
The ratio of Actin.
In a kind of preferred embodiment of the method provided by the invention, in the step 1 used by conversion processing
Reagent is bisulfites or bisulfite.
In a kind of preferred embodiment of the method provided by the invention, pcr amplification reaction program in the step 2
For：95 DEG C of denaturation 10min；50cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.
Compared to the prior art, provided by the present invention for drawing for colorectal cancer related gene Septin9 DNA methylation assays
Object is the advantageous effect of, kit and method：
First, the primer and probe high by designing specificity, and it is configured to the reliable reagent of easy to use and testing result
Box, the rational PCR reaction systems of the science of redesigning out so that the present invention has quick, high-throughput, sensitive and specific good spy
Point realizes quick to the methylation of colorectal cancer Septin9 genes and accurate measurement, to be carried out indirectly to colorectal cancer
In time, effective diagnose and treat reduces medical treatment cost, saves social resources.
2nd, the quality of sample is controlled using gene β-actin as reference gene, it is contemplated that house-keeping gene may
Situation about methylating selects the position on no CpG islands when designing internal control primer probe, is set for the sequence after its vulcanization
Meter ensures Quality Control of the house-keeping gene to sample.
3rd, after by carrying out bisulfite processing to sample, specific primer and probe expand nucleic acid, measure
With the maximally related CpG sites of colorectal cancer tumour in the promoter region of Septin9 genes, methylating for target gene is judged, and
Standard curve prepared by the positive sample and negative sample determined according to the clinic of synchronization process, preliminary judgement sample methylate
Degree, evaluation colorectal cancer patients are in different times Septin9 gene methylation degree；And the internal reference base in sample is measured simultaneously
Because of β-actin, can not only quality control be carried out to sample, but also the methylation level of sample can be evaluated to a certain extent.
The method can reach the purpose of quick detection Septin9 gene methylations, final to be provided indirectly for Human colorectal carcinoma early diagnosis
Effective information early finds that early treatment provides a kind of easy-to-use method for Human colorectal carcinoma.
Description of the drawings
Fig. 1 is methylation positive sample amplification curve diagram；
Fig. 2 is the negative sample amplification curve diagram that methylates；
Fig. 3 is the gDNA amplification curve diagrams without sulfiting.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, it is right
The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only to explain the present invention, and
It is not used in the restriction present invention.
Embodiment 1：The preparation of kit
First, the design and synthesis of primer and probe
For the promoter region of Septin9 genes in human genome and reference gene β-Actin (b-actin), (sequence is joined
See mankind's whole genome sequence disclosed in ncbi database), use Primer Premier 3.0 and Methyl Primer
Express v1.0 softwares, separately design a pair of of specific primer and probe.
Specific primer and probe sequence, it is as shown in the table：
Remarks：Y is to annex base, i.e. Y=C/T.
2nd, reference substance selects
Using Septin9 methylate standard items DNA for normal human peripheral blood's genomic DNA through I methylases of Sss handle,
The C in all CG sequences of genome can be made to methylate on C5 positions；The non-standard items DNA that methylates is normal human peripheral blood
Genomic DNA.
3rd, PCR reaction solution forms
PCR reaction solution including containing above-mentioned specific primer and probe, the PCR reaction solution include：
Septin9 forward primers：5'-AAATAATCCCATCCAACTA-3',
Septin9 reverse primers：5'-GATTYGTTGTTTATTAGTTATTATGT-3',
Septin9 detection probes：5'FAM-TTAACCGCGAAATCCGAC-3'MGB,
β-Actin forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3',
β-Actin reverse primers：5'-CCAAACTATAACCTCTACAACCTTCAAAA-3',
β-Actin detection probes：5'VIC-CCCATTAACTAAACACAACCT-3'MGB；
Remarks：Y is to annex base, i.e. Y=C/T；
10 × PCR buffer,
DNTP and nuclease-free water.
Wherein, 10 × PCR buffer, dNTP and nuclease-free water are purchased from Dalian precious biology (precious biology (Dalian)
Engineering Co., Ltd)；The nuclease-free water uses DEPC (Diethyl for (Nuclease-Free Water)
Pyrocarbonate, pyrocarbonic acid diethyl ester) the treated and ultra-pure water through autoclave sterilization.
The PCR reaction solution ingredient it is final concentration of：
1 × PCR buffer,
0.4 μM of (μm ol/L) Septin9 forward primer,
0.4 μM of Septin9 reverse primer,
0.4 μM of Septin9 detection probe；
0.4 μM of β-Actin forward primer,
0.4 μM of β-Actin reverse primer,
0.4 μM of β-Actin detection probe；
0.25mM(mmol/L)dNTP。
The kit further includes Ex Taq enzymes, purchased from the precious biology in Dalian (precious biology (Dalian) Engineering Co., Ltd)；Institute
The activity for stating Ex Taq enzymes remains more preferable, and longer segment can be effectively expanded compared with general T aq.
