CN110042159A - A kind of polymerase spiral amplimer group and detection kit based on Human colorectal carcinoma related gene SEPT9 DNA methylation assay - Google Patents
A kind of polymerase spiral amplimer group and detection kit based on Human colorectal carcinoma related gene SEPT9 DNA methylation assay Download PDFInfo
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Abstract
The present invention provides a kind of polymerase spiral amplimer group, kit and detection method based on Human colorectal carcinoma related gene SEPT9 DNA methylation assay.The present invention devises a pair of PSR primer and kit for the detection of Human colorectal carcinoma specific methylation for the region promoter V2 of SEPT9 gene in Human colorectal carcinoma genome.PSR detection is carried out to the genomic DNA of sulphite modified with the primer and detection architecture, rapid amplifying can be carried out in 40min, whether reaction terminates through the color change situation judgement sample of indicator to be Human colorectal carcinoma sample.Compared with conventional diagnosis of colorectal carcinoma method, method of the invention has the characteristics that quick, sensitive and specific good, can be used for the detection and clinical diagnosis of early stage Human colorectal carcinoma.
Description
Technical field
The invention belongs to technical field of gene detection, and in particular to one kind is based on Human colorectal carcinoma related gene SEPT9 first
The polymerase spiral amplimer group and detection kit of baseization detection.
Background technique
Colorectal cancer (Colorectal cancer, CRC) is that Colorectal mucosa epithelium is issued in the effect of a variety of carcinogenic factors
Raw malignant tumour, disease incidence occupy common cancer the 3rd, poor prognosis, and case fatality rate is higher.Five Nian Shengcun of early stage CRC
Rate is up to 90%, and advanced stage CRC is then reduced to 5%, and efficient CRC methods for screening can improve 5 years survival rates of patient, drop
Low actual.Therefore, early diagnosis, early treatment are the key that prevention and treatment CRC, can significantly reduce its death rate.
SEPT9 gene hyper-methylation level can be used as the biomarker of colorectal cancer early diagnosis.The DNA first of several genes
Generation, the development of base and CRC are closely related, wherein recall rate of the SEPT9 in CRC is much higher than other tumours.Clinical research
The same SEPT9 gene for confirming that high methylation can be detected in CRC sample, it is thin that excrement both contained the normal Colon and rectum epithelium to fall off
Born of the same parents and free DNA, also containing the cancer cell largely to fall off from colorectal cancer and free cancer DNA；Again since enteron aisle is alkali
Property environment, is conducive to the preservation of DNA, keeps the DNA mass extracted from excrement preferable.Therefore, faeces DNA is to carry out alimentary canal to swell
The best sample of tumor Molecular Detection, molecular diagnosis.
The early diagnosis effective ways of colorectal cancer have occult blood test, imageological examination, hemotoncus tumor markers, colon
Spectroscopy etc..In order to achieve the purpose that early diagnosis, the cure rate for improving colorectal cancer reduces the death rate, should widely popularize early stage
Screening.With the fast development of molecular biology, computer and image technology, genetic test and CTVC are checked and will be tied directly in early stage
It plays a greater and greater role in the diagnosis of intestinal cancer, and new a piece of new world will be provided for the diagnoses and treatment of colorectal cancer. PSR
(polymerase spiral detection method) is to be combined together PCR amplification and LAMP isothermal amplification technology, is realized under isothermal conditions
The detection of gene methylation.
Summary of the invention
For overcome common detection efficiency it is low, it is difficult operation, defect at high cost, the purpose of the present invention is to provide people's Colon and rectums
The polymerase spiral augmentation detection reagent of cancer associated gene SEPT9 DNA methylation assay, when being detected using the reagent, when detection
Between it is short, specificity and sensibility are high, easy to operate and at low cost.
The present invention provide it is a kind of based on Human colorectal carcinoma related gene SEPT9 DNA methylation assay polymerase spiral amplification draw
Object group, the primer sets are made of following primer:
Forward primer: 5-acgaattcgtacatagaagtatag-TATTAGTTATTATGTCGGATTTCGC-3；
Reverse primer: 5-gatatgaagatacatgcttaagca-GCCTAAATTAAAAATCCCGTC-3.
