CN107177679A - A kind of kit for detecting esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree - Google Patents
A kind of kit for detecting esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree Download PDFInfo
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Abstract
The present invention relates to a kind of kit for detecting esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree, include methylation-specific amplification forward primer, reverse primer and its non-methylation-specific amplification forward primer and reverse primer, reference gene ACTB amplification forward primers and reverse primer in TFPI2 and HSPB2 gene promoter CpG sites；TFPI2 gene promoter CpG sites are by interior at least one the CpG site included of TFPI2 gene start codons (ATG) upstream 300bp sequences；HSPB2 gene promoter CpG sites are by interior at least one the CpG site included of HSPB2 gene start codons (ATG) upstream 300bp sequences；Reference gene ACTB amplimers do not include any CpG sites in ACTB gene orders；The present invention is considerably increased the degree of accuracy and the specificity of detection, had broad application prospects in the clinical assistant diagnosis of esophageal squamous cell carcinoma using the method for two gene association detections.
Description
Technical field
The present invention relates to biological technical field, and in particular to one kind detection esophageal squamous cell carcinoma related gene TFPI2 and HSPB2
The kit of promoter methylation degree.
Background technology
The cancer of the esophagus (esophageal carcinoma, EC) is one of common cancer, and its incidence of disease is in the whole world with
Ranking the 8th and the 4th is distinguished by state, and the whole world has more than 480,000 new cases every year, causes about 400,000 people dead, and wherein half occurs
In China.Current esophageal carcinoma therapy method is extremely limited, and 5 years survival rates are 15-25% (Chen, Zheng et al.2016).
Esophageal squamous cell carcinoma (esophageal squamous cell carcinoma, ESCC) be the topmost cancer of the esophagus type in Asia (about
90%), the ESCC cases in the whole world about 70% occur in China.ESCC pathogenesis is still not clear at present, smoking, is drunk, is drunk
Dietary habits and family's medical history etc. are considered as most important influence factor, and data shows that these influence factors have significantly
Domain sex differernce, wherein eating habit and family's medical history are bigger (Chen, Zheng et al.2016) to Chinese influence.
The diagnosis to esophageal squamous cell carcinoma is still confined to late stage at present, and lacks effective early diagnosis means.In recent years,
With the extensive use of the progress of molecular diagnostic techniques, especially molecular diagnostic techniques in terms of liquid biopsy, colorectal cancer, lung
The early screening technology of the kinds cancers such as cancer, breast cancer achieves major progress.Existing Molecular Detection object focuses mostly in gene
Mutation, i.e. gene order or copy number variation, but this kind of mutation generally occurs in larger sequence context, gives detection work
Bring many difficulties (Warton and Samimi 2015).By contrast, tumour is used as using gene promoter hypermethylation
Mark has unique advantage, and this kind of methylate is limited in promoter CpG island, and detection range is smaller, and special with tumour
The opposite sex, can just be detected in morbidity early stage, therefore with huge Development volue.At present, based on detection SEPT9 promoters
The commercialization colorectal cancer detection kit of methylation has obtained U.S. FDA approval and has been applied to clinic, but esophageal squamous cell carcinoma dependency basis
Because DNA methylation assay field there is no corresponding product to come out.
At present, existing multiple research institutions deliver the relevant report of esophageal squamous cell carcinoma DNA methylation assay target gene, these targets
Marking gene includes TFPI2 (tissue factor pathway inhibitor) and HSPB2 (heat shock protein
B2).TFPI2 expresses downward in kinds of tumors, is a potential tumor suppressor gene, and research shows the specific of TFPI2 promoters
Methylate frequency of the CpG sites in early stage esophageal squamous cell carcinoma for 67%, and with tumour progression positive correlation (Jia, Yang et
al.2012).HSPB2 is heat shock protein family member, and its frequency that methylates in early stage esophageal squamous cell carcinoma is 95.7%
(Chang,Yamashita et al.2011).These all provide theoretical foundation, but state at present for esophageal squamous cell carcinoma auxiliary detection
It is inside and outside to there is no TFPI2 and HSPB2 promoter methylations detection kit and its aid in the report of context of detection in esophageal squamous cell carcinoma.
