CN108374041A - Primer, detection method and its application for the detection of FBXO32 gene promoter methylations - Google Patents
Primer, detection method and its application for the detection of FBXO32 gene promoter methylations Download PDFInfo
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Abstract
The invention discloses a kind of primer of DNA methylation assay, detection method and its applications, including：(1) 5 ' the end label biotins of specificity amplification primer SEQ NO 1 and SEQ NO 2, wherein SEQ NO 1, and specificity sequencing primer SEQ NO 3；(2) pcr amplification reaction reagent, sulphite reagent treatment and pyrosequencing related reagent.The present invention has detection cycle short, it is specific high, the advantages that accuracy height and low stain risk, can be used for judging the FBXO32 gene promoter methylation states of sample to be tested, while testing result can refer to guiding doctor and give birth to diagnosis, treatment and the prognosis evaluation for carrying out relevant disease.
Description
Technical field
The invention belongs to life sciences and biotechnology, are a kind of FBXO32 gene promoter methylations detection reagents
Box.
Background technology
DNA methylation becomes an important way of epigenetic modification, and the islands CpG of gene promoter area are in normal shape
It is generally non-under state to methylate, when it methylates, genetic transcription silence is often resulted in, makes some important genes as pressed down
The losses of function such as oncogene, DNA-repair gene regulate and control not normal and DNA damage not so as to cause the Growth and Differentiation of normal cell
It can be timely repaired, these mechanism form closely related with kinds of tumors.So far oneself find many malignant tumours there are one or
The islands multiple tumor suppressor gene CpG methylate.The islands these tumor suppressor genes CpG methylate through the base sequence for preventing promoter region
Row are combined with transcription factor complex and other transcription factors, or are enhanced indirectly by the mediation of Methylated CpG binding proteins
The effect of transcriptional repressor proteins causes the mRNA expressions of corresponding gene to reduce or lack, and then the expression of down-regulation protein,
It is out of control finally to there is cell growth differentiation, leads to the generation of cancer.
FBXO32, Skpl, Cullin l and Rocl/Rbxl/Hrtl are constitute ubiquitin protein ligase complex four
Subunit.FBXO32 is a member of F-box protein families, and latter three is the member of ubiquitin ligase SCF families, to target spot bottom
The degradation of object relies on ubiquitination.There is research that this ubiquitination is claimed to act through the adjusting of MKK6/p38MAPK signal paths.F-box
Albumen is connect by protein interaction region with special substrate, it by the regions F-box and Skpl bundle again with it is compound
Body is connected, and participates in various kinds of cell process such as signal transduction, transcription, cell cycle progression, differentiation, apoptosis, tumour and is formed and thin
Born of the same parents' ubiquitination etc..Research finds that FBXO32 can be such that its transcription is suppressed and may be with malignant tumour by table genetic mechanism
Occur and poor prognosis is related.There is scholar late to find that the frequency that methylates of FBXO32 is up to 29.3% in ovarian neoplasm,
And the hyper-methylation of FBX032 can influence the prognosis of patient.In addition researcher has found methylating for FBXO32 in cardia cancer
Frequency is up to 44.6%, and related with TNM stage.The early diagnosis that the methyl of detection FBXO32 turns to intestinal cancer provides foundation, together
When can provide reference to its therapeutic scheme, contribute to the development of accurate medical treatment.
Have at present for the conventional method of FBXO32 promoter methylations detection：Methylation status of PTEN promoter (MSP), sulfurous
Sour hydrogen salt sequencing PCR (BSP), methylation sensitive high-resolution melting curve analysis (MS-HRM), fluorescent quantitation.Wherein
MSP methods are to carry out judgement sample with the presence or absence of methylating using PCR amplification detection, and the method is practical and universal, but cannot accomplish quantitative
Detection, and there are higher false positive risks；BSP methods mainly combine sanger sequencing technologies to detect methyl by PCR
Change state, but since its is cumbersome, be not suitable for mass detection, while the number of selected clone may influence to examine
It surveys as a result, therefore BSP can only be semiquantitative method；MS-HRM is by the way that the difference of single base sequence is transformed into melting curve
Difference, to judge whether to methylate, requirement of this method to instrument is quite high, needs the fluorescence with HRM modules fixed
PCR instrument is measured, while the method can only analyze detection segment entirety methylation state, and methylating for each sites CpG cannot be specified
State；Fluorescent quantitation is the technology developed based on MSP, mainly adds TaqMan probe in the detection, to ensure that
Higher sensitivity and accuracy, but its detection for multiple methylation sites can only also accomplish that integration is analyzed, simultaneously
Probe cost is higher, therefore the method is relatively suitable for the great amount of samples detection of a small number of sites.