Embodiment 2：The method that colorectal cancer DNA methylation assay is carried out using mentioned reagent box
First, technical principle
The promoter region of Septin9 genes and reference gene β-Actin (b-actin) separately design one in human genome
To the primer and probe of the DNA methylation assay of specificity.Then sample of the amplification through sulphite conversion is removed with the primer and probe
DNA determines methylating for sample to be tested according to the relative fluorescence CT values of Septin9 and β-Actin gene PCR amplifications
Rate judges the risk of Colon and rectum canceration indirectly according to methyl rate.
2nd, detection method
Step 1：The DNA of sample extracting to be detected is taken, conversion processing is carried out to it, moulds of the DNA after conversion as PCR
Plate；
Wherein, reagent is bisulfites or bisulfite and other auxiliary (corresponding reagents used by conversion processing
Box is the EpiTect Fast DNA BisuLfite Kit purchased from German QIAGEN companies).
Step 2：The above-mentioned kit for colorectal cancer related gene Septin9 DNA methylation assays is provided, to the mould
Plate carries out PCR amplification；
Wherein, pcr amplification reaction program is：95 DEG C of denaturation 10min；50cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.
Step 3：It is to be detected to determine according to the relative fluorescence CT values of Septin9 and β-Actin gene PCR amplifications
The methyl rate of sample, i.e., Septin9/ β in the ratio divided by positive reference substance of Septin9/ β-Actin in sample to be detected-
The ratio of Actin.
Specific detection method is as follows：
One) biological sample is collected：
Suffer from selected from 30 Human colorectal carcinomas in the refined hospital admission in Hunan Province Hunan during in June, -2017 in October, 2016
Person's peripheral blood and 30 normal person's DNA nucleic acid.
Two) tissue DNA is extracted：
(section of formalin paraffin-embedded tissue extraction step 1-27, nonneoplastic lesion sample extraction step 7-27.)
1) paraffin section is taken, instills a little dimethylbenzene, moistens the paraffin section containing tissue, undertissue is scraped with scalpel, is turned
It moves on in 1.5mL centrifuge tubes；Can with a little dimethylbenzene will on scalpel and paraffin section on tissue be flushed to repeatedly 1.5mL from
In heart pipe；
2) after sample being transferred to 1.5mL centrifuge tubes, 1mL dimethylbenzene is added in, 1min is acutely vibrated, makes paraffin completely molten
Solution；
3) centrifuge tube containing sample is put into 15000g on centrifuge and centrifuges 2min, carefully abandon the dimethylbenzene suction in pipe,
It is careful not to be drawn onto tissue；
4) 1mL absolute ethyl alcohols are added in into the centrifuge tube containing sample, 15000g centrifuges 2min after vortex oscillation 30s, small
Ethyl alcohol is abandoned in heart suction, not be drawn onto tissue；
5) 1mL absolute ethyl alcohols are added in again into the centrifuge tube containing sample, and 15000g is centrifuged after vortex oscillation 30s
2min, careful inhale abandon ethyl alcohol, not be drawn onto tissue；
6) 5-10min is placed at room temperature for, ethyl alcohol is made to volatilize；
7) 200 μ L Lysis Buffer is taken to be added in sample；
8) 10000g centrifuges 15s, and solution is divided into two layers, is at the middle and upper levels oily liquids, lower floor is blue liquid；
9) plus 20 μ L Proteinase Ks are to lower floor's liquid, and with liquid-transfering gun mixing；
10) when 56 DEG C of heating 1 are small；
11) when 80 DEG C of heating 4 are small；
12) sample is cooled down in room temperature, brief centrifugation makes liquid fall into tube bottom after cooling；
13) plus 10 μ L RNase are to cytolysate liquid bottom, and with pipettor mixing；
14) (20-25 DEG C) placement 5min of room temperature；
15) plus 220 μ L BL Buffer are in cytolysate；
16) plus 240 μ L absolute ethyl alcohols are in pipe, vortex mixing；
17) 10000g centrifuges 15s, and solution is divided into two layers, is at the middle and upper levels oily liquids, lower floor is blue liquid；
18) each sample prepares a set of column and collecting pipe, and blue liquid in cytolysate is transferred completely into knot
In zygostyle, including some sediments that may be present, the lid of column is covered, oil reservoir in liquid will be combined and removed；
19) 10000g centrifuges 30s, abandons the waste liquid in collecting pipe, column is reinserted in collecting pipe；
20) 500 μ 1 × Wash of L solution (plus ethyl alcohol) are added in combining in pillar, cover lid；
21) 10000g centrifuges 30s, abandons the waste liquid in collecting pipe, column is reinserted in collecting pipe；
22) open and combine pillar lid, 16000g centrifuges 3min together with collecting pipe by column, makes liquid in pillar thorough
Bottom is by from getting off；
23) column is inserted into the centrifuge tube of clean 1.5mL, abandons collecting pipe；
24) plus 30-50Elution buffer are into column, cover the lid of column；
25) 16000g centrifuges 1min, abandons column；
26) centrifuge tube lid is covered, obtains DNA solution, in -30 DEG C to -10 DEG C preservations.
