CN106521007A - Reagent system and kit for detecting methylation of BMP3 genes and application of reagent system and kit - Google Patents
Reagent system and kit for detecting methylation of BMP3 genes and application of reagent system and kit Download PDFInfo
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Abstract
The invention relates to the technical field of medical examination, in particular to a reagent system and kit for detecting methylation of BMP3 genes and application of the reagent system and the kit. The reagent system comprises specific primers and a specific probe, wherein the specific primers are as shown in SEQ ID NO. 1 and SEQ ID NO. 2; and the specific probe is as shown in SEQ ID NO. 3. The specific primers SEQ ID NO. 1 and SEQ ID NO. 2 and the specific probe SEQ ID NO. 3 are applied to preparation of a reagent or kit for detecting methylation of BMP3 genes. The kit is qualified accurately, and has the advantages of high sensitivity of PCR, specificity of DNA hybridization and accurate and quantitative spectrum technologies. Results are visible, detecting speed is high, and change in a PCR process can be detected directly. Compared with common PCR, the reagent system has the characteristics that the results can be observed in real time, products do not require to be detected by gel electrophoresis, complete closed-tube operation is implemented, and pollution is reduced effectively.
Description
Technical field
The present invention relates to technical field of medical examination, and in particular to a kind of BMP3 gene methylations detection reagent system and
Kit and its application.
Background technology
Colorectal cancer is the higher malignant tumour of the current incidence of disease, and in European and American developed countries, its incidence of disease occupies malignant tumour
2nd, lung cancer is only second to, is also modal gastrointestinal cancer.In recent years, China's colorectal cancer incidence rate and the death rate
In continuous ascendant trend, the death rate is malignant tumour the 5th.Early stage colorectal cancer patients 5 years survival rates Jing after treatment are reachable
More than 90%, but often early stage colorectal cancer patients seldom have clinical symptoms, are difficult to be found.And advanced colorectal cancer patient
Survival rate is less than 10% within 5 years.Therefore, a test method for being effectively easy to examination and early diagnosis colorectal cancer is found, it is right
The treatment of colorectal cancer even healing has very important significance.Current conventional colorectal cancer examination, diagnostic method, such as excrement are hidden
There is the deficiency of specificity, sensitiveness or patient compliance in blood test and colonoscopy etc., limit which in colorectal cancer
Extensive application in examination and early diagnosis.
The generation of many tumours in tumor research is all accompanied by methylating for gene, the demethylation and suppression cancer of oncogene
The methylation state of gene, can cause activation, the inactivation of tumor suppressor gene of oncogene.The hypomethylation and tumor suppressor gene of oncogene
Hyper-methylation change be tumour cell a key character.Therefore the present invention have selected related to colorectal cancer generation
BMP3 gene methylation states are determining the generation of colorectal cancer.Methylating for BMP3 genes is determined through a large amount of clinical testings
The generation of colorectal cancer can be caused, by detecting the BMP3 gene methylation states come off in cancer cell in a large number in patient's enteron aisle,
So as to diagnose the generation of colorectal cancer.The present invention adopts advanced molecular diagnosis method, using fluorescent PCR method early stage, quick, letter
Just, sensitive, specific detection colorectal cancer.
Fluorescence PCR assay took the lead in succeeding in developing by PE companies of the U.S. in nineteen ninety-five, and it is miscellaneous that it has the high sensitivity of PCR, DNA concurrently
The advantage of the specificity and spectral technique accurate quantification of friendship, visual result, the change during energy direct detection PCR.With it is common
PCR is compared, its result can Real Time Observation, product do not need detected through gel electrophoresis, and complete stopped pipe operation significantly reduces dirt
Dye.
The content of the invention
The present invention need solve prior art problem be：Colorectal cancer early diagnosis is difficult, the diagnosis side of prior art
Method sensitiveness is low, positive rate is low and inconvenient, not yet has the examination that can quickly, easily detect BMP3 gene methylation states
Agent.
In order to solve the above problems, the invention provides a kind of BMP3 gene methylations detection reagent system and kit
And its application, difficult for solving existing diagnosis of colorectal carcinoma, sensitiveness is low, positive rate is low and inconvenient problem.
