CN108642180A - Detect the method and kit of SDC2 gene methylations - Google Patents
Detect the method and kit of SDC2 gene methylations Download PDFInfo
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Abstract
The present invention relates to a kind of methods and kit of detection SDC2 gene methylations.The method of SDC2 gene methylations in detection sample to be tested provided by the invention, including：DNA is extracted from the sample to be tested；The DNA sample is subjected to the processing that methylates, obtain methylating treated DNA；Using reference gene as standard, methylates that treated to described DNA carries out fluorescence quantitative PCR detection, determine testing result；Wherein, when carrying out the fluorescence quantitative PCR detection, α caseins are added in the fluorescence quantitative PCR detection system.A kind of kit of detection SDC2 gene methylations is additionally provided simultaneously.The present invention is detected using SDC2 gene methylations as reliable intestinal cancer marker, reaches 90% or more to the sensitivity of colorectal cancer detection, specificity is 93% or more.
Description
Technical field
The invention belongs to molecular biology fields, are related to a kind of method and kit of detection SDC2 gene methylations.
Background technology
Clinically colorectal cancer screening is mainly detected by feces occult blood experiment and two kinds of technologies of enteroscopy.Feces occult blood
Experiment be detection stool in hemoglobin, if repeatedly, constantly positive reaction indicate hemorrhage of digestive tract, should be further examined
To be vigilant the generation of intestinal canal tumour.The technology is convenient and efficient, but is easy to be influenced by diet etc., and then influences the standard of testing result
True property.In addition, enteroscopy is current most effective reliable diagnostic method, most early stage patients with bowel cancer can be by endoscopy
It was found that and making a definite diagnosis.But the diagnostic method is relatively elaborate time-consuming, and patient has certain pain, and inspection fee is high, it is more difficult to push away on a large scale
Extensively.
It also needs to be further improved for the early diagnosis and therapy of colorectal cancer.
Invention content
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, the present invention carries
A kind of method and kit of detection SDC2 gene methylations are supplied, for realizing the early diagnosis and therapy of colorectal cancer.Profit
With the method and kit of the present invention, for colorectal cancer detection high sensitivity up to 90% or more, specificity up to 93%
More than.
Specifically, according to the first aspect of the invention, the present invention provides SDC2 genes in a kind of detection sample to be tested
The method to methylate, including：DNA is extracted from the sample to be tested；The DNA sample is subjected to the processing that methylates, obtains first
Baseization treated DNA；Using reference gene as standard, methylates that treated to described DNA carries out quantitative fluorescent PCR inspection
It surveys, determines testing result；Wherein, when carrying out the fluorescence quantitative PCR detection, add in the fluorescence quantitative PCR detection system
Enter alpha-casein.
The method of detection SDC2 gene methylations provided by the invention can carry out colorectal cancer molecule diagnosis, right
Reach 90% or more in the sensitivity of colorectal cancer detection, specificity reaches 93% or more.And the method pair of the application present invention
SDC2 genes carry out DNA methylation assay, can be thin due to having the cancer that some fall off in excrement using excrement as detection sample
Born of the same parents, therefore by being detected to the molecular marked compound in fecal sample, the relevant information of intestinal cancer can be obtained.In view of excrement at
This is more complicated, and the mortifier of many Molecular Detections is contained in the inside, cause detection sensitivity in fecal sample and specificity compared with
Difference affects the accuracy of result.Then the discovery of the present inventor's creativeness adds in fluorescence quantitative PCR detection system
Enter alpha-casein, can effectively resist the interference for remaining mortifier in faeces DNA, improve stability and the sensitivity of amplification.From
And make detected level and the colorectal cancer of SDC2 genes in excrement there are high correspondence, obtain good sensitivity and
Specificity.
According to an embodiment of the invention, the method for SDC2 gene methylations can be further attached in the above detection sample to be tested
Add following technical characteristic：
According to an embodiment of the invention, the reference gene is ACTB genes or GADPH genes.ACTB is also known as β-
Actin is a house-keeping gene, and expression is stablized relatively in most cells.GADPH genes are also house keeper's base
Cause, almost in a organized way in all high expression.Using β-actin genes or GADPH genes as reference gene, to assess DNA
The quality of DNA after extraction, sulphite conversion, it can be ensured that the reliability of detection.
According to an embodiment of the invention, the alpha-casein in the fluorescence quantitative PCR detection system a concentration of 0.1
~1mg/mL, preferably 0.4mg/mL.Under the concentration, the alpha-casein, which can be resisted effectively, remains suppression in faeces DNA
The interference of object processed improves stability and the sensitivity of amplification.
According to an embodiment of the invention, the alpha-casein is cow's serum alpha-casein.