27) with the concentration of micro UV spectrophotometer measuring DNA, it is desirable that its absorbance (A) ratio (A260/A280)
About 1.80.
Three) DNA conversion process：
1) by the DNA configuration sulphite transformation system configurations of extraction：
| Ingredient | Reaction volume (μ L) |
| The DNA to be measured of extraction | 10μL |
| Ultra-pure water (RNAse-free Water) | 10μL |
| Solution of sodium bisulfite (BisuLfite solution) | 85μL |
| DNA protect Buffer | 35μL |
| It amounts to | 140μL |
2) operation architecture of sulphite conversion：
| Reaction temperature | Reaction time |
| 95℃ | 5min |
| 60℃ | 10min |
| 95℃ | 5min |
| 60℃ | 10min |
| 20℃ | ∞ |
3) DNA is purified after sulphite conversion：
DNA purifying agent formulations used are selected from German QIAGEN companies after being converted below to sulphite
EpiTect Fast DNA sodium hydrogensulfite kits.
A) product after sulfurous acid is converted is transferred in the EP pipes of 1.5ml；
B) being less than 100ng/ μ L DNA need to add in 1 μ L Carrier RNA to Buffer BL, if concentration is more than 100ng/ μ L
Carrier RNA need not then be added；
C) 310 μ L Buffer BL mixings are added；
D) 250 μ L absolute ethyl alcohols, mixing 15sec are added again；
E) above mixture is transferred to Filter column, 10000rpm centrifugations 1min；
F) filtrate is abandoned, adds 500 μ L Buffer BW, 10000rpm centrifugations 1min；
G) filtrate is abandoned, adds the static 15min of 500 μ L Buffer BD, 10000rpm centrifugations 1min；
H) filtrate is abandoned, adds 500 μ L Buffer BW, 10000rpm centrifugations 1min；
I) step h) is repeated；
J) filtrate is abandoned, adds 250 μ L absolute ethyl alcohols, 10000rpm centrifugations 1min；
K) filtrate, then 12000rpm idle running 1min are abandoned；
L) plus 15 μ L Buffer EB room temperatures static 1min, 10000rpm centrifuge 1min；
M) DNA of collection is stored in -20 DEG C.
Four) PCR flows
1st, the PCR instrument used (is purchased from Life Tech for 7500 type real-time fluorescence quantitative PCR systems of American AB I
(applied biosystems companies), reaction system are 20 μ L；
2nd, the preparation of PCR reaction systems and condition, it is as shown in the table：
PCR reaction systems：
| Ingredient | Reaction volume (μ L) |
| PCR reaction solution | 18.3μL |
| Ex Taq enzymes | 0.2μL |
| DNA profiling after sulphite conversion | 1.5μL |
PCR response procedures：
Five) interpretation of result
With in the ratio of Septin9/ β-Actin in sample to be tested divided by positive control (methylate standard items DNA)
The ratio of Septin9/ β-Actin methylates ratio to calculate Septin9.
Methyl rate calculation formula is borrows the methyl rate that the formula of relative quantification calculates sample to be detected, and formula is such as
Shown in lower.
2- △ △ CT=2Sample to be tested (Septin9CT- β-Actin CT)-permethylated sample (Septin9CT- β-Actin CT)
Kit testing result meets Quality Control requirement, and sample is judged according to testing result.Pattern detection result is
Septin9 gene C t values≤35, β-actin≤33, then judge sample for Septin9 methylation positives；Pattern detection result is
Septin9 gene C t values ＞ 35, β-actin≤33；Then sentence sample to methylate feminine gender for Septin9, β-actin ＞ 33, then PCR
React invalid.