Specifically, for the deficiencies in the prior art, the invention provides following technical scheme：
On the one hand, the invention provides a kind of BMP3 gene methylations detection reagent system, the reagent system includes
Specific primer and specific probe, the specific primer are as shown in SEQ ID NO.1 and SEQ ID NO.2, described special
Property probe is as shown in SEQ ID NO.3.
Preferably, fluorophor and quenching group are connected with the specific probe, wherein, described fluorophor choosing
From 6- Fluoresceincarboxylic acids, chlordene -6- methylfluoresceins, four chloro- 6- Fluoresceincarboxylic acids, two chloro- 6- carboxyls of 2,7- dimethyl -4,5-
Fluorescein, fluorescein isothiocynate, the one kind in Texas Red, Cy3, Cy5 or VIC；Described quenching group is selected from BHQ, 4-
(4- dislikes amino benzeneazo) benzoic acid) or carboxyl tetramethylrhodamine.
Preferably, the reagent system also includes positive criteria product, contains and insert BMP3 bases in the positive criteria product
Because of the recombinant plasmid of methylation-specific sequence SEQ ID NO.4.
Preferably, reagent system also includes negative standards' product, contains and be not inserted into BMP3 genes in described negative standards' product
The plasmid of methylation-specific sequence.
Preferably, the concentration of the recombinant plasmid is 1.0 × 103-1.0×104Copies/ μ L, in negative standards' product
Plasmid concentration is 1.0 × 103-1.0×104copies/μL。
Preferably, described plasmid or recombinant plasmid are identical, in pETs, pUCs, pGM-T, pBR322 or pMD-T
One kind, preferably pGM-T or pBR322.
The preparation method of the plasmid in recombinant plasmid and negative standards' product preferably, in the positive criteria product is such as
Under：
(1) with sequence SEQ ID NO.4 as template, expanded using primer sequence SEQ ID NO.5 and SEQ ID NO.6
Increase, obtain genes of interest；
(2) genes of interest is connected on plasmid vector, obtains connection product；
(3) connection product is transformed in host cell；
(4) screening contains the recombinant plasmid for building as positive criteria product；Recombinant plasmid conduct without genes of interest
Negative standards' product.
Preferably, described reagent system also includes buffer solution and dNTPs, and described buffer solution includes trihydroxy methyl amino
Methane, potassium chloride and magnesium chloride.
Preferably, the concentration of described trishydroxymethylaminomethane is 50～200mM；The concentration of potassium chloride be 300～
800mM；The concentration of magnesium chloride is 20～50mM.
Preferably, the concentration of described trishydroxymethylaminomethane is 100～150mM；The concentration of potassium chloride be 400～
600mM；The concentration of magnesium chloride is 30～40mM.
Preferably, described reagent system also includes archaeal dna polymerase.
Preferably, described archaeal dna polymerase is Taq DNA polymerase.
Second aspect, the invention provides a kind of BMP3 gene methylations detection kit, which includes any of the above item
Described reagent system.
The third aspect, the invention provides specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe
SEQ ID NO.3 are preparing BMP3 gene methylations detection reagent or the application in kit.
Preferably, the invention provides specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe
SEQ ID NO.3 are preparing BMP3 gene methylation colorectal cancer detection reagents or the application in kit.
Preferably, in use above, the using method of described BMP3 gene methylations detection reagent or kit includes
Following steps：
(1) process that methylates is carried out to sample DNA；
(2) it is using specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ IDNO.3, right
The sample DNA of the process that methylates that step (1) is obtained carries out quantitative fluorescent PCR reaction, detects the methylation level of sample DNA.
Preferably, in use above, the using method also includes：
The recombinant plasmid for inserting BMP3 gene methylation specific sequence SEQ ID NO.4 will be contained as positive criteria
Product, carry out quantitative fluorescent PCR reaction；
The plasmid for being not inserted into BMP3 gene methylation specific sequence SEQ ID NO.4 will be contained as negative standards' product,
Carry out quantitative fluorescent PCR reaction.
Preferably, in use above, the preparation method of the sample DNA by the process that methylates comprises the steps：
(1) genomic DNA is extracted from fecal sample；
(2) using bisulf iotate-treated genomic DNA, the sample DNA of the process that obtains methylating.