The content of the invention
It is contemplated that filling up the blank of esophageal squamous cell carcinoma related gene methylation detection kit, there is provided a kind of sensitivity
High, specific good, accuracy rate it is high and simple to operate can be used for detection esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter
The detection kit of methylation, the trouble of methylate frequency and the esophageal squamous cell carcinoma in the specific CpG sites of both gene promoters
Sick rate is proportionate, and can pass through this detection auxiliary diagnosis esophageal squamous cell carcinoma.
The present invention solve the technical scheme that is used of above-mentioned technical problem for：
A kind of kit that can be used for detecting esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree, bag
Include methylation-specific amplification forward primer, reverse primer and its non-methyl in TFPI2 and HSPB2 gene promoter CpG sites
Change specific amplification forward primer and reverse primer, wherein the methylation-specific in described TFPI2 gene promoter CpG sites
The nucleotide sequence of amplification forward primer is as follows：
5’-CGGTCGGACGTTCGTTTCGTATAAAGC-3’(SEQ ID NO.1)；
The nucleotide sequence of the methylation-specific amplification reverse primer in described TFPI2 gene promoter CpG sites is such as
Shown in lower：
5’-GAACGACCCGCTAAACAAAACGT-3’(SEQ ID NO.2)；
The nucleotide sequence of the non-methylation-specific amplification forward primer in described TFPI2 gene promoter CpG sites
As shown in SEQ ID NO.3：
5’-TTGGTTGGATGTTTGTTTTGTATAAAGTG-3’(SEQ ID NO.3)；
The nucleotide sequence of the non-methylation-specific amplification reverse primer in described TFPI2 gene promoter CpG sites
It is as follows：
5’-AAACAACCCACTAAACAAAACATC-3’(SEQ ID NO.4)。
The nucleotide sequence of the methylation-specific amplification forward primer in described HSPB2 gene promoter CpG sites is such as
Shown in lower：
5’-CGTGTAAATCGTTTGTGAGGGTTTTAGC-3’(SEQ ID NO.5)；
The nucleotide sequence of the methylation-specific amplification reverse primer in described HSPB2 gene promoter CpG sites is such as
Shown in lower：
5’-CGAAAATATAACCAACGCCGCCC-3’(SEQ ID NO.6)；
The nucleotide sequence of the non-methylation-specific amplification forward primer in described HSPB2 gene promoter CpG sites
It is as follows：
5’-TGTGTAAATTGTTTGTGAGGGTTTTAGTG-3’(SEQ ID NO.7)；
The nucleotide sequence of the non-methylation-specific amplification reverse primer in described HSPB2 gene promoter CpG sites
It is as follows：
5’-CAAAAATATAACCAACACCACCCAAACCC-3’(SEQ ID NO.8)；
The TFPI2 gene promoters CpG sites are in the 300bp sequences of TFPI2 gene start codons (ATG) upstream
Comprising at least one CpG site；The HSPB2 gene promoters CpG sites are HSPB2 gene start codons (ATG)
At least one the CpG site included in the 300bp sequences of upstream；The ACTB gene magnifications primer does not include ACTB gene sequences
Any CpG sites in row.
The kit also includes reference gene ATCB (β-Actin) amplification forward primers and reverse primer, wherein described
ATCB gene magnification forward primers nucleotide sequence it is as follows：
5’-GTGTTTAAGATAGTGTTGTGGGTG-3’(SEQ ID NO.9)；
The nucleotide sequence of described ATCB gene magnification reverse primers is as follows：
5’-CCTACTATACACCTACTTAATACACACTCC-3’(SEQ ID NO.10)。
The purity of the primer should reach electrophoresis level (PAGE) or HPLC grades.
Also include negative quality-control product, positive quality control product and no template control in the kit；
Preferably, the no template control refers to the reaction system without human genome DNA, it is further preferred that without mould
Plate control is to replace template DNA with sterilized water.
Preferably, the positive quality control product is bisulphite modified permethylated human genome DNA, the feminine gender
Quality-control product is the bisulphite modified non-human genome DNA that methylates.