Invention content
The object of the present invention is to provide a kind of primers for the detection of FBXO32 gene promoter methylations, including
5 ' the end label biotins of a pair of of specificity amplification primer SEQ NO 1 and SEQ NO 2, wherein SEQ NO1, and sequencing are drawn
Object SEQ NO 3, base sequence is as follows：
SEQ NO 1：5’-GGGGTAGTGAGYGAGGTTAGGAG-3’；
SEQ NO 2：5’-AACAAAACCAACCCCCCTCCTAC-3’；
SEQ NO 3：5’-CAACCAAATCAACCTCCC-3’.
The present invention also provides a kind of FBXO32 gene promoter methylations detection kits, including a pair of of specificity to expand
Increase 5 ' the end label biotins and sequencing primer SEQ NO 3 of primer SEQ NO 1 and SEQ NO 2, wherein SEQ NO1,
Base sequence is as follows：
SEQ NO 1：5’-GGGGTAGTGAGYGAGGTTAGGAG-3’；
SEQ NO 2：5’-AACAAAACCAACCCCCCTCCTAC-3’；
SEQ NO 3：5’-CAACCAAATCAACCTCCC-3’；
Further, SEQ NO 1：The ratio between use concentration of SEQ NO 2 is 0.5 μM：0.5μM.
Specifically, FBXO32 gene promoter methylation detection kits include following reagent：
(1) sulphite reagent treatment：Bisulfite Mix、RNase free water、DNA Protect
Buffer；
(2) PCR amplification reagent：10*PCR Buffer、2mM dNTP、5*Q Solution Buffer、5U/ul Taq
Enzyme, 10uM amplimers (SEQ NO1, SEQ NO 2) and aqua sterilisa；
(3) single-stranded purified reagent：Streptavidin beads, 70% (V/V) ethyl alcohol, 8mg/ml NaOH solutions, 1 × Wash
Buffer, combination buffer, annealing buffer；
(4) sequencing reagent：Archaeal dna polymerase, ATP sulfurylases, luciferase, apyrase, substrate
APS, fluorescein and dNTP and sequencing primer (SEQ NO3).
The present invention also provides a kind of application methods of the kit, include the following steps：
(i) DNA in sample is extracted；
(ii) template DNA is subjected to sulphite processing, and purifies recycling；
(iii) using the purifying recovery product in step (ii) as template, according to the specificity amplification primer (SEQ
NO1, SEQ NO 2) carry out two-wheeled PCR amplification；
(iiii) it after PCR product being carried out single-stranded purifying, places it in pyrosequencing instrument and is sequenced；
(iiiii) the FBXO32 promoter methylation states of sample are obtained according to related software.
Wherein, in step (ii), the sulphite processing and method for purifying and recycling according to
Bisulfite (QIAGEN, the U.S.) kit specification operates, and is as follows：
The sulphite processing reaction system of an example sample is 140 μ L：10μL DNA、10μL RNase free
water、85μL Bisulfite Mix、35μL DNA Protect Buffer。
Response procedures are：95 DEG C of 5min, 60 DEG C of 25min, 95 DEG C of 5min, 60 DEG C of 85min, 95 DEG C of 5min, 60 DEG C
175min。
The method for purifying and recycling of an example sample is as follows：310 μ L Buffer are added into above-mentioned sulphite reaction mixture
BL (wherein including 10 μ g/mL carrier RNA), vibrates mixing；250 μ L absolute ethyl alcohols are added, vibrate 15s, low-speed centrifugal
Mixing；Above-mentioned mixed liquor is moved into EpiTect spin columns, 1min is centrifuged at full speed, abandons waste liquid；500 μ L are added
Buffer BW centrifuge 1min, abandon waste liquid at full speed；500 μ L Buffer BD are added, is placed at room temperature for 15min, centrifuges at full speed
1min abandons waste liquid；500 μ L Buffer BW are added, centrifuges 1min at full speed, abandons waste liquid；Centrifugation 1min at full speed, abandons waste liquid；It opens
Centrifugal column lid, is placed in metal bath, 56 DEG C of 5min；Centrifugal column is placed on a clean 1.5mL centrifuge tube, to film
20 μ L Buffer EB, 12000 rpm 1min are added dropwise in center, and the liquid collected carries out -20 DEG C of next step PCR or placement
It preserves.