Preferably, in use above, described quantitative fluorescent PCR reaction condition is：94～95 DEG C of 2～5min of denaturation, 1
Individual circulation；94～95 DEG C of 15～20s, 53～55 DEG C of 30～50s, 58～60 DEG C of 30～50s, 40～50 circulations
Beneficial effects of the present invention are：
(1) kit of the present invention accurately, has the high sensitivity of PCR, the specificity of DNA hybridization and spectral technique essence concurrently
It is determined that the advantage of amount, visual result, the change during energy direct detection PCR, compared with regular-PCR, its result can be seen in real time
Examine, product does not need detected through gel electrophoresis, complete stopped pipe operation to significantly reduce pollution.
(2) kit detection sensitivity of the present invention and specificity are high, the dual fail-safe due to taking specific primer and probe
Design, sensitivity and specificity improve a lot, and can detect colorectal cancer before clinical symptoms occur.
(3) kit detection speed of the present invention is fast, only needs altogether 2 hours, and step is simple, can carry out high flux sample simultaneously
This detection.
Below in conjunction with the accompanying drawings with each specific embodiment, the present invention and its Advantageous Effects are described in detail,
Wherein：
Description of the drawings
Fig. 1 is the fluorescent PCR amplification curve of the colorectal cancer positive criteria product that the embodiment of the present invention one is provided and negative mark
The fluorescent PCR amplification curve of quasi- product.Wherein numbering 1 is positive criteria product, and numbering 2 is negative standards' product.
Fig. 2 is the fluorescent PCR amplification curve of volunteer's sample that the embodiment of the present invention two is provided.Wherein, numbering is 5,6,7
Pattern detection result be colorectal cancer sample, numbering be 3,4 pattern detection result be non-colorectal cancer sample.
Specific embodiment
As described above, the invention provides a kind of BMP3 gene methylations detection reagent system and kit, its purpose
It is to improve the sensitivity of tested crowd's diagnosis of colorectal carcinoma and specificity, there is provided the reliability of early diagnosis.
Specifically, the invention provides a kind of BMP3 gene methylations colorectal cancer detection reagent system, described
Kit includes specific primer SEQ ID the NO.1 and SEQ ID NO.2 and specific probe SEQ for colorectal cancer detection
ID NO.3。
Specifically, the invention provides the reagent system and kit of a kind of detection of BMP3 gene methylations, described
Contain reactant liquor and archaeal dna polymerase in reagent system.Contain specific primer SEQ ID NO.1 and SEQ ID in reactant liquor
NO.2, and specific probe SEQ ID NO.3；The 5 ' of specific probe and 3 ' ends are connected to fluorophor and fluorescence is quenched
Go out group.The group that the fluorophor and fluorescent quenching group are commonly used for those skilled in the art.When containing on probe simultaneously
When fluorescent reporter group and fluorescent quenching group, fluorescence is not sent；When specific probe is degraded by archaeal dna polymerase, fluorescence report
Accuse group and fluorescent quenching group to separate, send fluorescence.The solvent also needed containing PCR in reactant liquor,
Such as buffer solution and dNTPs.
Wherein in a preferred embodiment, the buffer solution in reactant liquor consists of 50～200mM trihydroxy methyl amino first
Alkane, 300～800mM potassium chloride and 10～30mM magnesium chlorides.Archaeal dna polymerase in reagent system is Taq DNA polymerase.
In another preferred embodiment, the invention provides a kind of restructuring matter containing BMP3 gene methylation sequences
Grain, as shown in SEQ ID NO.4, the plasmid is selected from pETs, pUCs, pGM-T, pBR322 to the BMP3 gene methylations sequence
With the one kind in pMD-T.In recombinant plasmid screening process, the restructuring matter successfully containing BMP3 gene methylation sequences is converted
Grain loads in positive criteria QC, as positive control；The empty plasmid for being not converted into work(loads in negative standards' QC, makees
For negative control.