In the kit, the final concentration composition of fluorescent PCR amplification reaction system is：1~10 × PCR buffer solutions, 0.1
~1mM dNTPs, 0.05~20ng/ μ l template DNAs, 0.01~0.10 Μ/μ l Taq archaeal dna polymerases, 1~5mM magnesium chlorides,
1~2 × SYBR Green nucleic acid dyes, 0.1~1 μM of primer.
Preferably, the final concentration composition of the fluorescent PCR amplification reaction system is：1~2 × PCR buffer solutions, 0.4~
0.8mM dNTPs, 0.2~0.5 μM of primer, 0.1~10ng/ μ l template DNAs, 0.05~0.10 Μ/μ l Taq DNA polymerizations
Enzyme, 2~3mM magnesium chlorides, 1~1.5 × SYBR Green nucleic acid dyes.
TrisCl, sodium chloride, potassium chloride and magnesium sulfate are included in the fluorescent PCR amplification reaction system.
In the kit, fluorescent PCR amplification condition is：Pre-degeneration：94~95 DEG C 30~60 seconds；Circulation：Denaturation 94~
95 DEG C 5~10 seconds, annealing 50~65 DEG C 10~30 seconds, extend 68~72 DEG C 15~60 seconds (collection fluorescence), totally 35~50 are followed
Ring；Melting curve：94~95 DEG C 5~10 seconds, 65 DEG C 60~90 seconds, be warming up to 94~95 DEG C with 0.1~0.5 DEG C/sec of speed
(continuous collecting fluorescence).
The present invention judges testing result by TFPI2, HSPB2 gene and reference gene ATCB PCR amplifications, carries
The high sensitiveness and reliability of testing result.The Cq values of PCR amplifications are can obtain by fluorescent PCR amplification curve, by melting curve
It can obtain the Tm values of PCR primer.TFPI2, HSPB2 gene and reference gene PCR amplifications refer to its Cq values, i.e. TFPI2,
The period that the fluorescence signal of HSPB2 genes and reference gene ATCB is undergone when reaching the threshold value of setting.By Cq values, then tie
Close melting curve and may determine that whether there is specific amplification, so as to learn the degree of TFPI2, HSPB2 promoter methylation in sample.
Compared with conventional art, beneficial outcomes of the invention have：First, esophageal squamous cell is detected present invention firstly discloses one kind
The kit of the methylation in cancer associated gene TFPI2 and HSPB2 promoter CpG site, is that esophageal squamous cell carcinoma auxiliary detection is carried
A new means are supplied.Secondly, set present invention is specifically directed to the peculiar CpG sites of the Chinese patients in TFPI2 and HSPB2 genes
Primer is counted, and experiments prove that these CpG sites and esophageal squamous cell carcinoma are closely related.Again, the present invention is using two genes
The method of joint-detection, considerably increases the degree of accuracy and the specificity of detection, has in the clinical assistant diagnosis of esophageal squamous cell carcinoma
Wide application prospect.
The key problem in technology point and pre-protection point of the present invention is esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 in the middle foundation of a nation
Distinctive promoter CpG site information in native patients with esophageal squamous cell carcinoma, and TFPI2 and HSPB2 methylate and non-methylation specific
Property amplimer sequence.
Embodiment
With reference to embodiment, 1 couple of present invention is described in further detail.
Embodiment 1
Material：Tumor tissues to be checked and cancer beside organism's sample, positive quality control product, negative quality-control product.The positive quality control product
For bisulphite modified permethylated human genome DNA, the negative quality-control product is bisulphite modified non-first
Base human genome DNA.Human esophageal squamous cell cancer related gene TFPI2 and HSPB2 promoter methylation detection kit, wherein wrapping
Include：The Methylation-specific primer in TFPI2 and HSPB2 gene promoter CpG sites；TFPI2 and HSPB2 gene promoters CpG
The non-Methylation-specific primer in site；Reference gene ACTB primer.