According to an embodiment of the invention, the processing that methylates is carried out to the DNA using bisulfites.Bisulfites energy
It is enough that all cytimidines not methylated are converted into uracil, and the cytimidine to methylate is constant, in sample to be tested
DNA carry out the processing that methylates.Sample genomic dna is methylated through bisulfites after processing, if in sample genome
Cytimidine (C) in SDC2 gene orders has been methylated, then the SDC2 genes after the treatment kits processing that methylates
Sequence is constant, and specific primer and specific probe can be in connection, during Fluorescence PCR, in the work of archaeal dna polymerase
With the fluorescent reporter group of 5 ' ends on lower probe and the fluorescent quenching group separation of 3 ' ends, fluorescence is sent out.
According to an embodiment of the invention, the sample to be tested is fecal sample.It is thin that the cancer that some fall off is had in excrement
Born of the same parents, therefore by being detected to the molecular marked compound in fecal sample, to intestinal cancer screening important role.
According to an embodiment of the invention, the first specific primer and the amplification of the first specific probe and detection are utilized respectively
SDC2 genes, first specific primer are selected from SEQ ID NO:1~SEQ ID NO:At least one set in 10, preferably
SEQ ID NO:3 and SEQ ID NO:4, the nucleic acid sequence of first specific probe is selected from SEQ ID NO:11~SEQ ID
NO:16, preferably SEQ ID NO:15.The methylation sites of SDC2 genes mostly occur on the islands CpG of promoter region, for SDC2
Primer sequence designed by the islands CpG of gene promoter area, designs different primer and probes, included in obtained amplified fragments
Methylation sites it is different, it is finally different to the sensitivity of colorectal cancer detection.Sequence SEQ ID NO:1~SEQ ID NO:10
Middle odd numbered sequences represent forward primer, and number is that the sequence of even number represents reverse primer, and the sequence for the number that is connected is drawn as a pair
Object, such as SEQ ID NO:1 and SEQ ID NO:2 are used as pair of primers, SEQ ID NO:3 and SEQ ID NO:4 as another
To primer, and so on.
According to an embodiment of the invention, the second specific primer and the amplification of the second specific probe and detection are utilized respectively
ACTB genes, second specific primer are selected from SEQ ID NO:17~SEQ ID NO:At least one set in 26, preferably
SEQ ID NO:21 and SEQ ID NO:22, second specific probe is selected from SEQ ID NO:28~SEQ ID NO:32,
Preferably SEQ ID NO:29.
According to an embodiment of the invention, it is connected separately on first specific probe and second specific probe
Fluorescent reporter group and quenching group, the fluorophor are selected from 6- Fluoresceincarboxylic acids, chlordene -6- methylfluoresceins, four chloro- 6-
Fluoresceincarboxylic acid, two chloro- 6- Fluoresceincarboxylic acids of 2,7- dimethyl -4,5-, fluorescein isothiocynate, Texas Red, Cys3, Cy5
Or one kind in VIC；The quenching group is selected from BHQ, 4- (4- dislikes amino benzeneazo) benzoic acid) or carboxyl tetramethyl Luo Dan
It is bright.
According to the second aspect of the invention, the present invention provides a kind of kits of detection SDC2 gene methylations, including
First specific primer and the first specific probe, first specific primer are selected from SEQ ID NO:1~SEQ ID NO:
At least one set in 10, preferably SEQ ID NO:3 and SEQ ID NO:4, the nucleic acid sequence choosing of first specific probe
From SEQ ID NO:11~SEQ ID NO:16, preferably SEQ ID NO:15.
According to an embodiment of the invention, the kit can further be appended below technical characteristic：
According to an embodiment of the invention, the kit further includes amplification and the second specificity of detection reference gene ACTB
Primer and the second specific probe, second specific primer are selected from SEQ ID NO:17~SEQ ID NO:26, preferably
SEQ ID NO:21 and SEQ ID NO:At least one set in 22, second specific probe are selected from SEQ ID NO:27~
SEQ ID NO:32, preferably SEQ ID NO:29.
According to an embodiment of the invention, the kit further includes alpha-casein.The alpha-casein is for resisting excrement
Just the interference that mortifier is remained in DNA, improves stability and the sensitivity of amplification.
According to an embodiment of the invention, the alpha-casein is cow's serum alpha-casein.
It is obtained by the present invention to have the beneficial effect that：
1. the present invention provides one kind using house-keeping gene as reference gene, the molecule diagnosis of detection SDC2 gene methylations
Kit.It reliably diagnoses colorectal cancer, and the sensitivity of detection reaches 90% or more, specificity reach 93% with
On.