Wherein, in step (iii), the PCR amplification method is as follows：
The first round PCR system of an example sample is 25 μ L：10*PCR Buffer 2.5μL、2mM dNTP 2.5μL、5*Q
5 μ L of Solution Buffer, 10 μM of primer SEQ NO10.5 μ L, 10 μM of 0.5 μ L of SEQ NO2,5U/ μ L Taq enzymes 0.125
μ L, 11.875 μ L of aqua sterilisa, 2 μ L of template.
First round PCR response procedures：95 DEG C of 15min pre-degenerations；94 DEG C of 30s, 64-56 DEG C of 30s often recycle (- 0.5 DEG C),
72 DEG C of 1min, 16 cycles；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 24 cycles, 72 DEG C of 10min.
Second wheel PCR system of an example sample is 50 μ L, and wherein template is 2 μ L of first round PCR product, other PCR reagents
Component is identical, and volume increases by 1 times；
Second wheel amplification condition is 95 DEG C of 15min pre-degenerations；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 40 cycles, 72
℃10min。
Wherein, described in step (iiii) the specific steps are：By 50 obtained μ L PCR products and 3 μ L streptavidins
Bead and the mixing of 47 μ L combination buffers, are incubated at room temperature 10~15min, during which shake 2~3 times, in case bead sinks.Then
Bead is drawn using the Vaccuum prep tool in Vaccuum prep workstation, it will be with bead's
Vaccuum prep tool put 70% (V/V) ethyl alcohol, Denaturation Solution, 1 × Wash Buffer into successively
Middle cleaning 10s or so, then Vaccuum prep tool are put into containing 49 μ L annealing buffers and 1 μ L sequencing primers (SEQ
NO3 in 96 orifice plates), bead is discharged, which is positioned over 80 DEG C of 2min, be cooled to room temperature to get to sequencing reaction
Required single-stranded purified product.
Wherein, the interpretation of result described in step (iiiii), specially：With sulphite treated FBXO32 promoters
Sequence is standard sequence, and is inputted DNA methylation assay software PyroMark CpG, is analyzed by the software and obtains sample
FBXO32 promoter methylation states.
The present invention also provides a kind of detection methods of FBXO32 gene promoter methylations, include the following steps：
(i) DNA in sample is extracted；
(ii) template DNA is subjected to sulphite processing, and purifies recycling；
(iii) using the purifying recovery product in step (ii) as template, according to the specificity amplification primer SEQ
NO1 and SEQ NO 2 carry out two-wheeled PCR amplification；
(iiii) it after PCR product being carried out single-stranded purifying, places it in pyrosequencing instrument and is sequenced；
(iiiii) the FBXO32 promoter methylation states of sample are obtained according to related software；The amplimer and survey
The sequence of sequence primer is as follows：
SEQ NO 1：5’-GGGGTAGTGAGYGAGGTTAGGAG-3’；
SEQ NO 2：5’-AACAAAACCAACCCCCCTCCTAC-3’；
SEQ NO 3：5’-CAACCAAATCAACCTCCC-3’.