The TFPI2 gene promoters CpG sites are in the 300bp sequences of TFPI2 gene start codons (ATG) upstream
Comprising at least one CpG site；The HSPB2 gene promoters CpG sites are HSPB2 gene start codons (ATG)
At least one the CpG site included in the 300bp sequences of upstream；The ACTB gene magnifications primer does not include ACTB gene sequences
Any CpG sites in row.(its is final concentration of for the Methylation-specific primer in the TFPI2 gene promoters CpG sites：0.1
~1 μM) include：
Forward primer：5’-CGGTCGGACGTTCGTTTCGTATAAAGC-3’(SEQ ID NO.1)；
Reverse primer：5’-GAACGACCCGCTAAACAAAACGT-3’(SEQ ID NO.2)；
(its is final concentration of for the non-Methylation-specific primer in the TFPI2 gene promoters CpG sites：0.1~1 μM) bag
Include：
Forward primer：5’-TTGGTTGGATGTTTGTTTTGTATAAAGTG-3’ (SEQ ID NO.3)；
Reverse primer：5’-AAACAACCCACTAAACAAAACATC-3’(SEQ ID NO.4)；
(its is final concentration of for the Methylation-specific primer in the HSPB2 gene promoters CpG sites：0.1~1 μM) bag
Include：
Forward primer：5’-CGTGTAAATCGTTTGTGAGGGTTTTAGC-3’ (SEQ ID NO.5)；
Reverse primer：5’-CGAAAATATAACCAACGCCGCCC-3’(SEQ ID NO.6)；
(its is final concentration of for the non-Methylation-specific primer in the HSPB2 gene promoters CpG sites：0.1~1 μM) bag
Include：
Forward primer：5’-TGTGTAAATTGTTTGTGAGGGTTTTAGTG-3’ (SEQ ID NO.7)；
Reverse primer：5’-CAAAAATATAACCAACACCACCCAAACCC-3’ (SEQ ID NO.8)；
(its is final concentration of for the ACTB reference genes amplimer：0.1~1 μM) include：
Forward primer：5’-GTGTTTAAGATAGTGTTGTGGGTG-3’(SEQ ID NO.9)；
Reverse primer：5’-CCTACTATACACCTACTTAATACACACTCC-3’ (SEQ ID NO.10).
All primer purity should reach electrophoresis level (PAGE) or HPLC grades, without miscellaneous band.The conjunction that combination mechanism is provided is provided
Quality inspection into product proves that such as PAGE electrophoresis results or HPLC analyze collection of illustrative plates, it was demonstrated that should have after purification using PAGE or HPLC
Substantially unimodal PAGE or HPLC purify collection of illustrative plates, and concentration is that 100pmol/L is standby.
Instrument：Roche Lightcycler 96, the silent winged Nanodrop 1000 of match, supercentrifuge, water-bath, whirlpool shake
Swing instrument, refrigerator, baking oven, autoclave.
Reagent：Blood/cell/tissue genome DNA extracting reagent kit (Tiangeng Products are used in the present embodiment),
Archaeal dna polymerase (Sai Mofei companies), 10 × PCR Buffer (Sai Mofei companies), MgCl2(Sai Mofei companies), dNTP (Sai Mo
Fly company), purified water.
1. the extraction of tumor tissues and cancer beside organism's genomic DNA
DNA is extracted using Beijing Tiangeng bio tech ltd blood/cell/tissue genome DNA extracting reagent kit,
Comprise the following steps that：
(1) tissue should first be smashed and be processed as cell suspension, and then 10,000rpm (~11,200 × g) centrifuges 1min, fall
Supernatant, plus 200 μ L buffer solution GA to the greatest extent, vibration to thorough suspension.4 μ L RNaseA (100mg/ml) solution can be added, are vibrated
15sec, room temperature places 5min.
(2) 20 μ L Proteinase K solution are added, are mixed.In 56 DEG C of placements, until histolysis, brief centrifugation with
The globule of cap wall is removed, then carries out next step.Biased sample is overturned per hour 2-3 times, also may be used with water bath chader.
(3) 200 μ L buffer solution GB are added, fully reverse to mix, 70 DEG C of placement 10min, solution strains limpid, brief centrifugation
To remove the globule of cap wall.
(4) plus the μ L absolute ethyl alcohols of people 200, fully vibration mixes 15sec, now it is possible that flocculent deposit, briefly from
The heart is to remove the globule of cap wall.
(5) previous step resulting solution and flocculent deposit all add in an adsorption column CB3 to (adsorption column is put into collecting pipe
In), centrifugation 30sec in 12,000rpm (~13,400 × g) outwells waste liquid, adsorption column CB3 is put back in collecting pipe.
