CN105331727B - The detection kit of 9 gene methylation of septin in a kind of human peripheral Circulating tumor DNA - Google Patents
The detection kit of 9 gene methylation of septin in a kind of human peripheral Circulating tumor DNA Download PDFInfo
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- CN105331727B CN105331727B CN201510864133.8A CN201510864133A CN105331727B CN 105331727 B CN105331727 B CN 105331727B CN 201510864133 A CN201510864133 A CN 201510864133A CN 105331727 B CN105331727 B CN 105331727B
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of detection kit of 9 gene methylation of septin in human peripheral Circulating tumor DNA, including: the methylation specific primer in septin9 gene purpose site；The dideoxycytidine Mdification primer of the non-methylation specific in septin9 gene purpose site；The hydrolysis probes of septin9 gene specific；The primer of reference gene and the hydrolysis probes of reference gene；At least one site CpG that the septin9 gene purpose site includes by 1 exon 1 septin9 gene V2 transcript γ；The reference gene is one or more of GAPDH, β-actin, B2M, SDHA, HPRT1.The component of kit of the present invention is arranged and its result interpretation scheme makes detection process easy, strong operability.Testing result is reliable, specificity is good.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to 9 gene of septin in a kind of human peripheral Circulating tumor DNA
The detection kit of methylation.
Background technique
Colorectal cancer is worldwide to lead to one of the major disease of human death.Cancer in Europe and the U.S. is new
Colorectal cancer comes second and third respectively in morbidity, and European colorectal cancer new cases in 2012 are about 447000 people, dead
Case is about 215000 people, and U.S.'s new cases in 2013 are about 142820 people, and death is about 50830 people and in China,
Colorectal cancer is about 400000 people in new cases in 2012, it has also become Chinese's disease incidence and the high disease of death rate third
Disease.
The early detection of colorectal cancer can be realized by routine screening, and then carries out early treatment to prevent disease progression very
To healing.But status is that the colorectal cancer patients of 60%-70% are just to be diagnosed discovery in mid-term or advanced stage, only close
40% patient is discovered in an early phase.From USPSTF (US Preventive Services Task Forceodic)
Data show and can avoid nearly 60% because dead caused by colorectal cancer by regularly screening, and 5 years of colorectal cancer patients
Survival rate also will be promoted to 73% by 46%.Therefore a kind of effective colorectal cancer methods for screening will improve 5 years of patient
Survival rate reduces the death rate.
Mainly there are fecal occult blood experiment (FOBT [guaiac method (gFOBT) and immuno-chemical method (iFOBT)]), knot straight at present
Two kinds of Screening Method for Colorectal Cancer of colonoscopy.As a kind of conventional screening method, guaiac method fecal occult blood tests (gFOBT) meeting
Because of diet, the influence of drug and other factors and lead to false positive, thus influence detection stability.Immuno-chemical method fecal occult blood
Test (iFOBT) on sensibility and specificity than traditional guaiac method fecal occult blood experiment (gFOBT) performance it is superior, but
IFOBT that most of clinical labororatories carry out or qualitatively and if sample from collecting between detection more than 5 days, examine
Extracting rate will substantially reduce.In contrast, Colon and rectum mirror is a kind of detection of intrusive mood and needs corresponding enteron aisle before detection
It is prepared to ensure that the visual field of the intracavitary satisfaction of large intestine.It is it is also possible to cause such as hemorrhage of digestive tract, enterobrosis and infection complication.
And with diseases such as adverse cardiac, cardio-pulmonary function obstacle, acute diarrhea, severe ulcerative colitis, Crohn disease, peritonitis
The patient and pregnant woman of disease not can be carried out colonoscopy.Therefore whether all there is patient in fecal occult blood experiment or Colon and rectum mirror
The bad problem of compliance.The screening rate that fecal occult blood is tested in the U.S. is only 10%, 50 years old or more 2008 crowd's Colon and rectum mirror
Screening rate is 50.2%.And in China, fecal occult blood experiment and the screening rate of Colon and rectum mirror are estimated less than 5% and 3% respectively.Due to
There are inefficiencies or invasive for the Screening Method for Colorectal Cancer of tradition hair, thus be now badly in need of it is a kind of more convenient, it is more accurate
Screening method improves the screening rate of colorectal cancer.
Recently, the colorectal cancer detection-SEPT9 gene methylation detection based on blood is expected to be applied to Clinical screening.It is more
The SEPT9 gene of a clinical test discovery methylation is the special biomarker of colorectal cancer early detection.In colorectal cancer
In early days, SEPT9 gene is discharged into peripheral blood by supermethylation, DNA by the tumour cell of necrosis or apoptosis.Detection can be passed through
The degree of SEPT9 gene methylation judges its risk for suffering from colorectal cancer in the peripheral blood of subject.Circulating DNA is found in
In human peripheral's serum, and its content in a variety of diseases such as rheumatism, tumour can sharply increase.The DNA of methylation is equally
It is found to be present in human serum.Lofton-Day and its colleague are in the blood sample of colorectal cancer patients and normal healthy controls crowd
Test 3 DNA methylation biomarkers.SEPT9 is proved to be the best candidates of specificity.Then, multiple cases pair
Confirm that SEPT9 can be used as the methylation biomarker of colorectal cancer detection according to research.The SEPT9 in these case-control studies
The sensibility and specificity of detection is respectively 67-79.3% and 86-99%.It is a kind of essence that these researchs, which demonstrate SEPT9 detection,
Really, properly, quick and minimally invasive colorectal cancer detection method.