Six) colon cancer tissue and the distribution of distal end normal structure Septin9 gene methylations
Septin9 gene methylations do not detect in 30 normal populations, are detected in 33 colorectal cancer peripheral bloods
30 (account for 90.9%, the P compared with normal group<0.001), the ratio that methylates of Septin9 genes is with the colorectal cancer course of disease
It aggravates and raises, it is as shown in the table：
The Septin9 gene methylation detection statistics of colon cancer tissue and distal end normal structure
Based on specific experiment data of the invention referring to Fig. 1-Fig. 3, wherein, Fig. 1 is methylation positive sample amplification curve
Figure；Fig. 2 is the negative sample amplification curve diagram that methylates；Fig. 3 is the gDNA amplification curve diagrams without sulfiting.It can draw
Difference methylates the amplification curve relation of sample Septin9 gene magnifications curve and β-Actin house-keeping genes, if Septin9
For gene with β-Actin house-keeping genes without amplification curve such as Fig. 3, detection or resampling need to be repeated by representing that sample is unqualified.
Provided by the present invention for primer pair, kit and the side of colorectal cancer related gene Septin9 DNA methylation assays
The advantageous effect of method is：
First, the primer and probe high by designing specificity, and it is configured to the reliable reagent of easy to use and testing result
Box, the rational PCR reaction systems of the science of redesigning out so that the present invention has quick, high-throughput, sensitive and specific good spy
Point realizes quick to the methylation of colorectal cancer Septin9 genes and accurate measurement, to be carried out indirectly to colorectal cancer
In time, effective diagnose and treat reduces medical treatment cost, saves social resources.
2nd, the quality of sample is controlled using gene β-actin as reference gene, it is contemplated that house-keeping gene may
Situation about methylating selects the position on no CpG islands when designing internal control primer probe, is set for the sequence after its vulcanization
Meter ensures Quality Control of the house-keeping gene to sample.
3rd, after by carrying out bisulfite processing to sample, specific primer and probe expand nucleic acid, measure
With the maximally related CpG sites of colorectal cancer tumour in the promoter region of Septin9 genes, methylating for target gene is judged, and
Standard curve prepared by the positive sample and negative sample determined according to the clinic of synchronization process, preliminary judgement sample methylate
Degree, evaluation colorectal cancer patients are in different times Septin9 gene methylation degree；And the internal reference base in sample is measured simultaneously
Because of β-actin, can not only quality control be carried out to sample, but also the methylation level of sample can be evaluated to a certain extent.
The method can reach the purpose of quick detection Septin9 gene methylations, final to be provided indirectly for Human colorectal carcinoma early diagnosis
Effective information early finds that early treatment provides a kind of easy-to-use method for Human colorectal carcinoma.
The foregoing is merely the embodiment of the present invention, are not intended to limit the scope of the invention, every to utilize this hair
The equivalent process transformation that bright description is made directly or indirectly is used in other relevant technical fields, similarly wraps
It includes in the scope of patent protection of the present invention.
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<213>Artificial sequence (Artificial Sequence)
<400> 6
cccattaact aaacacaacc t 21
Claims (8)
1. a kind of primer pair for colorectal cancer related gene Septin9 DNA methylation assays, which is characterized in that including being directed to
The promoter region of Septin9 genes and the specific primer and probe of β-Actin reference genes, it is as follows：
Septin9 forward primers：5'-AAATAATCCCATCCAACTA-3',
Septin9 reverse primers：5'-GATTYGTTGTTTATTAGTTATTATGT-3',
Septin9 detection probes：5'FAM-TTAACCGCGAAATCCGAC-3'MGB；
β-Actin forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3',
β-Actin reverse primers：5'-CCAAACTATAACCTCTACAACCTTCAAAA-3',
β-Actin detection probes：5'VIC-CCCATTAACTAAACACAACCT-3'MGB.
2. a kind of kit for colorectal cancer related gene Septin9 DNA methylation assays, which is characterized in that including containing such as
The PCR reaction solution of specific primer and probe described in claim 1, the PCR reaction solution include：Septin9 forward primers,
Septin9 reverse primers, Septin9 detection probes, β-Actin forward primers, β-Actin reverse primers, β-Actin detections are visited
Pin, 10 × PCR buffer, dNTP and nuclease-free water.
3. the kit according to claim 2 for colorectal cancer related gene Septin9 DNA methylation assays, feature
It is, the kit further includes Ex Taq enzymes.
4. the kit according to claim 2 for colorectal cancer related gene Septin9 DNA methylation assays, feature
It is, the ingredient of the PCR reaction solution is final concentration of：1 × PCR buffer, 0.4 μM of Septin9 forward primer, 0.4 μM
Septin9 reverse primers, 0.4 μM of Septin9 detection probe, 0.4 μM of β-Actin forward primer, 0.4 μM of β-Actin reversely draw
Object, 0.4 μM of β-Actin detection probe, 0.25mM dNTP.