Beneficial effects of the present invention：
The main technological route of the present invention is that PCR combines pyrosequencing, by primer amplified destination region,
And analyze sample FBXO32 promoter methylation states using pyrosequencing.The detection method that the present invention uses is combined for PCR
Pyrosequencing techniques.Pyrosequencing techniques are that the enzyme cascade chemiluminescence in the same reaction system by 4 kinds of enzymatics is anti-
It answers, principle is：After primer is annealed with template DNA, in archaeal dna polymerase (DNA polymerase), ATP sulfurylases, fluorescence
Under the synergistic effect of 4 kinds of enzymes of plain enzyme and apyrase, by the polymerization of each dNTP on primer and first order fluorescence
The release coupling of signal is got up, and by detecting release and the intensity of fluorescence, achievees the purpose that the real time measure DNA sequence dna.According to this
One principle when detecting only need to be according to the fluorescence signal intensity of C and T in single examined site, you can determine the first in the site
Base state so that testing result is more intuitive.Therefore this method not only can be qualitative, can also realize and quantify, or even combines related
Software can accurately analyze the methylation state of examined segment area.Meanwhile pyrosequencing instrument can be in 30min or so
The sequencing of 96 samples can be completed, therefore has the advantages such as accuracy is high, detection flux is high, detection cycle is short.
Since DNA sequence dna through sulphite after handling, part C bases can be changed into T, lead to CG in sequence area
Content and the generation of TM values largely make a variation, and then influence conventional primer design software obtains in its sequence and preferably draws
Object sequence.Therefore, the amplimer and sequencing primer that PCR is used in the present invention are designed, designed, such as SEQ NO 1, SEQ
NO 2 and SEQ NO 3.In another embodiment, the binding site at 3 end of sequencing primer is located immediately at examining first site
Previous base, to further shorten detection cycle.Meanwhile the use ratio of amplimer is real by repeatedly optimization
It tests, there is higher amplification efficiency.Further, since in sulphite processing procedure, sample DNA can by a degree of degradation,
To influence late detection, the agarose gel electrophoresis result of Fig. 2 and Fig. 3 and the pyrosequencing result of Fig. 4 in the present invention
Also demonstrating this point, the equal band in sample 1,3 and 5 is weaker in Fig. 2, causes pyrosequencing result background very high (Fig. 4), and
Band is very bright in Fig. 3, and pyrosequencing result background is very low, thus the present invention uses two-wheeled PCR, improves recall rate.
Therefore, qualitative and quantitative, the same phase to methylate for site not only can be achieved at the same time in detection method of the invention
Has detection extracting rate height to other common detection methods, detection cycle is short, and flux is high, accuracy height and low stain risk
Etc. advantages.Testing result will be helpful to the early stage of prediction related neoplasms disease, is conducive to clinician and preferably carries out related disease
The diagnosis and treatment of disease.Clinician can be instructed preferably to use alkylating agent drug according to the individual difference of sufferer simultaneously, realized
Individualized treatment.
Description of the drawings
Fig. 1 is FBXO32 gene promoters through sulphite treated partial sequence, and wherein grey mark is 3
The methylation sites examined.
Fig. 2 is the first round PCR product agarose gel electrophoresis result figure of present invention detection tissue samples 1-5.
Fig. 3 is the second wheel PCR product agarose gel electrophoresis result figure of present invention detection tissue samples 1-5.
Fig. 4 is 3 first round of tissue samples PCR product pyrosequencing result.Because PCR product is less, the pyrosequencing back of the body
Scape is very high.
Fig. 5 is the pyrosequencing result of tissue samples 2.The percentage that methylates that the sample examines 3 sites is respectively
52%, 8%, 7% (dash area is five detection sites in Fig. 5 Sequencing chromatograms, and wherein 3-5 is 3 sites for analysis,
Numerical value above dash area in corresponding frame is its percentage that methylates, similarly hereinafter)；The FBXO32 promoter methyl of the sample
It is 22% to change percentage.
Fig. 6 is the pyrosequencing result of tissue samples 4.The percentage difference that methylates for examining 3 sites of the sample
It is 0%, 0% and 12%；The FBXO32 promoter methylation percentages of the sample are 4%.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be noted that unaccounted normal in embodiment
Rule condition and method usually routinely use method according to fields experimenter：For example, Ao Sibai and James Kingston are edited
《Fine works molecular biology experiment guide》Fourth edition, or according to the step and condition proposed by manufacturer.
Embodiment 1
FBXO32 promoter methylation detection kits, including：A pair of of specificity amplification primer SEQ NO 1 and SEQ NO
5 ' the end label biotins and sequencing primer SEQ NO 3 of 2, wherein SEQ NO1, base sequence is as follows：
SEQ NO 1：5’-GGGGTAGTGAGYGAGGTTAGGAG-3’；
SEQ NO 2：5’-AACAAAACCAACCCCCCTCCTAC-3’；
SEQ NO 3：5’-CAACCAAATCAACCTCCC-3’；
Further, SEQ NO 1：The ratio between use concentration of SEQ NO 2 is 0.5 μM：0.5μM.