DNA methylation is the biochemical process that a methyl group is added on cytimidine or adenine, it is in cell
By stem cell division, be divided into specific tissue when change the transcript and expression of gene.Gene methylation result in cell is usual
Be it is permanent and unidirectional, to prevent cell from reverting to stem cell or be changed into other types tissue.The methylation of DNA usually by
Essence it is oval at when fallen by place to go to reach the epigenetics silencing during reproduction cell reprogramming and then keep genome complete
Whole property, and when cell continuously divides in growth course, establishes again.The reconstruction of this DNA methylation is one kind independently of warp
The genetic process of the mendel's law of allusion quotation expresses that specific gene in the special mode in close source.This process is referred to as
Genomic imprinting.Other than genomic imprinting, a lot of other genetic processes in DNA methylation or mammal normal development
Important component, such as: x chromosome inactivation, inhibition of repeat element etc..
DNA methylation occurs mainly in the site CpG.The site CpG of 60%-90% is in methylation state in mammal
's.The site CpG of the presence of the unmethylated frequent cluster in the site CpG, these clusters is referred to as the island CpG.And the island CpG is then often deposited
It is very polygenic 5 ' control region, especially promoter region.In cancer patient, the gene of promoter region high methylation
Transcription is silenced, and building up for DNA methylation also will lead to long-term gene silencing.DNA methylation is inhibited by two ways
The transcription of gene.The first, the combination of certain gene transcription factor may be blocked by DNA methylation.Second, the DNA of methylation
It may be combined with methyl-CpG- binding domain protein (MBDs) to recruit other albumen to its site, such as DNA methylase inhibitor
Enzyme and other chromatin remodeling proteins form the chromatin (heterochromatin) of fine and close inactivation.
DNA methylation is the important elements of cancer development, it is related to numerous malignant tumours.There is low-methoxyls in cancer
Two kinds of forms of base and supermethylation, they play different roles in cancer mechanism.Abnormal supermethylation warp
Often occur on the island CpG of the promoter region of gene, it can lead to the Transcriptional Silencing or inactivation of tumor suppressor gene.On the contrary, low-methoxyl base
It is then fixed and to lose genetic imprinting related to chromosome instability to change.DNA methylation has embryonic development and somatic cell division important
Meaning because methylation state substantially can high-fidelity pass to daughter cell.
Septin family is one group of highly conserved gtp binding protein in eucaryote.They are some skelemins,
Play the role of structural support in cell division and separating.13 kinds of septin genes are shared in the mankind.All septin
Family member can form heteromeric complexes and be further formed higher structure, such as: fibril, caged compound.These are unique
Structure be in cell division cellular processes control required for.Some researches show that in the mankind, septin albumen can be with recently
Cage structure is formed around pathogenetic bacteria to fix harmful microorganism, prevent them from invading other cells.
The SEPT9 assignment of genes gene mapping is in the position chromosome 17q25.3.The gene has 18 kinds of transcripts, encodes 15 polypeptides.Its
Actin dynamic, angiogenesis, cell movement, cell Proliferation, cell shape, cytokinesis, micro-pipe regulation, exocytosis etc.
Play important role in physiological activity.Recently studies have pointed out that the gene and breast cancer, colorectal cancer, oophoroma, neck
Cancer, leukaemia and lymthoma etc. are closely related, it is also possible to Down's syndrome, inherited neurological algodystrophy and
The pathogenic factor of bacterium infection.It is lethal that the gene is knocked out in mice embryonic.
Each member of Septin family is present in the stable different aggressiveness of 6-8 subunit core, wraps in the different aggressiveness of the type
Containing SEPT2, SEPT6 and SEPT7 subunit.It also include SEPT9 in the different aggressiveness be made of 6 or more subunits.There is research to refer to recently
SEPT9 occupies the end of eight dimeric complexes out, and key effect is played in the polymerization of subunit, and then keeps different aggressiveness more steady
It is fixed.It is also critically important to the separation of daughter cell in endochylema movement.Therefore, endochylema movement may because SEPT9 unconventionality expression or
It does not express and is severely impacted.When SEPT9 gene is transcribed by supermethylation to be suppressed, in septin compound
Important function forfeiture may be colorectal cancer mechanism key factor.