2. detection method provided by the invention and kit are by methylation level come detect and diagnose cancer, and methyl
Change is the earliest events in tumour generating process, and detection methylates and can carry out the early screening of cancer.
3. the present invention is using excrement as detection sample, sample is easier, and any pain will not be caused to patient
And inconvenience.
Description of the drawings
Fig. 1 is to utilize primer SEQ ID NO.1-10 to SDC2 gene magnifications according to what one embodiment of the present of invention provided
Curve graph.
Fig. 2 is the primer SEQ ID NO.1-10 for SDC2 gene magnifications provided according to one embodiment of the present of invention
Melting curve figure.
Fig. 3 is to utilize primer SEQ ID NO.17-27 to β-actin genes according to what one embodiment of the present of invention provided
The curve graph of amplification.
Fig. 4 is the primer SEQ ID for β-actin gene magnifications provided according to one embodiment of the present of invention
The melting curve of NO.17-27.
Fig. 5 is to utilize primer SEQ ID NO.3-4 and probe SEQ ID according to what one embodiment of the present of invention provided
The SDC2 fluorescent PCR amplification curve diagrams that NO.11-16 is obtained.
Fig. 6 is to utilize primer SEQ ID NO.21-22 and probe SEQ according to what one embodiment of the present of invention provided
β-actin fluorescent PCR the amplification curve diagrams that ID NO.28-32 are obtained.
Specific implementation mode
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.
Molecule diagnosis in recent years is quickly grown in terms of cancer diagnosis.Has a large amount of colorectal cancer molecular marked compound at present
It is organizing and is being studied and reports in blood sample.Such as：Gene methylation, KRAS mutation etc..A kind of participation of SDC2 gene expressions
Cell division, migration and the protein expressed in colon mesenchymal cell.With the SDC2 mesh in pairs of adjacent nonneoplastic tissue
Mark region is compared, the notable higher of the methylation level of the target areas SDC2 in tumor tissues.
The present invention provides a kind of method and kit that detection SDC2 methylates, may be implemented colorectal cancer screening and
Diagnosis.The method that SDC2 gene methylation states are detected using the method for the invention and kit is simple, high sensitivity, specifically
Property is strong；It can effectively judge whether sample to be tested is colorectal cancer patients by detecting SDC2 gene methylations.
Accession number of the heretofore described SDC2 genes on the Genbank in NCBI is 6383.
The method of SDC2 gene methylations in detection excrement provided by the invention, including：DNA is extracted from excrement；By institute
It states DNA sample and carries out the processing that methylates, obtain methylating treated DNA；Using reference gene as standard, methylate to described
Treated, and DNA carries out fluorescence quantitative PCR detection, determines testing result；Wherein, when carrying out the fluorescence quantitative PCR detection,
Alpha-casein is added in the fluorescence quantitative PCR detection system.
The present invention detects the method that SDC2 methylates, by quantitative fluorescent PCR (qPCR) technology, in pcr amplification reaction
The real-time detection of each circulation products fluorescence signal is to realize and qualitatively analysis quantitative to starting template.
According to an embodiment of the invention, the first specific primer and the amplification of the first specific probe and detection are utilized respectively
SDC2 genes, first specific primer are selected from SEQ ID NO:1~SEQ ID NO:10, preferably SEQ ID NO:3 Hes
SEQ ID NO:4；The nucleic acid sequence of first specific probe is selected from SEQ ID NO:11~SEQ ID NO:16, preferably
SEQ ID NO:15。
According to an embodiment of the invention, using ACTB genes as reference gene.According to an embodiment of the invention, sharp respectively
It is selected from SEQ with the second specific primer and the amplification of the second specific probe and detection ACTB genes, second specific primer
ID NO:17~SEQ ID NO:27, preferably SEQ ID NO:21 and SEQ ID NO:22, the second specific probe choosing
From SEQ ID NO:28~SEQ ID NO:32, preferably SEQ ID NO:29.
According to an embodiment of the invention, it is connected separately on first specific probe and second specific probe
Fluorescent reporter group and quenching group, the fluorophor are selected from 6- Fluoresceincarboxylic acids, chlordene -6- methylfluoresceins, four chloro- 6-
Fluoresceincarboxylic acid, two chloro- 6- Fluoresceincarboxylic acids of 2,7- dimethyl -4,5-, fluorescein isothiocynate, Texas Red, Cys3, Cy5
Or one kind in VIC；The quenching group is selected from BHQ, 4- (4- dislikes amino benzeneazo) benzoic acid) or carboxyl tetramethyl Luo Dan
It is bright.5 ' the ends and 3 ' ends of specific probe are connected separately with fluorophor and fluorescent quenching group in the present invention.It is described glimmering
Light reporter group and fluorescent quenching group are the common group of those skilled in the art.When on probe simultaneously contain fluorescence report base
When group and fluorescent quenching group, fluorescence is not sent out；When specific probe is degraded by archaeal dna polymerase, fluorescent reporter group and glimmering
Optical quenching group separates, and sends out fluorescence.