(6) 500 μ L buffer solutions GD (please first checked whether before use and added absolute ethyl alcohol) are added into adsorption column CB3,
12,000rpm (~13,400 × g) centrifuge 30sec, outwell waste liquid, adsorption column CB3 is put into collecting pipe.
(7) 600 μ L rinsing liquids PW (please first checked whether before use and added absolute ethyl alcohol) are added into adsorption column CB3,
12,000rpm (~13,400 × g) centrifuge 30sec, outwell waste liquid, adsorption column CB3 is put into collecting pipe.
(8) step 7 is repeated.
(9) adsorption column CB3 is put back in collecting pipe, 12,000rpm (~13,400 × g) centrifuge 2 min, outwell waste liquid.
Adsorption column CB3 is placed in into room temperature to place several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
(10) adsorption column CB3 is transferred in a clean centrifuge tube, 50- is vacantly added dropwise to the middle part of adsorbed film
200 μ L elution buffer TE, room temperature places 2-5min, 12,000rpm (~13,400 × g) centrifugation 2min, and solution is collected into
In centrifuge tube.It is required that the DNA concentration isolated and purified out is at least in more than 10ng/ μ L, A260/A280 ratios are more than 1.80.
DNA products should be stored in -20 DEG C, to prevent DNA degradation.
Bisulfites 2. (Bisulfite) modifying DNA
Using Qiagen companiesFast Bisulfite Conversion Kit (Cat.59824), specifically
Step is as follows：
(1) ensure that bisulfite solution (Bisulfite Solution) is completely dissolved.
(2) reaction each component is added according to table below：
| High concentration (1ng-2 μ g) | Low concentration (1-500ng) | |
| DNA | Most 20 μ L | Most 40 μ L |
| Ultra-pure water | ||
| Bisulfite solution | 85μL | 85μL |
| DNA protects liquid | 35μL | 15μL |
| Cumulative volume | 140μL | 140μL |
(3) acutely mix, be placed in room temperature.
(4) reaction condition is：95℃5min；60 DEG C of 1~20min, 2 circulations.
(5) of short duration centrifugation after the completion of reacting, reactant is fully transferred in 1.5mL centrifuge tubes.
(6) 310 μ L Buffer BL are added, concussion is mixed, of short duration centrifugation.
(7) 250 μ L absolute ethyl alcohols are added, vibration 15sec is mixed, of short duration centrifugation.
(8) MinElute DNA Spin Column are taken out from 4 DEG C of refrigerators, upper step reaction solution is all added into pillar
In.
(9) maximum (top) speed centrifugation 1min, abandons filtrate.
(10) 500 μ L Buffer BW, maximum (top) speed centrifugation 1min are added, filtrate is abandoned.
(11) 500 μ L Buffer BD incubations at room temperature 15min is added.
(12) maximum (top) speed centrifugation 1min, abandons filtrate.
(13) 500 μ L Buffer BW, maximum (top) speed centrifugation 1min are added, filtrate is abandoned.
(14) walked 1 time in repetition.
(15) 500 μ L absolute ethyl alcohols are added, maximum (top) speed centrifugation 1min abandons filtrate.
(16) pillar is put into 2ml collecting pipes, maximum (top) speed centrifugation 1min eliminates residual night.
(17) pillar is put into new 1.5ml centrifuge tubes, adds 15 μ L Buffer EB, froth lid.
(18) it is incubated at room temperature 1min.
(19) maximum (top) speed centrifugation 1min, collects DNA, -20 DEG C of preservations.
The system and reaction condition of 3.PCR reactions
The final concentration of the fluorescent PCR amplification reaction system is constituted：1~10 × PCR buffer solutions, 0.1~1mM
DNTPs, 0.1~1 μM of primer, 0.05~20ng/ μ L template DNAs, 0.01~0.10 Μ/μ L Taq archaeal dna polymerases, 1~2 ×
SYBR Green nucleic acid dyes, 1~5mM magnesium chlorides.
Preferably, the final concentration composition of the fluorescent PCR amplification system is：1~2 × PCR buffer solutions, 0.4~0.8mM
DNTPs, 0.2~0.5 μM of primer, 0.1~10ng/ μ L template DNAs, 0.05~0.10 Μ/μ L Taq archaeal dna polymerases, 1~
1.5 × SYBR Green nucleic acid dyes, 2~3mM magnesium chlorides.