Specifically, FBXO32 gene promoter methylation detection kits include following reagent：
(1) sulphite reagent treatment：Bisulfite Mix、RNase free water、DNA Protect
Buffer；
(2) PCR amplification reagent：10*PCR Buffer、2mM dNTP、5*Q Solution Buffer、5U/ul Taq
Enzyme, 10uM amplimers (SEQ NO1, SEQ NO 2) and aqua sterilisa；
(3) single-stranded purified reagent：Streptavidin beads, 70% (V/V) ethyl alcohol, 8mg/ml NaOH solutions, 1 × Wash
Buffer, combination buffer, annealing buffer；
(4) sequencing reagent：Archaeal dna polymerase, ATP sulfurylases, luciferase, apyrase, substrate
APS, fluorescein and dNTP and sequencing primer (SEQ NO3).
(5) positive reference substance：Containing through sulphite treated FBXO32 promoter DNAs；Negative controls：It is free of
Have through sulphite treated FBXO32 promoter DNAs
Embodiment 2：FBXO32 promoter methylation detection kit testing processes
1.FBXO32 the application method following steps of promoter methylation detection kit：
(1) DNA in sample is extracted；
(2) template DNA is subjected to sulphite processing, and purifies recycling；
(3) using the purifying recovery product in step (2) as template, according to the specificity amplification primer (SEQ NO1,
SEQ NO 2) carry out two-wheeled PCR amplification；
(4) it after PCR product being carried out single-stranded purifying, places it in pyrosequencing instrument and is sequenced；
(5) the FBXO32 promoter methylation states of sample are obtained according to related software.
2. step (1) DNA is extracted：Extracts reagent known to those skilled in the art can be selected to carry out DNA extractings,
The kit that biotech company develops can also be used to carry out DNA extractings.
Step 3. (2) sulphite processing and method for purifying and recycling according toBisulfite (QIAGEN,
The U.S.) kit specification operation, it is specific as follows：
The sulphite processing reaction system of an example sample is 140 μ L：10μL DNA、10μL RNase free
water、85μL Bisulfite Mix、35μL DNA Protect Buffer。
Response procedures are：95 DEG C of 5min, 60 DEG C of 25min, 95 DEG C of 5min, 60 DEG C of 85min, 95 DEG C of 5min, 60 DEG C
175min。
The method for purifying and recycling of an example sample is as follows：310 μ L are added into above-mentioned sulphite reaction mixture
Buffer BL (wherein including 10 μ g/mL carrier RNA), vibrate mixing；250 μ L absolute ethyl alcohols are added, vibrate 15s, it is low
Speed centrifugation mixing；Above-mentioned mixed liquor is moved into EpiTect spin columns, 1min is centrifuged at full speed, abandons waste liquid；500 μ are added
L Buffer BW centrifuge 1min, abandon waste liquid at full speed；500 μ L Buffer BD are added, is placed at room temperature for 15min, centrifuges at full speed
1min abandons waste liquid；500 μ L Buffer BW are added, centrifuges 1min at full speed, abandons waste liquid；Centrifugation 1min at full speed, abandons waste liquid；It opens
Centrifugal column lid, is placed in metal bath, 56 DEG C of 5min；Centrifugal column is placed on a clean 1.5mL centrifuge tube, to film
20 μ L Buffer EB, 12000 rpm 1min are added dropwise in center, and the liquid collected carries out -20 DEG C of next step PCR or placement
It preserves.
4. the PCR amplification method described in step (3) is as follows：
The first round PCR system of an example sample is 25 μ L：10*PCR Buffer 2.5μL、2mM dNTP 2.5μL、5*Q
5 μ L of Solution Buffer, 10 μM of primer SEQ NO10.5 μ L, 10 μM of 0.5 μ L of SEQ NO2,5U/ μ L Taq enzymes 0.125
μ L, 11.875 μ L of aqua sterilisa, 2 μ L of template.