Before fluorescence quantitative PCR detection, DNA is extracted from excrement first.Those skilled in the art can be utilized common
Method extracts the DNA in excrement.For example, the DNA in commercialized kit extraction excrement can be utilized.As a result, may be used
Ensure to obtain the DNA of high yield from fecal sample, and can efficiently remove the PCR inhibitor in fecal sample, under being suitable for
The detection of trip.Then the processing that methylates is carried out in the DNA to extraction, obtains the DNA handled through Hypermethylation.Such as it can utilize
Commercialized DNA methylation kit is handled.Thus, it is possible to further decrease methylate conversion process in host DNA
Loss late improves the detection sensitivity that low-copy host DNA methylates.
Kit
The kit that the present invention provides a kind of to detecting SDC2 gene methylations.In the kit in addition to comprising with
Except the specific primer of SDC genes and reference gene, specific probe, can also include：Hot Start Taq enzymes,
DNTPs, PCR amplification buffer solution etc..
According to an embodiment of the invention, the primer concentration is 500nM；A concentration of 250nM of TaqMan probe；It is described
A concentration of 1U/ μ L's of Taq enzyme；A concentration of 0.2mM of dNTPs；The excrement host DNA amount through Hypermethylation is 1~20 μ
L；Inventor obtains above-mentioned more excellent concentration proportioning by many experiments, as a result, with further increase specific amplification, stability or
Sensitivity.
Kit of the present invention can be used for detecting the first of SDC2 genes after fluorescence quantitative PCR detection is combined
Base state.SDC2 gene hypermethylations are the important molecule marks of colorectal cancer, therefore kit of the present invention is being tied
It is of great significance in carcinoma of the rectum diagnosis.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it is carried out according to technology or condition described in document in the art or according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
The extraction of 1 fecal sample of embodiment
The DNA in people source in excrement is extracted using kit, the extraction of used people's source DNA is to use commercial kit
QIAamp Fast DNA Stool Mini Kit, article No. 51604.The fecal sample is through the detection of clinical colonoscopy and pathology
Screening is determined as the fecal sample of the patient of colorectal cancer.Its specific extraction flow can refer to the specification of commercial reagents box into
Row.
According to QIAamp Fast DNA Stool Mini Kit kit standard operating processes：1mL is added into sample
To the abundant homogeneous of sample, 16000g centrifuges 1min by InhibitEX Buffer, vortex 1min；Take 25 μ L Proteinase Ks to it is new from
Heart pipe, and 600 μ L backs supernatants are drawn to Proteinase K pipe, 600 μ L Buffer AL and the 15s that is vortexed is added, is placed in 70 DEG C and incubates
Educate 10min；600 μ L ethyl alcohol, and the mixing that is vortexed is added, in three times QIAamp spin column, is centrifuged every time in 16000g
1min；500 μ L Buffer AW1 are carefully added on column, centrifuges 1min in 16000g, removes and collect liquid in pipe；To on column
500 μ L Buffer AW2 are added, centrifuges 3min in 16000g, removes and collect liquid in pipe；Continue to centrifuge 3min in 16000g,
QIAamp spin column are moved to new 1.5ml centrifuge tubes, 200 μ L Buffer ATE are added, are placed at room temperature for 1min, and
1min is centrifuged in 16000g, the DNA extracted from fecal sample is obtained, is used for subsequent operation.
2 sulphite of embodiment converts
The processing that methylates is carried out by the host DNA extracted in excrement to what is obtained in embodiment 1.The DNA methylation
Processing uses the EZ DNA Methylation Kit that commercial kit produces for ZYMO Research companies, article No.
D5005.During methylate processing, all Cytosines not methylated can be by bisulfites
Uracil, and the reaction does not occur for the cytimidine to methylate.Specifically, if in SDC2 gene orders in sample genome
Cytimidine (C) is not methylated, then after the treatment kits processing that methylates, the C in SDC2 gene orders sports U, then
After Hypermethylation is handled, subsequently during carrying out quantitative fluorescent PCR, specific probe can not be with place with specific primer
Sample DNA after reason combines, and during Fluorescence PCR, probe keeps complete, due to being connected with 5 ' fluorescent reporter groups simultaneously
With 3 ' fluorescent quenching groups, Fluorescence PCR does not send out fluorescence in the process.I.e. after the treatment kits processing that methylates, methyl
The SDC2 genes of change can send out fluorescence during fluorescence quantitative PCR detection, and the SDC2 genes without methylating do not send out glimmering
Light.