In another preferred embodiment, the invention provides a kind of BMP3 gene methylations detection kit, described
Kit includes the positive criteria product as positive control, used as negative standards' product of negative control, reactant liquor and DNA polymerizations
Enzyme.Reactant liquor is identical with the reactant liquor in the reagent system that aforementioned BMP3 gene methylations are detected, equally draws containing specificity
Thing SEQ ID NO.1 and SEQ ID NO.2, and specific probe SEQ ID NO.3, reactant liquor are consisted of：Every 16 μ L are anti-
The SEQ ID NO.1 that contain in answering liquid, SEQ ID NO.2, the amount of SEQ ID NO.3 and dNTP are followed successively by 10-15 μM, 10-15 μ
M, 10-15 μM and 2.5-3.0mM.
Sample cell in kit built with from excrement extract, and Jing methylate process after DNA sample.As the positive
The recombinant plasmid of standard items and the plasmid screening technique as negative standards' product are comprised the following steps：
(1) with sequence SEQ ID NO.4 as template, performing PCR amplification, the primer sequence such as SEQ ID are entered using primer
Shown in NO.5 and SEQ ID NO.6；
(2) pcr amplification product is connected on plasmid；
(3) it is transformed in Escherichia coli；
(4) screening loads in positive criteria QC containing the recombinant plasmid for building, and the loading without recombinant plasmid is negative
In control QC.
The situation of BMP3 gene methylations is detected using kit of the present invention, detection method is comprised the following steps：Sample
Product are pre-processed, and the sample after process is added in sample cell.Solution to be measured is prepared, i.e., toward positive criteria QC, negative standards' product
Reactant liquor and archaeal dna polymerase are separately added in pipe and sample cell, are mixed；Sample size or positive criteria product in each group solution to be measured
Content or negative standards' product content are identical, and the content of reactant liquor and archaeal dna polymerase is also identical；Generally, solution middle-jiao yang, function of the spleen and stomach to be measured
Property standard items, negative standards' product or sample and reactant liquor, the ratio of archaeal dna polymerase are：(1-2)μL：(18-20)μL：(5-20)
U.Solution to be measured is put in quantitative real time PCR Instrument and is measured, the condition that PCR is determined is：94～95 DEG C of denaturations 2～
5min, 1 circulation；94～95 DEG C of 15～20s, 53～55 DEG C of 30～50s, 58～60 DEG C of 30～50s, totally 40～50 circulations；
Whether ultimate analysis quantitative fluorescent PCR measurement result, judgement sample supplier suffer from colorectal cancer.
Sample treatment before detection includes, is extracted first with genome DNA extracting reagent kit from fecal sample
DNA, recycles genomic DNA methylation level treatment kits to process and extracts the DNA for obtaining.The principle of the treatment kits that methylate is
Bisulfite method methylates process, and the DNA Jing after the treatment kits that methylate are processed is used as sample.Sample in the present invention
DNA derives from the faeces DNA of people through the methylated process of bisulfite method.Sample genomic dna Jing methylates and processes examination
After the process of agent box, if the cytimidine (C) in the BMP3 gene orders in sample genome is methylated, Jing methylates place
Reason kit process after BMP3 gene orders it is constant, as shown in SEQ ID NO.4, specific primer SEQ ID NO.1 and
SEQ ID NO.2 and specific probe SEQ ID NO.3 can be combined with SEQ ID NO.4, during Fluorescence PCR,
5 ' fluorophors in the presence of archaeal dna polymerase on probe SEQ ID NO.3 are separated with 3 ' quenching groups, send fluorescence；
If the cytimidine (C) in the BMP3 gene orders in sample genome is no methylated, Jing methylates process
After kit process, the C in BMP3 gene orders becomes U, that is, methylate BMP3 gene orders and SEQ ID NO.4 after processing
Difference, specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ ID NO.3 cannot with process after
Sample DNA combine, during Fluorescence PCR, probe SEQ ID NO.3 keep complete, due to being connected with 5 ' fluorescence simultaneously
Group and 3 ' quenching groups, do not send fluorescence during Fluorescence PCR；
I.e. Jing after the treatment kits that methylate, methylated BMP3 genes can be sent during fluorescence quantitative PCR detection
Fluorescence, does not fluoresce without methylated BMP3 genes.Therefore, reagent system of the present invention and kit are fixed with fluorescence
After amount PCR detections combine, can be used for detecting the methylation state of BMP3 genes.BMP3 gene hypermethylations are Colon and rectums
The important molecule mark of cancer, therefore reagent system of the present invention and kit and its use have weight in diagnosis of colorectal carcinoma
Want meaning.