First round PCR response procedures：95 DEG C of 15min pre-degenerations；94 DEG C of 30s, 64-56 DEG C of 30s often recycle (- 0.5 DEG C),
72 DEG C of 1min, 16 cycles；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 24 cycles, 72 DEG C of 10min.
Second wheel PCR system of an example sample is 50 μ L, and wherein template is 2 μ L of first round PCR product, other PCR reagents
Component is identical；
Second wheel amplification condition is 95 DEG C of 15min pre-degenerations；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 40 cycles, 72
℃10min。
5. described in step (4) the specific steps are：By 50 obtained μ L PCR products and 3 μ L streptavidins bead and 47 μ
L combination buffers mix, and are incubated at room temperature 10~15min, during which shake 2~3 times, in case bead sinks.Then it uses
Vaccuum prep tool in Vaccuum prep workstation draw bead, by the Vaccuum with bead
Prep tool put into 70% (V/V) ethyl alcohol, Denaturation Solution, 1 × Wash Buffer and clean 10s successively
Left and right, then Vaccuum prep tool are put into 96 holes containing 49 μ L annealing buffers and 1 μ L sequencing primers (SEQ NO3)
In plate, bead is discharged, which is positioned over 80 DEG C of 2min, is cooled to room temperature to get to single-stranded needed for sequencing reaction
Purified product.
6. the interpretation of result described in step (5), specially：With sulphite, treated that FBXO32 promoter sequences are
Standard sequence, and DNA methylation assay software PyroMark CpG are inputted, the FBXO32 for obtaining sample is analyzed by the software
Promoter methylation state.
Embodiment 3：Peripheral blood sample detects
10 Patients with Peripheral blood samples are selected, are detected using the kit of the present invention.According to the method in embodiment 2 into
The extraction of row sample DNA, sulfiting, purifying recycling, PCR amplification, pyrosequencing and interpretation of result, 10 samples
FBXO32 gene promoter methylation state outcomes are as shown in table 1：
1 peripheral blood sample testing result of table
Note：Each sample methylates the numerical value on one column of percentage in table 1, indicates in FBXO32 promoters 3 detection sites
Average methyl percent value, the value provide after being analyzed by software PyroMark CpG.
Embodiment 4：Clinical sample detects
10, clinical tissue sample to be checked is taken, FBXO32 gene promoter methyl is carried out according to the method in embodiment 2
Change state analysis, the results are shown in Table 2.
2 tumor sample testing result of table
It, can by the result of embodiment 3 and example 4 it is found that combining the detection method of pyrosequencing using the PCR of the present invention
The methylation state of success and accurate detection sample FBXO32 gene promoters.Therefore, detection method of the invention can be real simultaneously
It methylates referring now to site qualitative and quantitative, while by the primer usage ratio and two-wheeled PCR amplification after optimization, protecting
Higher PCR amplification success rate is demonstrate,proved, further, since pyrosequencing techniques are used in combination, so that this detection method has
Standby high accuracy, high throughput, detection cycle be short and advantages, the final analysis result such as low stain risk can be conducive to clinic
Doctor preferably carries out the diagnosis, treatment and prognosis prediction of relevant disease.
Sequence table
<110>Nanjing Co., Ltd of Ai Dikang medical tests institute
<120>Primer, detection method and its application for the detection of FBXO32 gene promoter methylations
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggggtagtga gygaggttag gag 23
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aacaaaacca acccccctcc tac 23
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
caaccaaatc aacctccc 18
Claims (10)
1. being used for FBXO32 gene promoter methylation detection primers, which is characterized in that including a pair of of specificity amplification primer SEQ
5 ' the end label biotins and sequencing primer SEQ NO 3 of NO 1 and SEQ NO 2, wherein SEQ NO1, base sequence is such as
Shown in lower：
SEQ NO 1：5’-GGGGTAGTGAGYGAGGTTAGGAG-3’；
SEQ NO 2：5’-AACAAAACCAACCCCCCTCCTAC-3’；
SEQ NO 3：5’-CAACCAAATCAACCTCCC-3’.
2. primer as described in claim 1, which is characterized in that SEQ NO 1：The ratio between use concentration of SEQ NO 2 is 0.5 μ
M：0.5μM.