The present invention provides a kind of Human colorectal carcinoma related gene SEPT9 DNA methylation assay reagent, and the reagent includes right
It is required that primer sets described in 1.
Preferably, the reagent also comprises the following components: 10 × reaction buffer, dNTPs, MgSO4, Bst DNA it is poly-
Synthase, deionized water, colour reagent and template DNA.
Preferably, Mg in the reagent2+Concentration be 6mM.
Preferably, the concentration of dNTPs is 1.5mM in the reagent.
Preferably, the reagent further includes positive control and negative control, the positive control is SEPT9 methylation mark
Quasi- product DNA；The negative control is the non-methylation standard items DNA of SEPT9.
The present invention provides a kind of Human colorectal carcinoma related gene SEPT9 methylation detection kit, and the kit includes
The described in any item Human colorectal carcinoma related gene SEPT9 DNA methylation assay reagents of claim 2-6.
The present invention provide above-mentioned detection reagent or detection kit it is following it is any in application:
(1) detection not for the purpose of the diagnosing and treating of disease or auxiliary detection Human colorectal carcinoma related gene SEPT9
Methylation；
(2) product for detecting or assisting detection Human colorectal carcinoma related gene SEPT9 methylation is prepared.
The present invention provides a kind of method detected or auxiliary detection Human colorectal carcinoma related gene SEPT9 methylates, the party
Method is not for the purpose of the diagnosing and treating of disease, comprising the following steps:
(1) genome is extracted from fecal specimens, genomic DNA is used as template by bisulphite modified, then adopts
Polymerase spiral expansion is carried out with the described in any item detection reagents of claim 2-6 or detection kit as claimed in claim 7
Increase, obtains amplified production；
(2) determine whether sample to be tested occurs SEPT9 gene methylation according to following either method:
A, glassy yellow then SEPT9 gene generation methylation change is presented in amplified production；Amplified production is presented purple and does not go out then
Existing SEPT9 gene methylation changes；
B, amplified production is subjected to agarose gel electrophoresis, electrophoretic band is distributed in scalariform, then methyl occurs for SEPT9 gene
Change and changes；An only primer band does not occur the change of SEPT9 gene methylation then.
Preferably, the reaction condition of the polymerase spiral amplification is 61 DEG C of effect 40min of constant temperature in step (1).
Beneficial effects of the present invention are as follows:
The polymerase spiral kit that the present invention detects SEPT9 gene methylation has high sensitivity: will be handled
SEPT9 gene methylation DNA profiling is used as PSR reaction template after carrying out 10 times of gradient dilutions, with same template concentrations PCR
Method is detected, the results showed that PSR method is at least 10 times higher than the method for standard PCR amplification.It can be seen that PSR of the present invention
Method has higher sensitivity.
The polymerase spiral kit that the present invention detects SEPT9 gene methylation has high specific: by the colon cancer positive
Feces of Patients sample, intestinal polyposis human faecal mass sample, chronic enteritis Feces of Patients sample and normal adults fecal specimens difference
It as test object, is detected with method of the invention, only SPET9 is the positive, shows that this method has high specific.
The polymerase spiral reagent that the present invention detects SEPT9 gene methylation has rapidity: opposite common PCR reaction number
The reaction time of hour and detection time, detection method only need 40min that entire reaction and result judgement can be completed.
The polymerase spiral reagent that the present invention detects SEPT9 gene methylation has operability: relative to Standard PCR,
SEPT9 gene methylation polymerase spiral expands detection reagent and does not need expensive PCR instrument, it is only necessary to a simple water bath with thermostatic control
Reaction can be completed in pot；And result detection directly can visually determine, without specific apparatus such as gel electrophoresises, therefore the present invention tries
Agent box has stronger operability.
Detection method is easy to operate, low in cost, can be widely applied.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:
Fig. 1 is STEP9-PSR amplification figure.