The method that BMP3 gene methylation states are detected using kit of the present invention is simple, and sensitivity is high, specificity
By force；By detecting whether BMP3 gene methylations can judgement sample donor be effectively colorectal cancer patients.
It should be noted that unaccounted normal condition and method in embodiment, generally according to art experimenter
Conventional method, specifically can refer to Pehanorm Brooker and Russell chief editor《Molecular Cloning:A Laboratory guide》The third edition, or according to
Step and condition proposed by manufacturer.
The present invention will be further described in detail with specific embodiment below in conjunction with the accompanying drawings, used wherein in embodiment
Reagent and instrument producer it is as follows：
Quantitative real time PCR Instrument, producer：American AB I real-time fluorescence quantitative PCR instrument 7500, the U.S.；
DNTPs, producer：TAKARA, Japan；
Taq archaeal dna polymerases, producer：TAKARA, Japan；
SAP enzyme EF0511, producer：Thermo Fisher Scientific Inc.；
ExoI enzymes, producer：TAKARA, Japan；
Genome DNA extracting reagent kit, purchased from Tiangeng company；
Genomic DNA methylation level treatment kits, purchased from QIAGEN companies；
PGM-T plasmid vectors, pUC18 plasmid vectors, pMD18-T plasmid vectors are purchased from Tiangeng biochemical technology (Beijing)
Co., Ltd；
The specific primer used in the present invention, and specific probe sequence is by the limited public affairs of Shanghai English fine horse biotechnology
Department's synthesis, wherein, the fluorophor connected on specific probe and quenching group are in the lump by the limited public affairs of the handsome biotechnology in Shanghai
Department's synthesis.
The preparation of one kit of embodiment
(1) preparation of positive criteria product and negative standards' product
Compared by the DNA sequence dna to a large amount of clinic colorectal cancer patients and normal population, design is obtained
One sequence relevant with colorectal cancer disease, BMP3 gene methylation sequence SEQ ID NO.4, SEQ ID NO.4 sequences are such as
Under：
TTATTTCGTT GTATTCGGTC GCGTTTCGGG TTTCGTGCGT TTTCGTTTTA GTTGGTTTGG 60
AGTTTAATTT TCGGTTTCGT CGTCGGTTTT TTGCGTTTTC GGAGTGTTTC GTAGCGACGT 120
CGGGAGTCGA CGCGTCGCGC GGGTATTTAG TTATGGTT 158
Artificial synthesized SEQ ID NO.4 sequences, then with SEQ ID NO.4 sequences as template, using Standard PCR technology,
With the DNA (SEQ ID NO.4 sequences) after the process of colorectal cancer cell genomic methylation as template, BMP3 gene target sequences are expanded
Row, PCR reaction systems are shown in Table 1.Wherein primer sequence SEQ ID NO.5 are：5’-TTATTTCGTTGTATTCGGTCGCG-3’；
Primer sequence SEQ ID NO.6 are：5 '-AACCATAACTAAATACCCGCGCGA-3 ',
1 PCR reaction systems of table
Tris-HCl buffer solution of the buffer solution in table 1 for pH8.0.
After amplification terminates, purified PCR primer is connected on pGM-T plasmid vectors, obtains connecting purposeful base
The plasmid of cause；The purification system of the PCR primer is as shown in table 2：
2 PCR primer purification system of table
| Component | Volume (μ L) |
| PCR primer | 20 |
| SAP enzymes | 0.85(1U/μL) |
| ExoI enzymes | 0.375(10U/μL) |
| ddH2O | 3.775 |
| Cumulative volume | 25 |
PCR primer purification system is positioned in PCR amplification instrument and is reacted, reaction condition be 37 DEG C, 20min, 73 DEG C,
20min。
Then product is converted into Escherichia coli TOP10 competent cells, by the recombinant plasmid Jing for building two-way DNA sequencing mirror
Fixed, the recombinant plasmid of genes of interest and plasmid successful connection extracts plasmid as positive criteria product, and ultraviolet specrophotometer is fixed
Amount, and positive criteria product is diluted to into 103The concentration of per microlitre of copy, is stored in -20 DEG C.