The detection gene is TFPI2 and HSPB2 genes, and the reference gene is ACTB (β-actin).
The dNTPs includes 10mM dATP, 10mM dCTP, 10mM dTTP and 10mM dGTP.
TrisCl, sodium chloride, potassium chloride, magnesium sulfate and magnesium chloride are included in the fluorescent PCR amplification reaction system.
The detailed process of the fluorescent PCR amplified reaction is：94~95 DEG C of 30~60sec of pre-degeneration；Amplification stage, 94
~95 DEG C of 5~10s of denaturation, 50~65 DEG C of 10~30s of annealing, 68~72 DEG C of 15~60sec of extension (collection fluorescence) carry out 35
~50 circulations；Melting curve：94~95 DEG C 5~10 seconds, 65 DEG C 60~90 seconds, be warming up to 94 with 0.1~0.5 DEG C/sec of speed
~95 DEG C (continuous collecting fluorescence).
The sample-adding layout of 4.PCR reactions
DNA sample to be measured, negative quality-control product, positive quality control product and no template control carry out 2 multiple holes detections, PCR instrument
96 orifice plates sample-adding layout see the table below 1.PC represents positive quality control product (Positive Control) in table, and NC represents negative quality-control product
(Negative Control), NTC represents no template control (No template control), and S represents detection sample
(Sample)。
Table 1
After 5. experiment terminates, the analysis of testing result is carried out according to the following steps, is judged
The amplification curve analysis of no template control (NTC), TFPI2, HSPB2 and reference gene ACTB should be without amplification curves
Obvious amplified signal, illustrates that experiment is pollution-free, can continue analysis.
The reference gene ACTB of quality-control product should have amplified signal, and S-type amplification curve, quality-control product reference gene ACTB
Cq values should meet the parameter area of quality-control product in table 2；TFPI2, HSPB2 of negative quality-control product should become without obvious amplification curve
Change, the Cq values of positive quality control product TFPI2, HSPB2 should meet the parameter area of quality-control product in table 2.More than quality-control product detection is met
During condition, it was demonstrated that experiment is effective, analysis can be continued.
Table 2
| Quality-control product | TFPI2 or HSPB2 Cq values | ACTB Cq values |
| Positive quality control product | Cq value≤40 | Cq value≤32 |
| Negative quality-control product | Without amplified signal | Cq value≤32 |
The reference gene ACTB of sample should have amplified signal, and a S-type amplification curve, and sample PCR testing results should be by
Interpretation is carried out according to the standard of table 3.
Table 3
If not meeting the 1st in table 3,2 requirements, it is recommended that re-starting PCR detections.Will if not meeting in table 3 the 3rd
Ask, then advise extracting tissue DNA again.
The experimental method of unreceipted actual conditions in the embodiment above, according to normal condition, such as according to molecule gram
Condition described in grand experiment guide (third edition, J. Pehanorm Brookers etc. write) or the condition proposed by manufacturer are grasped
Make.
Bibliography
1.Chang,X.,et al.(2011)."Promoter methylation of heat shock protein
B2in human esophageal squamous cell carcinoma."Int J Oncol 38(4):1129-1135.
2.Chen,W.,et al.(2016)."Cancer statistics in China,2015."CA Cancer J Clin 66(2): 115-132.
3.Jia,Y.,et al.(2012)."Methylation of TFPI-2 is an early event of
esophageal carcinogenesis."Epigenomics 4(2):135-146.
4.Warton,K.and G.Samimi(2015)."Methylation of cell-free circuLating
DNA in the diagnosis of cancer."Front Mol Biosci 2:13.
The content not being described in detail in this specification belongs to prior art known to professional and technical personnel in the field.