5. according to any reagent for colorectal cancer related gene Septin9 DNA methylation assays in claim 2-4
Box, which is characterized in that further include positive reference substance and negative controls, the positive reference substance methylates standard for Septin9
Product DNA, the negative controls are the non-standard items DNA that methylates.
A kind of 6. method for colorectal cancer related gene Septin9 DNA methylation assays, which is characterized in that including walking as follows
Suddenly：
Step 1：The DNA of sample extracting to be detected is taken, conversion processing is carried out to it, templates of the DNA after conversion as PCR；
Step 2：Kit as claimed in claim 5 is provided, PCR amplification is carried out to the template；
Step 3：Sample to be detected is determined according to the relative fluorescence CT values of Septin9 and β-Actin gene PCR amplifications
Methyl rate, i.e., Septin9/ β-Actin in the ratio divided by positive reference substance of Septin9/ β-Actin in sample to be detected
Ratio.
7. the method according to claim 6 for colorectal cancer related gene Septin9 DNA methylation assays, feature exists
In reagent is bisulfites or bisulfite used by conversion processing in the step 1.
8. the method according to claim 7 for colorectal cancer related gene Septin9 DNA methylation assays, feature exists
In pcr amplification reaction program is in the step 2：95 DEG C of denaturation 10min；50cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.
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| CN108424968A (en) * | 2018-06-01 | 2018-08-21 | 深圳市太科健康科技有限公司 | The DNA methylation marker of colorectal cancer and the method and kit for utilizing its detection colorectal cancer |
| CN109055552A (en) * | 2018-08-23 | 2018-12-21 | 北京迈基诺基因科技股份有限公司 | The method and its special complete reagent whether detection Septin9 gene promoter methylates |
| CN109811056A (en) * | 2019-02-28 | 2019-05-28 | 苏州唯善生物科技有限公司 | For colorectal cancer and its primed probe group and kit of precancerous lesion early diagnosis, detection or screening |
| CN110846418A (en) * | 2019-12-16 | 2020-02-28 | 苏州璞瑞卓越生物科技有限公司 | Primer group, probe group, kit and application for methylation detection of colorectal cancer Septin9 gene |
| CN111647655A (en) * | 2020-04-01 | 2020-09-11 | 南京普派医疗科技有限公司 | Kit for detecting colorectal cancer by DNA methylation of multi-target feces and detection method thereof |
| CN112501297A (en) * | 2020-12-03 | 2021-03-16 | 广东辉锦创兴生物医学科技有限公司 | Fluorescent quantitative PCR detection kit for detecting methylation of Septin9 gene and application thereof |
| CN113215258A (en) * | 2021-06-04 | 2021-08-06 | 杭州圣庭医疗科技有限公司 | Nucleic acid composition, kit and detection method for detecting methylation of colorectal cancer related genes |
| CN114085904A (en) * | 2020-08-24 | 2022-02-25 | 广州达健生物科技有限公司 | Colorectal cancer gene methylation detection primer probe combination, kit and application thereof |
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| CN110846418A (en) * | 2019-12-16 | 2020-02-28 | 苏州璞瑞卓越生物科技有限公司 | Primer group, probe group, kit and application for methylation detection of colorectal cancer Septin9 gene |
| CN111647655A (en) * | 2020-04-01 | 2020-09-11 | 南京普派医疗科技有限公司 | Kit for detecting colorectal cancer by DNA methylation of multi-target feces and detection method thereof |
| CN114085904A (en) * | 2020-08-24 | 2022-02-25 | 广州达健生物科技有限公司 | Colorectal cancer gene methylation detection primer probe combination, kit and application thereof |
| CN114085904B (en) * | 2020-08-24 | 2024-02-23 | 广州达健生物科技有限公司 | Colorectal cancer gene methylation detection primer probe combination, kit and application thereof |
| CN112501297A (en) * | 2020-12-03 | 2021-03-16 | 广东辉锦创兴生物医学科技有限公司 | Fluorescent quantitative PCR detection kit for detecting methylation of Septin9 gene and application thereof |
| CN113215258A (en) * | 2021-06-04 | 2021-08-06 | 杭州圣庭医疗科技有限公司 | Nucleic acid composition, kit and detection method for detecting methylation of colorectal cancer related genes |
| CN114561452A (en) * | 2022-03-10 | 2022-05-31 | 天津市人民医院 | Application of mSEPT9 as marker for predicting postoperative recurrence risk of colorectal cancer patient and for evaluating effectiveness of chemotherapy regimen |
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