3. a kind of FBXO32 gene promoter methylations detection kit, which is characterized in that including following reagent：
(1) sulphite reagent treatment；
(2) PCR amplification reagent：Including specificity amplification primer SEQ NO1 and SEQ NO2；
(3) single-stranded purified reagent；
(4) pyrosequencing reagent：Including sequencing primer SEQ NO3；
The sequence of the amplimer and sequencing primer is as follows：
SEQ NO 1：5’-GGGGTAGTGAGYGAGGTTAGGAG-3’；
SEQ NO 2：5’-AACAAAACCAACCCCCCTCCTAC-3’；
SEQ NO 3：5’-CAACCAAATCAACCTCCC-3’.
4. detection kit as claimed in claim 3, which is characterized in that the sulphite reagent treatment includes
Bisulfite Mix, RNase free water and DNA Protect Buffer.
5. detection kit as claimed in claim 3, which is characterized in that the single-stranded purified reagent includes streptavidin
Beads, 70% (V/V) ethyl alcohol, 8mg/ml NaOH solutions, 1 × Wash Buffer, combination buffer and annealing buffer.
6. the application method of FBXO32 gene promoter methylations detection kit shown in one of claim 3 to 5 includes：
(i) DNA in sample is extracted；
(ii) template DNA is subjected to sulphite processing, and purifies recycling；
(iii) using the purifying recovery product in step (ii) as template, according to the specificity amplification primer SEQ NO1 and
SEQ NO 2 carry out two-wheeled PCR amplification；
(iiii) it after PCR product being carried out single-stranded purifying, places it in pyrosequencing instrument and is sequenced；
(iiiii) the FBXO32 promoter methylation states of sample are obtained according to related software；
The sequence of the amplimer and sequencing primer is as follows：
SEQ NO 1：5’-GGGGTAGTGAGYGAGGTTAGGAG-3’；
SEQ NO 2：5’-AACAAAACCAACCCCCCTCCTAC-3’；
SEQ NO 3：5’-CAACCAAATCAACCTCCC-3’.
7. application method as claimed in claim 6, which is characterized in that the first round amplification condition of step (iii) is：95
DEG C 15min pre-degenerations；94 DEG C of 30s, 64-56 DEG C of 30s often recycle (- 0.5 DEG C), 72 DEG C of 1min, 16 cycles；94 DEG C of 30s, 60 DEG C
30s, 72 DEG C of 1min, 24 cycles, 72 DEG C of 10min；The second wheel amplification condition is 95 DEG C of 15min pre-degenerations；94℃
30s, 60 DEG C of 30s, 72 DEG C of 1min, 40 cycles, 72 DEG C of 10min.
8. application method as claimed in claim 7, which is characterized in that step (iiiii), including treated with sulphite
FBXO32 promoter sequences are standard sequence, and are inputted DNA methylation assay software PyroMark CpG, pass through the software point
Analysis obtains the FBXO32 promoter methylation states of sample.
9. a kind of detection method of FBXO32 gene promoter methylations, includes the following steps：
(i) DNA in sample is extracted；
(ii) template DNA is subjected to sulphite processing, and purifies recycling；
(iii) using the purifying recovery product in step (ii) as template, according to the specificity amplification primer SEQ NO1 and
SEQ NO 2 carry out two-wheeled PCR amplification；
(iiii) it after PCR product being carried out single-stranded purifying, places it in pyrosequencing instrument and is sequenced；
(iiiii) the FBXO32 promoter methylation states of sample are obtained according to related software；
The sequence of the amplimer and sequencing primer is as follows：
SEQ NO 1：5’-GGGGTAGTGAGYGAGGTTAGGAG-3’；
SEQ NO 2：5’-AACAAAACCAACCCCCCTCCTAC-3’；
SEQ NO 3：5’-CAACCAAATCAACCTCCC-3’.
10. detection method as claimed in claim 9, which is characterized in that it is characterized in that, the first round of step (iii)
Amplification condition is：95 DEG C of 15min pre-degenerations；94 DEG C of 30s, 64-56 DEG C of 30s often recycle (- 0.5) DEG C, and 72 DEG C of 1min, 16 are followed
Ring；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 24 cycles, 72 DEG C of 10min；The second wheel amplification condition is 95 DEG C
15min pre-degenerations；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 40 cycles, 72 DEG C of 10min.
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