Fig. 2 is time conditions optimum results.
Fig. 3 is temperature condition optimizing result.
Fig. 4 is SEP9PSR atopic Test Drawing.
Fig. 5 is SEP9PSR reaction sensitivity Test Drawing.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
Embodiment 1
1, reagent associated materials
Stool DNA Extraction Kit excrement extracts kit (Beijing Quanshijin Biotechnology Co., Ltd),
Betaine (SIGMA company), BstDNA polymerase and 10 × reaction buffer (500mM KCl, 100mM (NH4)2SO4, 8mM
Betaine, 1%Tween 20), dNTPs (clontech company), Mg2+And DL2000 (TAKARA company).Colour reagent is
2.0mM is phenol red and the dyed blended liquid of 0.5mM cresol red, is purchased from Solarbio company.
2, the design and synthesis of PSR primer
It is utilized with reference to the SEPT9 gene promoter V2 zone methylation sequence delivered at present for specific conservative region
Photographing On-line software Oligo 7.37 separately designs 2 primers, and primer includes upstream primer SEPT9-SF, downstream primer
SEPT9-SR, sequence difference are as follows:
SEPT9-SF:5-acgaattcgtacatagaagtatag-ATTAGTTATTATGTCGGATT TCGC-3；
SEPT9-SR:5-gatatgaagatacatgcttaagca-GCCTAAATTAAAAATCCCGT C-3.
The primer that design is completed is synthesized by Beijing AudioCodes bio-engineering corporation, and the primer after synthesis is diluted to 10 with ultrapure water
Mmol/mL solution, -20 DEG C of preservations.
3, the extraction of genomic DNA
Take the excrement of 100~200mg with stool DNA Extraction Kit extract DNA, operation to specifications into
Row extracts completion UV spectrophotometer measuring purity and content.
Then by DNA with bisulphite modified.DNA is after bisulphite modified, unmethylated cytimidine in CpG
It will be converted into uracil, and the cytimidine to methylate does not change then.Sample gene group DNA is referring to " Wizard DNA
Clean-up system " kit is modified；The sodium hydrogensulfite modification of blood, excrement genomic DNA: referring to " EZ DNA
MethylationTM-Direct Kit " kit specification carries out.
4, the foundation of SEPT9 gene methylation PSR reaction system
SEPT9 gene methylation detection reagent includes following component:
Wherein, positive control is the genomic DNA of SEPT9 gene methylation sample.
It is placed in after said components are mixed in PCR instrument or 61 DEG C of effect 40min of constant temperature in water-bath；Reaction terminates
Afterwards, can by amplified production 2% Ago-Gel (ethidium bromide containing 0.5 μ g/mL) electrophoresis detection, deposition condition 80V,
30min；The discoloration of its color can also be directly visually observed, the two determines that result is consistent.When visually observing, positive findings are shown as yellow
Color indicates that SEPT9 gene occurs methylation and changes；Negative findings are shown as purple, indicate that methylation does not occur and changes for SEPT9 gene
Become.
Electrophoresis detection result is consistent with the theoretical results, expression SEPT9 as shown in Figure 1, positive electrophoretic band is distributed in scalariform
Gene occurs methylation and changes；The amplification of negative control only has primer dimer appearance, indicates that first does not occur for SEPT9 gene
Baseization changes.
Fig. 1 is STEP9-PSR amplification figure.Wherein (a) amplification: M:DNA molecular weight standard DL2000；1: positive sample
Amplification；2: negative control.(b) color change figure :+: positive amplification；: negative control.
5, the optimization of SEP9PSR detection method condition
The optimization of 5.1 reaction time
Reaction system is configured, respectively in thermostat water bath, at 61 DEG C of constant temperature, reacts 20min, 30min, 40 respectively
It is taken out after min, 50min, 60min, after reaction product is all taken out, respectively takes 5 μ L products, (contain 0.5 μ with 2% Ago-Gel
The ethidium bromide of g/mL) electrophoresis detects reaction product.