The pGM-T plasmids for not occurred to recombinate are diluted to 103The concentration of per microlitre of copy is used as negative standards' product.
(2) preparation of reactant liquor
According to the hyper-methylation sequence SEQ ID NO.4 of BMP3 genes, specific primer SEQ ID NO.1 and SEQ are designed
ID NO.2 and specific probe SEQ ID NO.3,
Wherein, forward primer SEQ ID NO.1 are 5 '-GTTTAATTTTCGGTTTCGTCGTC-3 '；
Reverse primer SEQ ID NO.2 are 5 '-CTCCCGACGTCGCTACG-3 '；
Specific probe SEQ ID NO.3 are 5 '-TTTTTTGCGTTTTCGGAGTGTTTCG-3 ', are contained on specific probe
There are fluorescent reporter group FAM and fluorescent quenching group BHQ, FAM and BHQ to be connected to specific probe sequence SEQ ID
The 5 ' of NO.3 and 3 ' ends.
According to the formula shown in table 3, reactant liquor is prepared, it is standby.
The formula of 3 reactant liquor of table
| Reactant liquor composition | Volume (μ L) |
| 10*buffer | 2 |
| Primer SEQ ID NO.1 (10 μM) | 0.4 |
| Primer SEQ ID NO.2 (10 μM) | 0.4 |
| Specific probe SEQ ID NO.3 (10 μM) | 0.4 |
| dNTPs(2.5mM) | 0.8 |
| Water | 14 |
| Cumulative volume | 18 |
Wherein, the formula of buffer solution (buffer) solution is as follows：Tris-HCl 100mM、KCl 500mM、MgCl2
The pH value of 25mM, wherein Tris-HCl is 8.5.
(3) composition of kit
The positive criteria product for obtaining made above, negative standards' product, reactant liquor and archaeal dna polymerase are attached separately to different
In ep pipes, the kit described in the present embodiment is prepared, wherein, containing the spy for colorectal cancer detection in the reactant liquor
Specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ ID NO.3.
The capacity of positive criteria QC and negative standards' QC is 200 μ L, is respectively provided with concentration for 103copies/μL
Positive criteria product and negative standards' product.The volume of reaction liquid pipe is 500 μ L, and the reactant liquor cumulative volume in pipe is 380 μ L.DNA
The volume of polymerase pipe is 0.65mL, and which is equipped with 22 μ L of Taq archaeal dna polymerases, and its concentration is 0.5U/ μ L.
The use of two kit of embodiment
The BMP3 gene methylation levels of the kit detection sample prepared using embodiment one.
(1) sample preprocessing
The fecal sample of clinical colorectal cancer patients or potential patient is obtained from Tianjin hospital general.By fecal sample (sample
7) this numbering respectively sample 3, sample 4, sample 5, sample 6 and sample (is purchased from according to excrement genome DNA extracting reagent kit
QIAGEN companies) on operating instruction extract excrement in colorectal cancer DNA.Then again with the process examination that methylates of QIAGEN
Agent box (being purchased from QIAGEN companies) processes the genomic DNA that said extracted is arrived, stand-by.
(2) pattern detection
New reaction tube is taken, sample detection group, positive controls and negative control group is respectively designated as, respectively according to table 4
Each experiment group of formula prepares solution, uniform with liquid-transfering gun piping and druming, is measured in being subsequently placed in quantitative real time PCR Instrument.
4 each experiment group of formula of table
| Detection group | Sample detection group | Positive controls | Negative control group |
| Reactant liquor | 18μL | 18μL | 18μL |
| Template DNA | 1μL | — | — |
| Positive criteria product | — | 1μL | — |
| Negative standards' product | — | — | 1μL |
| Archaeal dna polymerase (0.5U/ μ L) | 1μL | 1μL | 1μL |
(3) response procedures
Fluorescent reporter group FAM on probe SEQ ID NO.3 sends green fluorescence, when the sequence in probe cannot with treat
When the DNA profiling surveyed in solution is matched, probe keeps complete, i.e., contain fluorescent reporter group FAM and fluorescent quenching group simultaneously
Fluorescence cannot be detected after BHQ, PCR amplification；And when probe can occur base pair complementarity with the DNA in solution to be measured,
Probe is degraded by Taq DNA polymerase, and the reporter group of probe 5 ' is separated with 3 ' quenching group, can be examined in PCR amplification procedures
Measure fluorescence.Probe sequence SEQ ID NO.3 are with the DNA sequence dna after BMP3 gene hyper-methylations as stencil design, if PCR
Amplification procedure can detect the sequence after fluorescence then has BMP3 gene hyper-methylations in explanation system, colorectal cancer testing result
It is possible for the positive；If fluorescence cannot be detected in PCR amplification procedures, Colon and rectum testing result may be feminine gender；Specifically
Colorectal cancer testing result judges to also need to the quantitative analysis after expanding with reference to PCR.