Effect of the SEPT9 in elaboration of tumour mechanism is found in for the first time in Combination lymthoma, research shows that Combination is drenched
The proto-oncogene of bar tumor is SEPT9 to be merged with other genes.Breast cancer, the heterozygosis of oophoroma lose the normal areas in phenomenon
In also have SEPT9 gene, this illustrates that the gene is a possible tumor suppressor gene.The SEPT9 in some tumor cell lines
V4 transcript expression deletion or decrease, and can by 5-azacitidine handle regain activity, this discovery is also demonstrate,proved
Real above-mentioned viewpoint.Further, the DNA supermethylation of the promoter region of SEPT9_V2 transcript and the expression of the gene
The generation for leading to colorectal cancer is lowered, also illustrates that the gene is a tumor suppressor gene.People are in Colon and rectum biopsy, laser
Find the mrna expression amount of SEPT9 gene in the progress from adenoma to dysplasia to cancer in micro-dissections epithelial cell and matrix
Gradually decline in the process, and compared with normal control, the expression in colorectal cancer is significantly lowered.The supermethylation of SEPT9
Show that the expression of SEPT9 lowers and may can explain colorectal cancer with the significant correlation for losing its mRNA expression in colorectal cancer
From benign to the pathogenesis of malignant change.But, there are also research discoveries in breast cancer, prostate cancer, oophoroma and liver
The certain hypotypes of SEPT9 are to be overexpressed in cancerous tissue.With in colorectal cancer on the contrary, these discoveries show certain specific SEPT9
Transcript is oncogene.For example, there are the expression of a variety of SEPT9 gene hypotypes in oophoroma, and variant hypotype
Tissue specificity is presented in expression.The change of these Action Specifications completely contradicted methylation state of DNA in different tumours of SEPT9
Change may be a kind of common phenomenon, and DNA methylation is the intrinsic mechanism for adjusting different SEPT9 subtype expressions.
Recent research indicate that the main methylation state of SEPT9_V2 transcript changes in Colon and rectum adenoma and cancerous tissue
It is only limitted to occur on the 3rd this island CpG of the island CpG (CGI3).The region include SEPT9_V2 transcript promoter region and
ATG initiation codon.The region shows different methylation status in adenoma and colorectal cancer: the methyl in colorectal cancer
Change is that 5 ' ends are extended to from the core space of CGI3, and in adenoma, the 5 ' ends of CGI3 do not methylate, this may illustrate methyl
It is the late incident in colorectal cancer process that the expansion for changing range, which is by adenoma development,.The methylation shape of this CGI3 nucleus
The difference of formula may represent the conversion of methylation status, and reaction finishes the cellular processes of mucous membrane of rectum deterioration.The region CGI3
Supermethylation may inhibit the normal expression of SEPT9_V2 transcript, and then upset the formation of structure fibril and the cell function of key
Energy.
In Apoptosis, necrosis and breeding, intracellular DNA, which can be discharged or be secreted, becomes trip in the blood circulation of island
From Circulating DNA (cfcDNA).Its content, which is found in the serum of cancer patient, to be increased.CfcDNA makees in measurement serum
For tumor markers trial all because the unstability of cfcDNA concentration abnormal in serum and its content vulnerable to wound, inflammation,
The influence of many factors such as apoplexy, even strenuous exercise and fail.Until the mutation of tomour specific in cfcDNA is found,
Early diagnosis of tumor based on mutation analysis is just possibly realized.CfcDNA abrupt climatic change is become a real diagnostic products to deposit
In some obstacles, such as: in the low content of the early stage mutant nucleotide sequence of disease development；It can not be through the position of abrupt climatic change instruction tumour
And the technical problem of abrupt climatic change itself.But, the base mutation of tomour specific is not unique sequence that cfcDNA is carried
Information.The DNA methylation of epigenetics information, such as certain segments rich in GC sequence also can be from the detection and analysis of cfcDNA
It obtains.
A kind of cfcDNA methylation signature to become a kind of disorder in screening or diagnostic products need to meet several conditions, first
First, which will be continual and steady enough and can be detected.Second, which should have
Unique, stable methylation patterns, in order to detect and be different from the relevant methylation of other diseases.Third, the label are sensitive
Property and specificity must be optimized to the methylation state of the methylation state and normal tissue that can distinguish tumour.4th, the label
The disturbing factors such as level is infected, pregnancy, other tumours and anti-inflammation drugs influence.Finally, should have can for test itself
Operability, analyticity, simply with it is convenient the features such as.
The peripheral blood dissociative DNA in colorectal cancer early detection is confirmed in multiple case-control studies in past 5 years
SEPT9 gene methylation detected representation goes out stable hypersensitivity (67-79.3%) and high specific (86-99%), thus may be used
See, the SEPT9 methylation status for detecting dissociative DNA can be used as colorectal cancer and early diagnose a preferable molecular indexes.
At present detection methylation method mainly include the following types:
(1) direct sequencing (bisulfite sequencing PCR, BSP).The principle of direct sequencing is: weight sulfurous
Hydrochlorate makes the cytosine deamination not methylated in DNA be transformed into uracil, and the cytimidine to methylate remains unchanged,
After PCR amplification, uracil is completely converted into thymidine, finally to PCR product carry out sequencing and with untreated sequence
Compare, judges whether the site CpG methylates.Firstly the need of design of primers is carried out, (each gene designs one to the experimental method
To primer, primer location avoids the site CG in DNA as far as possible), it then needs to carry out gel extraction to purpose band；Recycling
PCR product afterwards connects cloning vector；It selects positive colony sequencing and sequencing result is analyzed.But the method experimentation
It is long, a large amount of cloning and sequencing is needed, process is relatively complicated, tests the costly of detection.