Fig. 2 is time conditions optimum results.Wherein, M:DL2000marker；N: negative control；1-5 swimming lane: it is respectively
20min、30min、40min、50min、60min。
The optimization of 5.2 reaction temperatures
Reaction system is configured, constant temperature is anti-in 60 DEG C, 61 DEG C, 61 DEG C, 63 DEG C, 64 DEG C, 65 DEG C of five reaction temperatures respectively
After answering 1 hour, 5 μ L products is respectively taken to carry out the inspection of 2% Ago-Gel (ethidium bromide containing 0.5 μ g/mL) electrophoresis after reaction
It surveys.
Fig. 3 is temperature condition optimizing result.Wherein, M:DL2000marker；1-6 swimming lane: being respectively 60 DEG C, 61 DEG C, 61
℃,63℃,64℃,65℃；N: negative control.
The optimization of each important component concentration in 5.3 reactions
In 25 μ L reaction systems, enzyme unit needed for primer concentration, reaction and template used concentration are constant, change component
MgSO4Concentration, grope MgSO4Influence of the concentration to amplification efficiency, additional amount be followed successively by for 7mM, 6mM, 5mM, 4mM, 3mM,
3 repetitions are arranged in 2mM, 1mM, 0mM, each concentration gradient.After reaction to all concentration gradients, it is solidifying to carry out 2% agarose
Gel electrophoresis detection；The reaction system optimization of different dNTPs concentration is set, gradient be set as 1.8mM, 1.5mM, 1.2mM,
0.9mM, 0.6mM, all grouping reactions all set negative control using deionized water as template, and 3 weights are arranged in each concentration gradient
It is multiple.After reaction to all concentration gradients, 2% agarose gel electrophoresis detection is carried out.
6, the optimum results of SEPT9 gene methylation PSR detection method
6.1 reaction time optimum results: when different between when reacted, it is more and more brighter electrophoretic band to be extended with the time
It is aobvious, when reacting 40min in thermostat water bath, as a result most preferably.
6.2 reaction temperature optimum results: when reaction temperature difference, it may appear that electrophoretic band luminance difference, at 61 DEG C
When reaction, effect is best.
The optimum results of each important component concentration in 6.3 reactions:
Work as MgSO4When concentration difference, it may appear that electrophoretic band luminance difference works as MgSO4When concentration is 6mM, effect is best.
When dNTPs concentration difference, it may appear that electrophoretic band luminance difference, when dNTPs concentration is 1.5mM, effect is most
It is good.
6.4 optimization systems and condition
By the optimization of above-mentioned condition, the last optimization of SEPT9 gene methylation polymerase spiral amplification detection kit
System are as follows:
The reaction condition of polymerase spiral amplification is 61 DEG C of effect 40min.
Kit further includes positive control and negative control, and positive control is SEPT9 methylation standard items DNA；It is negative right
According to for the non-methylation standard items DNA of SEPT9.
7, the specific test of PSR
With colon cancer positive patients fecal specimens, intestinal polyposis human faecal mass sample, chronic enteritis Feces of Patients sample and strong
Health adult's fecal specimens are test object, carry out PSR detection with SEPT9 primer, to verify the specificity of its reaction, and are arranged
Positive control, as a result referring to fig. 4.
The reaction system of PSR detection includes following component:
Amplification shows that only ladder with shape band occurs in colon cancer positive control, other 3 kinds of samples do not occur, explanation
There is no positive amplification when using other fecal specimens as template, the SEPT9-PSR primer that the present invention designs has the special of height
Property.
Fig. 4 is SEP9PSR atopic Test Drawing.Wherein M:DL2000marker；1: intestinal cancer positive patients excrement sample
Product；2: intestinal polyposis human faecal mass sample；3: chronic enteritis Feces of Patients sample；4: normal adults fecal specimens；N: negative right
According to.