Parameter window is opened, setting program is：95 DEG C 5 minutes, 1 circulation；95 DEG C 15 seconds, 53 DEG C 40 seconds, 58 DEG C 40 seconds,
Totally 40 circulations.After being provided with, file, operation program are preserved.
(4) reaction result judges
Fluorescent quantitative PCR result is analyzed using software, according to the initial value of image adjustment baseline parameter after analysis,
(user can be voluntarily adjusted according to actual conditions, and initial value can be 5～20 in 1～10, stop value for stop value and threshold value
Scope is selected), the canonical plotting under calibration curve window is reached most preferably.The analysis result under quantitative analysis menu, according to
Specimens point determines sample concentration in the position of calibration curve.
Wherein, if the not S-type curve of growth curve or Ct values>=36, then judgement sample is always containing for negative or sample
Amount is less than detectable limit.
If the S-type curve of growth curve and Ct values<36, then the diagnostic result of sample is positive.
After the completion of the operation of fluorescent PCR instrument program, its fluorescence when choosing positive criteria product and negative standards' product in software
Curve map is should be shown in Fig. 1.The clinical sample fluorescence curve figure of this experiment is as shown in Figure 2.
It will be seen from figure 1 that negative standards' product (numbering 2) are the horizontal linear that a CT value is 40, positive criteria product is
S type curve of the CT values less than 36.Result shown in Fig. 1 shows that the kit of the present invention is working properly simultaneously.
Testing results of the Fig. 2 for clinical sample, can be seen that the sample that sample number into spectrum is 5,6 and 7 from the testing result of Fig. 2
This, has suffered from colorectal cancer, and it is feminine gender that sample number into spectrum is 3 and 4 sample, does not suffer from colorectal cancer.
Present pre-ferred embodiments are the foregoing is only, the limitation present invention is not used to, it is all in the spiritual and former of the present invention
Modification, equivalent and improvement for being made within then etc., are required to be included within the protection domain of invention.
SEQUENCE LISTING
<110>Tianjin train of thought bio tech ltd
<120>BMP3 gene methylation detection reagent systems and kit and its application
<130> OICN160091
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<400> 1
gtttaatttt cggtttcgtc gtc 23
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<400> 2
ctcccgacgt cgctacg 17
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
ttttttgcgt tttcggagtg tttcg 25
<210> 4
<211> 158
<212> DNA
<213>Artificial sequence
<400> 4
ttatttcgtt gtattcggtc gcgtttcggg tttcgtgcgt tttcgtttta gttggtttgg 60
agtttaattt tcggtttcgt cgtcggtttt ttgcgttttc ggagtgtttc gtagcgacgt 120
cgggagtcga cgcgtcgcgc gggtatttag ttatggtt 158
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence
<400> 5
ttatttcgtt gtattcggtc gcg 23
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<400> 6
aaccataact aaatacccgc gcga 24
Claims (16)
1. a kind of BMP3 gene methylations detection reagent system, it is characterised in that the reagent system includes specific primer
And specific probe, the specific primer as shown in SEQ ID NO.1 and SEQ ID NO.2, the specific probe such as SEQ
Shown in ID NO.3.
2. reagent system according to claim 1, it is characterised in that：Be connected with the specific probe fluorophor and
Quenching group, wherein, described fluorophor is glimmering selected from 6- Fluoresceincarboxylic acids, chlordene -6- methylfluoresceins, four chloro- 6- carboxyls
Light element, two chloro- 6- Fluoresceincarboxylic acids of 2,7- dimethyl -4,5-, fluorescein isothiocynate, in Texas Red, Cy3, Cy5 or VIC
One kind；Described quenching group is selected from BHQ, 4- (4- dislikes amino benzeneazo) benzoic acid) or carboxyl tetramethylrhodamine.