(2) methylation status of PTEN promoter method (Methylmion Specific PCR, MSP).This method is with weight sulfurous acid
Salt makes Cytosines uracil, carry out design of primers (each gene designs two pairs of primers, be respectively as follows: methylated primers M,
Non-genomic primer U, the target fragment of corresponding amplification methylation and non-methylation), design nested primer is needed sometimes；PCR amplification:
The target fragment of methylation and non-methylation is expanded respectively, grads PCR instrument should be used as far as possible at this time, while using not
Same annealing temperature, to screen suitable annealing temperature；To PCR product electrophoresis.This method detection time is relatively long, cannot
Testing result is obtained in time.
(3) methylation sensitive curve analysis method (MS-High Resolution Melting Curve, MS-
HRM).This method is poor using the base between the methylate DNA and non-methylate DNA after bisulf iotate-treated (modification)
It is different, the variation of product melting temperature and melting curve difference can be caused in conjunction with base variation and thermal denaturation melts after the completion of reaction
The visibly different principle of curve graph can draw normal gradients curve in conjunction with gradient methylene standard items.Compare the heat of measuring samples
Region of the denaturation curve in standard curve, the methylation level section of you can get it the sample.This method needs different methyl
The standard items of change degree, experimental result read complicated cumbersome.And the fluorescent dye of this method is saturated fluorescence dyestuff, as a result
A fluorescence signal channel can only be detected, the amplification situation of sample reference gene cannot be analyzed simultaneously, cannot infer phosphorothioate
Effect, be easy to appear bisulfite and handle incomplete problem and false negative.
(4) methylation sensitive restriction enzyme (methylation-sensitive restriction Endonuc
Lease, MS-RE method).This method using methylation sensitive restriction enzyme to methylation area the characteristic that do not cut,
DNA is digested to be analyzed again after different size of segment.The restriction enzyme for the methyl-sensitive being often used has
HpaII-MspI (identification sequence C CGG) and SmaI-XmaI (CCCGGG) etc..Since the base number of the latter's identification is relatively more,
The probability that base sequence occurs in vivo is relatively low, so i.e. HpaII-MspI is more often used with the former.Wherein HpaII and MspI
It can identify CCGG sequence, however when the cytimidine in sequence methylates, HpaII is not cut, and utilizes HpaII-MspI
This attribute handle DNA, then carry out Southern or PCR amplification separation product, specify methylation state.This is a kind of warp
The methylation detecting technique of allusion quotation, its advantage is that: relatively easy, low in cost, methylation sites are clear, and experimental result is easily explained；
The disadvantage is that: 1. since CG is not limited only in CCGG sequence, and the CG in the non-sequence will be ignored；2. only detecting and turning
When recording the methylation state in relevant key site, the result of the detection method is just significant；3. in contrast, Southern
Method is more complex, and needs the amount of sample big；4. the problem of there is false positives caused by enzyme incomplete digestion；5. not being suitable for
Mixing sample.
Summary of the invention
The present invention is directed to overcome the shortcomings of above-mentioned technology, provides and recycled in a kind of human peripheral for diagnosis of colorectal carcinoma
The kit that septin9 gene methylation detects in Tumour DNA,.
In order to achieve the above object, the technical scheme is that
It in the detection kit of 9 gene methylation of septin include: septin9 in the human peripheral Circulating tumor DNA
The methylation specific primer in gene purpose site；The dideoxycytidine of the non-methylation specific in septin9 gene purpose site is repaired
Adorn primer；The hydrolysis probes of septin9 gene specific；The primer of reference gene and the hydrolysis probes of reference gene；It is described
At least one site CpG that the gene purpose site septin9 includes by 1 exon 1 septin9 gene V2 transcript γ；Institute
Stating reference gene is one or more of GAPDH, β-actin, B2M, SDHA, HPRT1.
Preferably, methylation specific primer (its is final concentration of: the 0.1~1uM) packet in the septin9 gene purpose site
It includes:
Forward primer: 5 '-ggctcttgagcccacaggc-3 ' (SEQ ID NO.1)；
Reverse primer: 5 '-tttgggcagccaaacaaagttctctgt-3 ' (SEQ ID NO.2)；
The non-methylation specific in the septin9 gene purpose site dideoxycytidine Mdification primer (its is final concentration of:
0.1~1uM) include:
Forward primer: 5 '-gactcttgaacccacagac-3 ' (SEQ ID NO.3), 3 ' end quilts of the forward primer
It is double
Deoxycytidine modification；
Reverse primer: 5 '-tttgggcagccaaacaaagttctctgt-3 ' (SEQ ID NO.4)；
The hydrolysis probes of the septin9 gene specific (its is final concentration of: 0.1~1uM) are as follows:
5’-accgccgccgcgcgctcta-3’(SEQ ID NO.5)。
Preferably, the primer of the reference gene and the hydrolysis probes of reference gene are as follows:
GAPDH:
Forward primer: 5 '-ttggaaaaggatatttttattgtaaaa-3 ' (SEQ ID NO.6)；
Reverse primer: 5 '-aattccccaaaacacccaaacaccca-3 ' (SEQ ID NO.7)；
Probe: 5 '-ctaacctttaaaatcaaat-3 ' (SEQ ID NO.8)；
β-actin:
Forward primer: 5 '-taatttgtgtgtgtgttttggggtttg-3 ' (SEQ ID NO.9)；
Reverse primer: 5 '-cctccacccaattcaaacaacaccaa-3 ' (SEQ ID NO.10)；
Probe: 5 '-aaatacacacaaacaccca-3 ' (SEQ ID NO.11)；
B2M:
Forward primer: 5 '-tttatttgattataatggaaagtatg-3 ' (SEQ ID NO.12)；
Reverse primer: 5 '-atttcataaactaaaaaacaaaaaaac-3 ' (SEQ ID NO.13)；
Probe: 5 '-aacaaataccttaaataat-3 ' (SEQ ID NO.14)；
SDHA:
Forward primer: 5 '-gtggttagagttaatattatatagtatg-3 ' (SEQ ID NO.15)；
Reverse primer: 5 '-aaatataatattaacaataaccaccat-3 ' (SEQ ID NO.16)；
Probe: 5 '-taatattaacaataaccac-3 ' (SEQ ID NO.17)；
HPRT1:
Forward primer: 5 '-gattatagataggggtgagagaaaga-3 ' (SEQ ID NO.18)；
Reverse primer: 5 '-cttatagataggggtgagagaaaga-3 ' (SEQ ID NO.19)；
Probe: 5 '-accacaaataatacacatcc-3 ' (SEQ ID NO.20).