Wherein utilize methylating reagent box processing procedure as follows：
Zymo Research kit conversion process：130 μ L CT are added in 20 good μ L DNA samples of said extracted
Conversion Reagent (now with the current), after vortex mixing according to (98 DEG C, 10min；64 DEG C, 2.5h) condition turned
Change reaction；600 μ L M-Binding Buffer are added in adsorption column, then overturn number by the sample addition system of conversion again
Secondary mixing, 10000rpm centrifuge 30s, absorb lower layer's solution；100 μ L M-Wash Buffer are added into adsorption column,
10000rpm centrifuges 1min, absorbs lower layer's solution；Into adsorption column, 200 μ L M-Desulphonation Buffer, room temperature are put
20min is set, then 10000rpm centrifuges 1min, absorbs lower layer's solution；200 μ L M-Wash Buffer are added into adsorption column,
10000rpm centrifuges 1min, absorbs lower layer's solution；It repeats the above steps；Adsorption column is transferred to new collecting pipe, adds 15 μ L M-
After being placed at room temperature for 5min, 1min is centrifuged in 10000rpm by Elution Buffer.DNA sample is for subsequently examining after being converted
It surveys.
Embodiment 3qPCR detection process
The DNA after bisulfite conversion that embodiment 2 obtains is subjected to quantitative fluorescent PCR reaction (qPCR),
The system and program of middle qPCR is respectively as shown in table 1, table 2.Simultaneously in order to screen optimal specific primer, it is utilized respectively difference
Specific primer SDC2 genes and β-actin genes are expanded respectively, obtain corresponding amplification curve and melting
Curve, it is as shown in Figure 1 to 4 respectively.Wherein used specific primer sequence is as shown in table 3.
1 qPCR detection architectures of table
| Ingredient | Addition (uL) |
| SDC2 forward primers (10 μM) | 2 |
| SDC2 reverse primers (10 μM) | 2 |
| β-actin forward primers (10 μM) | 2 |
| β-actin reverse primers (10 μM) | 2 |
| 5U/μL HotStarTaq Plus DNA Polymerase | 2.5 |
| 5 × PCR amplification buffer solution | 10 |
| DNA profiling after conversion | 10 |
| 10×SYBR Green | 5 |
| Sterile deionized water | 14.5 |
| It is total | 50 |
Wherein, the ingredient of 5 × PCR amplification buffer solution is：50mM Tris-HCl(pH8.3)、250mM KCl、12.5mM
MgCl2, 2mg/ml alpha-caseins, 1mM dNTPs, 100mM tetramethyl ammonium chlorides, 2.5%Tween-20.
2 qPCR programs of table
3 SDC2＆ β-actin primer sequences of table
In table 3, odd number represents forward primer, and even number represents reverse primer.Include wherein adjacent odd number per pair of primers
And even number, i.e. SEQ ID NO:1 and SEQ ID NO:2 be pair of primers；SEQ ID NO:3 and SEQ ID NO:4 be another pair
Primer, and so on.Wherein Fig. 1 and 2 is the amplification curve and melting curve of NO.1~10 SDC2 gene primer SEQ ID；From
It can be seen from the figure that SEQ ID NO.3, SEQ ID NO.4 amplification efficiencies are good and without non-specific amplification.
Fig. 3 and 4 is the amplification curve and melting curve of NO.17~26 ACTB gene primer SEQ ID；It can from figure
Go out, amplification efficiency is preferably and the primer sequence without non-specific amplification is SEQ ID NO.21,22.
The optimization of 4 probe of embodiment
In order to further verify the probe of SDC2 and reference gene, according to fluorescent quantitative PCR result respectively to the probe of table 5
Sequence is screened.The wherein system of qPCR and program is respectively as shown in table 4, table 2.Probe sequence difference for SDC2 detections
For SEQ ID NO:11~SEQ ID NO:16, and fluorescent reporter group FAM and fluorescence are connected separately on the specific probe
Quenching group BHQ1, FAM and BHQ are connected to the 5 ' ends and 3 ' ends of specific probe sequence.Fluorescence on specific probe
Reporter group FAM sends out green fluorescence, and when the sequence in probe can not match clock synchronization with the DNA profiling in solution to be measured, probe is protected
Guarantee is held, i.e., contains fluorescent reporter group FAM and fluorescent quenching group BHQ1 simultaneously, fluorescence can not be detected after PCR amplification；And
When with the DNA in solution to be measured base pair complementarity can occur for probe, probe is degraded by Taq archaeal dna polymerases, probe 5 '
The reporter group at end is detached with the quenching group at 3 ' ends, and fluorescence is able to detect that during PCR amplification.Probe sequence is with SDC2
DNA sequence dna after gene hyper-methylation is stencil design,
Similarly, it is respectively SEQ ID NO.28~SEQ ID NO for Gene A CTB probes:32, and the specificity is visited
It is connected separately with fluorescent reporter group VIC on needle and fluorescent quenching group MGB, VIC and MGB are connected to specific probe sequence
5 ends of row ' and 3 ' ends.