8, the sensitivity tests of PSR
Modulus plate DNA product carries out 10 times of progressive dilutions, and is divided into two parts, and a part is for the PSR method after optimizing
Detection, directly visually observes after phenol red dyeing liquor is added, measures the sensibility of this method；Another part is examined with Standard PCR method
It surveys, the two testing result is compared, scheme referring to a and c of Fig. 5, to verify the sensibility of its reaction.B figure is PSR addition
Color change results (this dyestuff is addition before reaction) figure of nucleic acid dye.
The result shows that: PSR method is at least 10 times higher than the method for standard PCR amplification.It can be seen that PSR method of the present invention
With higher sensibility.
Fig. 5 is SEP9PSR reaction sensitivity Test Drawing.Wherein in a figure and c figure, M:DNA molecular criteria amount standard
DL2000；1-7 swimming lane is respectively that template is diluted to 10-1、10-2、10-3、10-4、10-5、10-6、10-7, n is negative control.B figure
In, it is from left to right followed successively by template and is diluted to 10-1、10-2、10-3、10-4、10-5、10-6、10-7。
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (10)
1. a kind of polymerase spiral amplimer group based on Human colorectal carcinoma related gene SEPT9 DNA methylation assay, feature
Be: the primer sets are made of following primer:
Forward primer: 5-acgaattcgtacatagaagtatag-TATTAGTTATTATGTCGGATTTCGC-3；
Reverse primer: 5-gatatgaagatacatgcttaagca-GCCTAAATTAAAAATCCCGTC-3.
2. a kind of Human colorectal carcinoma related gene SEPT9 DNA methylation assay reagent, it is characterised in that: the reagent includes that right is wanted
Primer sets described in asking 1.
3. reagent according to claim 2, it is characterised in that: the reagent also comprises the following components: 10 × reaction buffering
Liquid, dNTPs, MgSO4, Bst archaeal dna polymerase, deionized water, colour reagent and template DNA.
4. reagent according to claim 3, it is characterised in that: Mg in the reagent2+Concentration be 6mM.
5. reagent according to claim 3, it is characterised in that: the concentration of dNTPs is 1.5mM in the reagent.
6. reagent according to claim 3, it is characterised in that: the reagent further includes positive control and negative control, institute
Stating positive control is SEPT9 methylation standard items DNA；The negative control is the non-methylation standard items DNA of SEPT9.
7. a kind of Human colorectal carcinoma related gene SEPT9 methylation detection kit, it is characterised in that: the kit includes power
Benefit requires the described in any item Human colorectal carcinoma related gene SEPT9 DNA methylation assay reagents of 2-6.
8. the described in any item detection reagents of claim 2-6 or detection kit as claimed in claim 7 it is following it is any in
Application:
(1) detection not for the purpose of the diagnosing and treating of disease or auxiliary detection Human colorectal carcinoma related gene SEPT9 methyl
Change；
(2) product for detecting or assisting detection Human colorectal carcinoma related gene SEPT9 methylation is prepared.
9. a kind of method of detection or auxiliary detection Human colorectal carcinoma related gene SEPT9 methylation, this method is not with disease
For the purpose of diagnosing and treating, it is characterised in that: the following steps are included:
(1) genome is extracted from fecal specimens, genomic DNA is used as template by bisulphite modified, then using power
Benefit requires the described in any item detection reagents of 2-6 or detection kit as claimed in claim 7 to carry out the amplification of polymerase spiral, obtains
To amplified production；
(2) determine whether sample to be tested occurs SEPT9 gene methylation according to following either method:
A, glassy yellow then SEPT9 gene generation methylation change is presented in amplified production；Purple, which is presented, in amplified production does not occur then
SEPT9 gene methylation changes；
B, amplified production is subjected to agarose gel electrophoresis, electrophoretic band is distributed in scalariform, then SEPT9 gene occurs to methylate and change
Become；An only primer band does not occur the change of SEPT9 gene methylation then.
10. according to the method described in claim 9, it is characterized by: in step (1), the reaction of the polymerase spiral amplification
Condition is 61 DEG C of effect 40min of constant temperature.
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