Preferably, the end the probe 5' reporter fluorescence group of the probe of the septin9 gene and reference gene be FAM,
One of TET, HEX, TAMRA, Texas Red, Rox, Cy5 or a variety of；The probe and reference gene of the septin9 gene
The end probe 3' quencher be BHQ1, BHQ2, TAMRA, DABCYL in it is one or more.
It preferably, further include negative quality-control product, positive quality control product and no template control, the no template in the kit
Control refers to the reaction system without human genome DNA.Preferably, the positive quality control product is bisulphite modified complete
Methylate human genome DNA, and the feminine gender quality-control product is bisulphite modified non-methylation human genome DNA.
In the kit, the final concentration of pcr amplification reaction system is formed are as follows: 1~10 × PCR buffer, 0.1~1mM
DNTPs, 0.01~0.10U/ul Taq DNA polymerase, 1~5mM magnesium chloride.
The present invention passes through the calculating of septin9 gene and reference gene PCR amplification result, sentences to provide to testing result
It is fixed, improve the sensibility and reliability of testing result.Septin9 gene and reference gene PCR amplification result refer to its Ct value, i.e.,
The fluorescence signal of septin9 gene and reference gene reaches recurring number experienced when the threshold value of setting.Pass through septin9 gene
Calculating with reference gene PCR amplification result is come the mode for providing judgement to testing result: septin9 gene C t≤32 are sentenced
Determine result: methylation positive；Septin9 gene C t > 32, septin9 gene C t- reference gene Ct≤8 determine result: methyl
Change positive；Ct > 32, septin9 gene C t- reference gene Ct > 8 determines result: methylation is negative.
The present invention has blocked the non-of the non-methylation template in septin9 gene purpose site using dideoxycytidine Mdification primer
Specific amplification improves its sensitivity, and septin9 gene methylation situation, operation letter can be detected in Circulating tumor DNA
It is single.By the calculating of target gene and reference gene PCR amplification result, to provide judgement to testing result, detection knot is improved
The sensibility and reliability of fruit.
The application and direct sequencing are the difference is that this application method therefor is directly to pass through PCR (polymerase chain
Formula reaction) obtained fluorescence signal directly judges the state of " methylation in the site CpG "；And direct sequencing needs to pass through PCR
The state of " methylation in the site CpG " can be obtained to sequencing reaction is carried out after the amplification of target area.
The component of kit of the present invention is arranged and its result interpretation scheme makes detection process easy, operability
By force.Testing result is reliable, specificity is good.
Specific embodiment
Embodiment 1
Material: plasma sample to be checked, positive quality control product, negative quality-control product.The positive quality control product is repaired for bisulfites
The permethylated human genome DNA of decorations, the feminine gender quality-control product is bisulphite modified non-methylation human genome
DNA。
The detection kit of 9 gene methylation of septin in human peripheral Circulating tumor DNA, including: septin9
The methylation specific primer in gene purpose site；The dideoxycytidine of the non-methylation specific in septin9 gene purpose site is repaired
Adorn primer；The hydrolysis probes of septin9 gene specific；The primer of reference gene and the hydrolysis probes of reference gene；It is described
At least one site CpG that the gene purpose site septin9 includes by 1 exon 1 septin9 gene V2 transcript γ；Institute
Stating reference gene is one or more of GAPDH, β-actin, B2M, SDHA, HPRT1.