4 qPCR detection architectures of table
| Ingredient | Addition (uL) |
| SDC2 forward primers (10 μM) | 2 |
| SDC2 reverse primers (10 μM) | 2 |
| SDC2 probes (10 μM) | 1 |
| β-actin forward primers (10 μM) | 2 |
| β-actin reverse primers (10 μM) | 2 |
| β-actin probes (10 μM) | 1 |
| 5U/μL HotStarTaq Plus DNA Polymerase | 2.5 |
| 5 × PCR amplification buffer solution | 10 |
| DNA profiling after conversion | 10 |
| Sterile deionized water | 17.5 |
| It is total | 50 |
Wherein, the ingredient of 5 × PCR amplification buffer solution is：50mM Tris-HCl(pH8.3)、250mM KCl、12.5mM
MgCl2, 2mg/ml alpha-caseins, 1mM dNTPs, 100mM tetramethyl ammonium chlorides, 2.5%Tween-20.
5 probe sequence of table
Fig. 5, which gives, utilizes specific primer SEQ ID NO:3 and SEQ ID NO:4 are expanded, and are then utilized respectively
Specific probe SEQ ID NO:11~SEQ ID NO:16 carry out the result of quantitative detection.As seen from Figure 5, probe SEQ
NO.11~16 ID can ensure that PCR processes are normally carried out, compare for, probe SEQ ID NO.15 amplification efficiencies are high by one
A bit.
Fig. 6, which gives, utilizes specific primer SEQ ID NO.21 and SEQ ID NO:22 are expanded, then sharp respectively
With specific probe SEQ ID NO.28~SEQ ID NO:32 carry out the result of quantitative detection.By result it is found that probe SEQ
NO.28~32 ID can ensure that PCR processes are normally carried out, compare for, the fluorescence signal higher of probe SEQ ID NO.29
Some, help to improve the recall rate of sample.
To sum up, for the primer sequence and probe sequence designed by the islands CpG of the gene promoter areas SDC2, work as primer sequence
When different, the methylation sites difference for including in obtained amplified fragments and the number etc. to methylate are all different, finally
It is different to the sensitivity of colorectal cancer detection.
5 pattern detection of embodiment
Clinic 30 colonoscopies of collection and pathologic finding are diagnosed as the fecal sample of the patient of intestinal cancer, and 30 enteroscopies are made a definite diagnosis
For the fecal sample of normal subject.DNA extractions are carried out to above-mentioned fecal sample according to the method in embodiment 1, according to reality
The method applied in example 2 carries out sulphite conversion to the DNA after extraction, is then utilized respectively specific primer SEQ ID NO:3
With SEQ ID NO:4 and specific probe SEQ ID NO:15 pairs of SDC2 genes carry out fluorescence quantitative PCR detections, using special
Property primer SEQ ID NO:21 and SEQ ID NO:22 and specific probe SEQ ID NO:29 pairs of β-actin genes carry out glimmering
Fluorescent Quantitative PCR detects.
Wherein, sample number 1-30 comes from patients with bowel cancer, and sample number 31-60 comes from normal subject, detection
The results are shown in table below：
6 SDC2 testing results of table are compared with clinical effectiveness
Then specificity and sensitivity is calculated as follows,
Specificity=true negative number/(true negative number+false positive number)) * 100%=28/30*100%=
93.3%
Sensitivity=true negative number/(true negative number+false positive number)) * 100%=27/30*100=90%
According to result of calculation it is found that for colorectal cancer, the detection sensitivity of SDC2 genes is 90%, and specificity is
93.3%.
In the description of the present invention, it is to be understood that, term "center", " longitudinal direction ", " transverse direction ", " length ", " width ",
" thickness ", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom" "inner", "outside", " up time
The orientation or positional relationship of the instructions such as needle ", " counterclockwise ", " axial direction ", " radial direction ", " circumferential direction " be orientation based on ... shown in the drawings or
Position relationship is merely for convenience of description of the present invention and simplification of the description, and does not indicate or imply the indicated device or element must
There must be specific orientation, with specific azimuth configuration and operation, therefore be not considered as limiting the invention.