3. reagent system according to claim 1 and 2, it is characterised in that the reagent system also includes positive criteria product,
Containing the recombinant plasmid for inserting BMP3 gene methylation specific sequence SEQ ID NO.4 in the positive criteria product.
4. the reagent system according to any one of claim 1-3, it is characterised in that：Reagent system also includes negative standards
Product, containing the plasmid for being not inserted into BMP3 gene methylation specific sequences in described negative standards' product.
5. the reagent system according to claim 3 or 4, it is characterised in that：The concentration of the recombinant plasmid is 1.0 × 103-
1.0×104Copies/ μ L, in negative standards' product, plasmid concentration is 1.0 × 103-1.0×104copies/μL。
6. the reagent system according to any one of claim 3-5, it is characterised in that：Described plasmid or recombinant plasmid phase
Together, the one kind in pETs, pUCs, pGM-T, pBR322 or pMD-T, preferably pGM-T or pBR322.
7. the reagent system according to any one of claim 3-6, it is characterised in that the restructuring in the positive criteria product
The preparation method of the plasmid in plasmid and negative standards' product is as follows：
(1) with sequence SEQ ID NO.4 as template, expanded using primer sequence SEQ ID NO.5 and SEQ ID NO.6,
Obtain genes of interest；
(2) genes of interest is connected on plasmid vector, obtains connection product；
(3) connection product is transformed in host cell；
(4) screening contains the recombinant plasmid for building as positive criteria product；Recombinant plasmid without genes of interest is used as feminine gender
Standard items.
8. the reagent system according to any one of claim 1-7, it is characterised in that：Described reagent system also includes slow
Liquid and dNTPs are rushed, described buffer solution includes trishydroxymethylaminomethane, potassium chloride and magnesium chloride.
9. reagent system according to claim 8, it is characterised in that：The concentration of described trishydroxymethylaminomethane is 50
～200mM, preferably 100～150mM；The concentration of potassium chloride is 300～800mM, preferably 400～600mM；Magnesium chloride it is dense
Spend for 20～50mM, preferably 30～40mM.
10. the reagent system according to any one of claim 1-9, it is characterised in that：Described reagent system also includes
Archaeal dna polymerase, preferably Taq DNA polymerase.
11. a kind of BMP3 gene methylations detection kits, it is characterised in that comprising any one of claim 1-10
Reagent system.
12. specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ ID NO.3 are preparing BMP3
Application in gene methylation detection reagent or kit, is preferably preparing BMP3 gene methylation colorectal cancer detection reagents
Or the application in kit.
13. applications according to claim 12, it is characterised in that described BMP3 gene methylations detection reagent or reagent
The using method of box comprises the steps：
(1) process that methylates is carried out to sample DNA；
(2) using specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ IDNO.3, to step
(1) sample DNA of the process that methylates for obtaining carries out quantitative fluorescent PCR reaction, detects the methylation level of sample DNA.
14. applications according to claim 13, it is characterised in that the using method also includes：
The recombinant plasmid for inserting BMP3 gene methylation specific sequence SEQ ID NO.4 will be contained as positive criteria product,
Carry out quantitative fluorescent PCR reaction；
, carry out containing the plasmid for being not inserted into BMP3 gene methylation specific sequence SEQ ID NO.4 as negative standards' product
Quantitative fluorescent PCR reacts.
15. applications according to claim 13 or 14, it is characterised in that the system of the sample DNA by the process that methylates
Preparation Method comprises the steps：
(1) genomic DNA is extracted from fecal sample；
(2) using bisulf iotate-treated genomic DNA, the sample DNA of the process that obtains methylating.
16. applications according to any one of claim 13-15, it is characterised in that described quantitative fluorescent PCR reaction bar
Part is：94～95 DEG C of 2～5min of denaturation, 1 circulation；94～95 DEG C of 15～20s, 53～55 DEG C of 30～50s, 58～60 DEG C 30
～50s, 40～50 circulations.
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Application publication date: 20170322 |