The methylation specific primer (its is final concentration of: 0.1~1uM) in the septin9 gene purpose site includes:
Forward primer: 5 '-ggctcttgagcccacaggc-3 ' (SEQ ID NO.1)；
Reverse primer: 5 '-tttgggcagccaaacaaagttctctgt-3 ' (SEQ ID NO.2)；
The non-methylation specific in the septin9 gene purpose site dideoxycytidine Mdification primer (its is final concentration of:
0.1~1uM) include:
Forward primer: 5 '-gactcttgaacccacagac-3 ' (SEQ ID NO.3), 3 ' end quilts of the forward primer
It is double
Deoxycytidine modification；
Reverse primer: 5 '-tttgggcagccaaacaaagttctctgt-3 ' (SEQ ID NO.4)；
The hydrolysis probes of the septin9 gene specific (its is final concentration of: 0.1~1uM) are as follows:
5’-accgccgccgcgcgctcta-3’(SEQ ID NO.5)。
The primer of the reference gene and the hydrolysis probes of reference gene are as follows:
GAPDH:
Forward primer: 5 '-ttggaaaaggatatttttattgtaaaa-3 ' (SEQ ID NO.6)；
Reverse primer: 5 '-aattccccaaaacacccaaacaccca-3 ' (SEQ ID NO.7)；
Probe: 5 '-ctaacctttaaaatcaaat-3 ' (SEQ ID NO.8)；
β-actin:
Forward primer: 5 '-taatttgtgtgtgtgttttggggtttg-3 ' (SEQ ID NO.9)；
Reverse primer: 5 '-cctccacccaattcaaacaacaccaa-3 ' (SEQ ID NO.10)；
Probe: 5 '-aaatacacacaaacaccca-3 ' (SEQ ID NO.11)；
B2M:
Forward primer: 5 '-tttatttgattataatggaaagtatg-3 ' (SEQ ID NO.12)；
Reverse primer: 5 '-atttcataaactaaaaaacaaaaaaac-3 ' (SEQ ID NO.13)；
Probe: 5 '-aacaaataccttaaataat-3 ' (SEQ ID NO.14)；
SDHA:
Forward primer: 5 '-gtggttagagttaatattatatagtatg-3 ' (SEQ ID NO.15)；
Reverse primer: 5 '-aaatataatattaacaataaccaccat-3 ' (SEQ ID NO.16)；
Probe: 5 '-taatattaacaataaccac-3 ' (SEQ ID NO.17)；
HPRT1:
Forward primer: 5 '-gattatagataggggtgagagaaaga-3 ' (SEQ ID NO.18)；
Reverse primer: 5 '-cttatagataggggtgagagaaaga-3 ' (SEQ ID NO.19)；
Probe: 5 '-accacaaataatacacatcc-3 ' (SEQ ID NO.20).
Instrument: Lightcycler 480, nanodrop 1000, supercentrifuge, water-bath, whirlpool concussion instrument, refrigerator,
Baking oven, autoclave.
Reagent: Circulating tumor DNA extracts kit (Shanghai Yi Maisai section), archaeal dna polymerase (Roche Holding Ag), 10 × PCR
Buffer (Roche Holding Ag), MgCl2 (Roche Holding Ag), dNTP (TaKaRa), purified water.
Primer: all primer purity should reach electrophoresis grade (PAGE) or HPLC grades, be free of miscellaneous band.Combination mechanism is provided to provide
The quality inspection of synthetic product prove, as PAGE electrophoresis result or HPLC analyze map, it was demonstrated that after purification using PAGE or HPLC
There should be obvious unimodal PAGE or HPLC purifying map, concentration is that 10ng/ μ l is spare.
(1) extraction of Circulating tumor DNA
Patient's Circulating tumor DNA is extracted using the Circulating tumor DNA extracts kit of Shanghai Emerging Therapeutics Co., Ltd.,
Specific step is as follows:
A, plasma sample is added proteopepsis liquid A, Proteinase K, 37 DEG C of constant temperature and incubates 30min into 2ml centrifuge tube.
B, nucleic acid combination liquid, DNA extraction magnetic bead is added, SolutionER is added after mixing, is placed in rotation and mixes instrument, mildly
Mix 25min.
C, magnetic bead 1min is adsorbed with Magneto separate frame, abandons supernatant.
D, cleaning solution I washing magnetic bead is added twice.
E, cleaning solution II washing magnetic bead is added twice.
F, magnetic bead is adsorbed, stands 3-6min after abandoning supernatant.
G, eluent is added, is placed in room temperature, is mixed evenly elution 5min.
H, magnetic bead is adsorbed in magnetic field, recycles the eluent containing genomic DNA.
I, the DNA content obtained in sample is quantitative determined by the way that minim DNA is quantitative.
B, using the Circulating tumor DNA of above-mentioned acquisition as template
(2) system and reaction condition of PCR reaction
The final concentration of the pcr amplification reaction system forms are as follows: 1~10 × PCR buffer, 0.1~1mM dNTPs, 0.1
~1uM septin9 methylated primers, 0.1~1uM septin9 non-methylation dideoxycytidine primer 0.1~1uM internal reference base
Because of 0.05~2ng/ul of primer template DNA, 0.1~1uM septin9Taqman hydrolysis probes, 0.1~1uM reference gene
Taqman hydrolysis probes, 0.01~0.10U/ul Taq DNA polymerase, 1~5mM magnesium chloride.
The detection gene is septin9 gene, septin9 methylated genes, and the reference gene is GAPDH, β-
One or more of actin (ACTB), B2M, SDHA, HPRT1.The probe of the septin9 gene and reference gene
The end probe 5' reporter fluorescence group is one of FAM, TET, HEX, TAMRA, Texas Red, Rox, Cy5 or a variety of；It is described
The quencher at the end probe 3' of the probe and reference gene of septin9 gene is a kind of in BHQ1, BHQ2, TAMRA, DABCYL
Or it is a variety of.
It include 10mM dATP, 10mM dCTP, 10mM dTTP and 10mM dGTP in the dNTPs, in the PCR system
Include TrisCl, potassium chloride, magnesium sulfate and magnesium chloride.