In addition, term " first ", " second " are used for description purposes only, it is not understood to indicate or imply relative importance
Or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be expressed or
Implicitly include at least one this feature.In the description of the present invention, the meaning of " plurality " is at least two, such as two, three
It is a etc., unless otherwise specifically defined.
In the present invention unless specifically defined or limited otherwise, term " installation ", " connected ", " connection ", " fixation " etc.
Term shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or integral；Can be that machinery connects
It connects, can also be to be electrically connected or can communicate each other；It can be directly connected, can also indirectly connected through an intermediary, it can be with
It is the interaction relationship of the connection or two elements inside two elements, unless otherwise restricted clearly.For this field
For those of ordinary skill, the specific meanings of the above terms in the present invention can be understood according to specific conditions.
In the present invention unless specifically defined or limited otherwise, fisrt feature can be with "above" or "below" second feature
It is that the first and second features are in direct contact or the first and second features pass through intermediary mediate contact.Moreover, fisrt feature exists
Second feature " on ", " top " and " above " but fisrt feature be directly above or diagonally above the second feature, or be merely representative of
Fisrt feature level height is higher than second feature.Fisrt feature second feature " under ", " lower section " and " below " can be
One feature is directly under or diagonally below the second feature, or is merely representative of fisrt feature level height and is less than second feature.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
It can be combined in any suitable manner in a or multiple embodiments or example.In addition, without conflicting with each other, the technology of this field
The feature of different embodiments or examples described in this specification and different embodiments or examples can be combined by personnel
And combination.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
SEQUENCE LISTING
<110>The Shanghai bio tech ltd Rui Yi
<120>Detect the method and kit of SDC2 gene methylations
<130> PIDC4180063
<160> 32
<170> PatentIn version 3.5
<210> 1
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<212> DNA
<213>Artificial sequence
<400> 1
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aatcgttgcg gtattttgtt tcgga 25
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ccgcgcacac gaatccgaaa caaaataccg c 31
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Claims (10)
1. a kind of method of SDC2 gene methylations in detection sample to be tested, which is characterized in that including：
DNA is extracted from the sample to be tested；
The DNA sample is subjected to the processing that methylates, obtain methylating treated DNA；
Using reference gene as standard, methylates that treated to described DNA carries out fluorescence quantitative PCR detection, determine detection knot
Fruit；
Wherein, when carrying out the fluorescence quantitative PCR detection, alpha-casein is added in the fluorescence quantitative PCR detection system.
2. according to the method described in claim 1, it is characterized in that, the reference gene be ACTB genes or GADPH genes,
Preferably ACTB genes.
3. method according to claim 1 or 2, which is characterized in that the alpha-casein is in the fluorescence quantitative PCR detection
A concentration of 0.1~1mg/mL in system, preferably 0.4mg/mL.
4. method described in any one of claim 1 to 3, which is characterized in that the alpha-casein is cow's serum α-junket
Albumen.
5. method according to any one of claims 1 to 4, which is characterized in that carried out to the DNA using sulphite
Methylate processing.
6. method according to any one of claims 1 to 5, which is characterized in that the sample to be tested is fecal sample.
7. according to method according to any one of claims 1 to 6, which is characterized in that be utilized respectively the first specific primer and
First specific probe expands and detection SDC2 genes, first specific primer are selected from SEQ ID NO:1~SEQ ID
NO:At least one set in 10, preferably SEQ ID NO:3 and SEQ ID NO:4,
The nucleic acid sequence of first specific probe is selected from SEQ ID NO:11~SEQ ID NO:16, preferably SEQ ID
NO:15。
8. according to the method described in claim 2, it is characterized in that, being utilized respectively the second specific primer and the second specificity spy
Needle expands and detection ACTB genes, second specific primer are selected from SEQ ID NO:17~SEQ ID NO:26, preferably
SEQ ID NO:21 and SEQ ID NO:At least one set in 22, second specific probe are selected from SEQ ID NO:27~
SEQ ID NO:32, preferably SEQ ID NO:29.
9. a kind of kit of detection SDC2 gene methylations, which is characterized in that special including the first specific primer and first
Property probe, first specific primer be selected from SEQ ID NO:1~SEQ ID NO:At least one set in 10, preferably SEQ
ID NO:3 and SEQ ID NO:4,
The nucleic acid sequence of first specific probe is selected from SEQ ID NO:11~SEQ ID NO:16, preferably SEQ ID
NO:15；
Optionally, further include the second specific primer and the second specific probe for expanding and detecting reference gene ACTB,
Second specific primer is selected from SEQ ID NO:17~SEQ ID NO:At least one set in 26, preferably SEQ ID
NO:21 and SEQ ID NO:22,
Second specific probe is selected from SEQ ID NO:27~SEQ ID NO:32, preferably SEQ ID NO:29.