The detailed process of the pcr amplification reaction are as follows: 92~97 DEG C initial denaturation 5~35 minutes, then carry out polymerase chain
It reacts the amplification stage, 92~97 DEG C of denaturation 10~30s, 50~65 DEG C of 10~30s of annealing, and carries out 35~55 circulations.
(3) sample-adding of PCR reaction is laid out (being shown in Table 1)
DNA sample, negative quality-control product, positive quality control product and no template control to be measured carry out 3 multiple holes detections, PCR instrument
96 orifice plates sample-adding layout see the table below.PC is represented positive quality control product (Positive Control) in table, and NC represents negative quality-control product
(Negative Control), NTC are represented no template control (No template control), and S represents detection sample
(sample)。
Table 1
| 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 | S... | S... | S... | ||||||
| E | S3 | S3 | S3 | NTC | NTC | NTC | ||||||
| F | S4 | S4 | S4 | |||||||||
| G | S5 | S5 | S5 | |||||||||
| H | S6 | S6 | S6 |
(4) after experiment terminates, the analysis of testing result is carried out according to the following steps, determines (being shown in Table 2):
The amplification curve analysis of no template control (NTC) is carried out, septin9 and reference gene (most preferably: ACTB) are at this time
It should illustrate to test pollution-free without the obvious amplified signal of amplification curve, can continue to analyze.
Reference gene (ACTB) Ying Junyou amplified signal of quality-control product, and S-type amplification curve, quality-control product reference gene
(ACTB) CP value should meet parameter area in following table；The septin9 of negative quality-control product should change without obvious amplification curve, positive
The CP value of quality-control product septin9 should meet the parameter area of quality-control product in following table.When quality-control product detection meets conditions above, it was demonstrated that
Experiment effectively, can continue to analyze.
Table 2
Reference gene (ACTB) Ying Junyou amplified signal of sample, and S-type amplification curve, the single PCR detection knot of sample
Fruit should be according to the single PCR result interpretation of 3 pattern detection of table-.
Table 3
If 1 in table 3,2 requirements are not met, it is recommended that detecting again.
Test method without specific conditions in the embodiment above, according to normal conditions, such as according to molecule gram
Condition proposed by condition described in grand experiment guide (third edition, J. Pehanorm Brooker etc. write) or manufacturer is grasped
Make.
SEQUENCE LISTING
<110>Hunan Hong Ya gene technology Co., Ltd
<120>in a kind of human peripheral Circulating tumor DNA 9 gene methylation of septin detection kit
<130> 1
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial synthesized
<400> 1
ggctcttgag cccacaggc 19
<210> 2
<211> 27
<212> DNA
<213>artificial synthesized
<400> 2
tttgggcagc caaacaaagt tctctgt 27
<210> 3
<211> 19
<212> DNA
<213>artificial synthesized
<400> 3
gactcttgaa cccacagac 19
<210> 4
<211> 27
<212> DNA
<213>artificial synthesized
<400> 4
tttgggcagc caaacaaagt tctctgt 27
<210> 5
<211> 19
<212> DNA
<213>artificial synthesized
<400> 5
accgccgccg cgcgctcta 19
<210> 6
<211> 27
<212> DNA
<213>artificial synthesized
<400> 6
ttggaaaagg atatttttat tgtaaaa 27
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<400> 7
aattccccaa aacacccaaa caccca 26
<210> 8
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<212> DNA
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<400> 8
ctaaccttta aaatcaaat 19
<210> 9
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<212> DNA
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<400> 9
taatttgtgt gtgtgttttg gggtttg 27
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cctccaccca attcaaacaa caccaa 26
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<212> DNA
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<400> 11
aaatacacac aaacaccca 19
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<212> DNA
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<400> 12
tttatttgat tataatggaa agtatg 26
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<212> DNA
<213>artificial synthesized
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atttcataaa ctaaaaaaca aaaaaac 27
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aacaaatacc ttaaataat 19
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<212> DNA
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gtggttagag ttaatattat atagtatg 28
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<212> DNA
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<400> 16
aaatataata ttaacaataa ccaccat 27
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<212> DNA
<213>artificial synthesized
<400> 17
taatattaac aataaccac 19
<210> 18
<211> 26
<212> DNA
<213>artificial synthesized
<400> 18
gattatagat aggggtgaga gaaaga 26
<210> 19
<211> 24
<212> DNA
<213>artificial synthesized
<400> 19
cttatagata ggggtgagag aaag 24
<210> 20
<211> 20
<212> DNA
<213>artificial synthesized
<400> 20
accacaaata atacacatcc 20
Claims (4)
1. the detection kit of 9 gene methylation of septin in a kind of human peripheral Circulating tumor DNA, which is characterized in that described
It include: the methylation specific primer in septin9 gene purpose site in kit；The non-methyl in septin9 gene purpose site
Change special dideoxycytidine Mdification primer；The hydrolysis probes of septin9 gene specific；The primer and reference gene of reference gene
Hydrolysis probes；The septin9 gene purpose site by 1 exon 1 septin9 gene V2 transcript γ include to
A few site CpG；The reference gene is one or more of GAPDH, β-actin, B2M, SDHA, HPRT1；Institute
The methylation specific primer for stating septin9 gene purpose site includes:
Forward primer: 5 '-ggctcttgagcccacaggc-3 '；
Reverse primer: 5 '-tttgggcagccaaacaaagttctctgt-3 '；
The dideoxycytidine Mdification primer of the non-methylation specific in the septin9 gene purpose site includes:
Forward primer: the 3 ' ends of 5 '-gactcttgaacccacagac -3 ', the forward primer are modified by dideoxycytidine；
Reverse primer: 5 '-tttgggcagccaaacaaagttctctgt-3 '；
The hydrolysis probes of the septin9 gene specific are as follows: 5 '-accgccgccgcgcgctcta-3 '；
The primer of the reference gene and the hydrolysis probes of reference gene are as follows:
GAPDH:
Forward primer: 5 '-ttggaaaaggatatttttattgtaaaa-3 '；
Reverse primer: 5 '-aattccccaaaacacccaaacaccca-3 '；
Probe: 5 '-ctaacctttaaaatcaaat-3 '；
β-actin:
Forward primer: 5 '-taatttgtgtgtgtgttttggggtttg-3 '；
Reverse primer: 5 '-cctccacccaattcaaacaacaccaa-3 '；
Probe: 5 '-aaatacacacaaacaccca-3 '；
B2M:
Forward primer: 5 '-tttatttgattataatggaaagtatg-3 '；
Reverse primer: 5 '-atttcataaactaaaaaacaaaaaaac-3 '；
Probe: 5 '-aacaaataccttaaataat-3 '；
SDHA:
Forward primer: 5 '-gtggttagagttaatattatatagtatg-3 '；
Reverse primer: 5 '-aaatataatattaacaataaccaccat-3 '；
Probe: 5 '-taatattaacaataaccac-3 '；
HPRT1:
Forward primer: 5 '-gattatagataggggtgagagaaaga-3 '；
Reverse primer: 5 '-cttatagataggggtgagagaaaga-3 '；
Probe: 5 '-accacaaataatacacatcc-3 '.
2. kit as described in claim 1, which is characterized in that the spy of the probe and reference gene of the septin9 gene
The end needle 5' reporter fluorescence group is one of FAM, TET, HEX, TAMRA, Texas Red, Rox, Cy5 or a variety of；It is described
The quencher at the end probe 3' of the probe and reference gene of septin9 gene is a kind of in BHQ1, BHQ2, TAMRA, DABCYL
Or it is a variety of.
3. kit as described in claim 1, which is characterized in that further include negative quality-control product, positive matter in the kit
Control product and no template control.
4. kit as claimed in claim 3, which is characterized in that the positive quality control product is bisulphite modified full first
Base human genome DNA, the feminine gender quality-control product is bisulphite modified non-methylation human genome DNA.
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| CN105861672A (en) * | 2016-04-26 | 2016-08-17 | 杭州壹锋生物科技有限公司 | Detection kit and detection method for methylation of septin9 gene in human peripheral blood cell-free DNA |
| CN106011239A (en) * | 2016-05-19 | 2016-10-12 | 北京安必奇生物科技有限公司 | Kit for amplifying Her2 (Human epidermal growth factor receptor-2) gene in human peripheral blood circulating tumor DNA (Deoxyribonucleic Acid) |
| CN106222267A (en) * | 2016-08-01 | 2016-12-14 | 博尔诚（北京）科技有限公司 | Compositions of detection hepatocarcinoma and application thereof |
| CN106244724A (en) * | 2016-10-26 | 2016-12-21 | 北京鑫诺美迪基因检测技术有限公司 | The primer of detection septin9 gene methylation and test kit |
| CN115786517A (en) * | 2019-02-14 | 2023-03-14 | 博尔诚（北京）科技有限公司 | Composition for screening cancers of intestinal cancers and precancers and application of composition |
| CN112159844B (en) * | 2020-05-25 | 2022-05-03 | 浙江中创生物医药有限公司 | Method and reagent for detecting DNA methylation of colorectal cancer |
| CN114277150A (en) * | 2021-12-30 | 2022-04-05 | 苏州睿明医学检验实验室有限公司 | Internal reference detection method suitable for colorectal tissues and/or tumors and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1544309A1 (en) * | 2003-12-16 | 2005-06-22 | Bayer HealthCare LLC | Assay for detecting methylation status by methylation specific primer extension (MSPE) |
| CN101889096A (en) * | 2007-10-04 | 2010-11-17 | 联邦科学及工业研究组织 | Nucleic acid amplification |
| CN104131070A (en) * | 2014-05-22 | 2014-11-05 | 苏州工业园区为真生物医药科技有限公司 | Gene methylation detection method |
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| EP2914753A4 (en) * | 2012-10-31 | 2016-06-08 | Becton Dickinson Co | Selective amplification and real-time pcr detection of rare mutations |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1544309A1 (en) * | 2003-12-16 | 2005-06-22 | Bayer HealthCare LLC | Assay for detecting methylation status by methylation specific primer extension (MSPE) |
| CN101889096A (en) * | 2007-10-04 | 2010-11-17 | 联邦科学及工业研究组织 | Nucleic acid amplification |
| CN104131070A (en) * | 2014-05-22 | 2014-11-05 | 苏州工业园区为真生物医药科技有限公司 | Gene methylation detection method |
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