10. kit according to claim 9, which is characterized in that further include alpha-casein, preferably cow's serum α-junket egg
In vain.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699437A (en) * | 2019-11-11 | 2020-01-17 | 益善生物技术股份有限公司 | Human SDC2 gene methylation detection kit |
| CN113981046A (en) * | 2021-11-05 | 2022-01-28 | 朱运峰 | DNA methylation detection method based on quantitative PCR technology and kit thereof |
| CN114008219A (en) * | 2019-06-18 | 2022-02-01 | 基因特力株式会社 | Method for detecting methylation of SDC2 gene |
| CN114207153A (en) * | 2020-03-20 | 2022-03-18 | 上海鹍远健康科技有限公司 | Method and kit for screening colorectal tumors |
| CN114672565A (en) * | 2022-04-01 | 2022-06-28 | 山东鲁抗好丽友生物技术开发有限公司 | Double-probe composition for detecting colorectal cancer and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104812899A (en) * | 2012-11-07 | 2015-07-29 | 基因特力株式会社 | Method for detecting gastric polyp and gastric cancer using marker gene of gastric polyp and gastric cancer-specific methylation |
| CN105452485A (en) * | 2013-08-14 | 2016-03-30 | 基因特力株式会社 | Method for detecting precancerous lesions |
| CN105543354A (en) * | 2015-12-31 | 2016-05-04 | 广州市康立明生物科技有限责任公司 | Tumor molecular detection/diagnosis reagent |
| CN106893777A (en) * | 2017-03-02 | 2017-06-27 | 武汉艾米森生命科技有限公司 | For detecting many site methylating reagent boxes of colorectal cancer related gene and application |
-
2018
- 2018-05-21 CN CN201810490724.7A patent/CN108642180A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104812899A (en) * | 2012-11-07 | 2015-07-29 | 基因特力株式会社 | Method for detecting gastric polyp and gastric cancer using marker gene of gastric polyp and gastric cancer-specific methylation |
| CN105452485A (en) * | 2013-08-14 | 2016-03-30 | 基因特力株式会社 | Method for detecting precancerous lesions |
| CN105543354A (en) * | 2015-12-31 | 2016-05-04 | 广州市康立明生物科技有限责任公司 | Tumor molecular detection/diagnosis reagent |
| CN106893777A (en) * | 2017-03-02 | 2017-06-27 | 武汉艾米森生命科技有限公司 | For detecting many site methylating reagent boxes of colorectal cancer related gene and application |
Non-Patent Citations (4)
| Title |
|---|
| BARBARA KINGA BARTAK 等: "Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1, SFRP2,SDC2, and PRIMA1 in plasma samples", 《EPIGENETICS》 * |
| FENG NIU 等: "Stool DNA Test of Methylated Syndecan-2 for the Early Detection of Colorectal Neoplasia", 《CANCER EPIDEMIOL BIOMARKERS PREV》 * |
| TAE JEONG OH 等: "Feasibility of quantifying SDC2 methylation in stool DNA for early detection of colorectal cancer", 《CLINICAL EPIGENETICS》 * |
| 卢洪洲 等: "《医学真菌检验与图解》", 31 January 2018, 上海科学技术出版社 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114008219A (en) * | 2019-06-18 | 2022-02-01 | 基因特力株式会社 | Method for detecting methylation of SDC2 gene |
| CN110699437A (en) * | 2019-11-11 | 2020-01-17 | 益善生物技术股份有限公司 | Human SDC2 gene methylation detection kit |
| CN114207153A (en) * | 2020-03-20 | 2022-03-18 | 上海鹍远健康科技有限公司 | Method and kit for screening colorectal tumors |
| EP3911756A4 (en) * | 2020-03-20 | 2022-08-10 | Singlera Health Technologies (Shanghai) Ltd. | Methods and kits for screening colorectal neoplasm |
| CN113981046A (en) * | 2021-11-05 | 2022-01-28 | 朱运峰 | DNA methylation detection method based on quantitative PCR technology and kit thereof |
| CN114672565A (en) * | 2022-04-01 | 2022-06-28 | 山东鲁抗好丽友生物技术开发有限公司 | Double-probe composition for detecting colorectal cancer and application thereof |
| CN114672565B (en) * | 2022-04-01 | 2023-01-20 | 山东鲁抗好丽友生物技术开发有限公司 | Double-probe composition for detecting colorectal cancer and application thereof |
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