<110>Beijing is in promise medical science and technology Co., Ltd
<120>A kind of kit for detecting esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree
<160> 10
<170> PatentIn version 3.3
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5’-CGGTCGGACGTTCGTTTCGTATAAAGC-3’
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5’-GAACGACCCGCTAAACAAAACGT-3’
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5’-TTGGTTGGATGTTTGTTTTGTATAAAGTG-3’
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5’-AAACAACCCACTAAACAAAACATC-3’
<210> 5
<211> 28
<212> DNA
<213>Artificial sequence
<400> 5
5’-CGTGTAAATCGTTTGTGAGGGTTTTAGC-3’
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence
<400> 6
5’-CGAAAATATAACCAACGCCGCCC-3’
<210> 7
<211> 29
<212> DNA
<213>Artificial sequence
<400> 7
5’-TGTGTAAATTGTTTGTGAGGGTTTTAGTG-3’
<210> 8
<211> 29
<212> DNA
<213>Artificial sequence
<400> 8
5’-CAAAAATATAACCAACACCACCCAAACCC-3’
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence
<400> 9
5’-GTGTTTAAGATAGTGTTGTGGGTG-3’
<210> 10
<211> 30
<212> DNA
<213>Artificial sequence
<400> 10
5’-CCTACTATACACCTACTTAATACACACTCC-3’
Claims (9)
1. a kind of kit for detecting esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree, its feature exists
In：Methylation-specific amplification forward primer, reverse primer including TFPI2 and HSPB2 gene promoter CpG sites and its non-
Methylation-specific amplification forward primer and reverse primer, reference gene ACTB amplification forward primers and reverse primer；
The TFPI2 gene promoters CpG sites are by including in the 300bp sequences of TFPI2 gene start codon ATG upstreams
At least one CpG site；The HSPB2 gene promoters CpG sites are HSPB2 gene start codon ATG upstreams 300bp sequences
At least one the CpG site included in row；The reference gene ACTB amplimers do not include any in ACTB gene orders
CpG sites；
The nucleotide sequence such as SEQ of the methylation-specific amplification forward primer in described TFPI2 gene promoter CpG sites
Shown in ID NO.1：
5’-CGGTCGGACGTTCGTTTCGTATAAAGC-3’；
The nucleotide sequence such as SEQ of the methylation-specific amplification reverse primer in described TFPI2 gene promoter CpG sites
Shown in ID NO.2：
5’-GAACGACCCGCTAAACAAAACGT-3’；
The nucleotide sequence such as SEQ of the non-methylation-specific amplification forward primer in described TFPI2 gene promoter CpG sites
Shown in ID NO.3：
5’-TTGGTTGGATGTTTGTTTTGTATAAAGTG-3’；
The nucleotide sequence such as SEQ of the non-methylation-specific amplification reverse primer in described TFPI2 gene promoter CpG sites
Shown in ID NO.4：
5’-AAACAACCCACTAAACAAAACATC-3’；
The nucleotide sequence such as SEQ of the methylation-specific amplification forward primer in described HSPB2 gene promoter CpG sites
Shown in ID NO.5：
5’-CGTGTAAATCGTTTGTGAGGGTTTTAGC-3’；
The nucleotide sequence such as SEQ of the methylation-specific amplification reverse primer in described HSPB2 gene promoter CpG sites
Shown in ID NO.6：
5’-CGAAAATATAACCAACGCCGCCC-3’；
The nucleotide sequence such as SEQ of the non-methylation-specific amplification forward primer in described HSPB2 gene promoter CpG sites
Shown in ID NO.7：
5’-TGTGTAAATTGTTTGTGAGGGTTTTAGTG-3’；
The nucleotide sequence such as SEQ of the non-methylation-specific amplification reverse primer in described HSPB2 gene promoter CpG sites
Shown in ID NO.8：
5’-CAAAAATATAACCAACACCACCCAAACCC-3’；
The nucleotide sequence of described ATCB gene magnification forward primers is as shown in SEQ ID NO.9：
5’-GTGTTTAAGATAGTGTTGTGGGTG-3’；
The nucleotide sequence of described ATCB gene magnification reverse primers is as shown in SEQ ID NO.10：
5’-CCTACTATACACCTACTTAATACACACTCC-3’。
2. the reagent of esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree is detected as claimed in claim 1
Box, it is characterised in that：Also include negative quality-control product, positive quality control product and no template control in the kit.
3. the reagent of esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree is detected as claimed in claim 2
Box, it is characterised in that：The positive quality control product is bisulphite modified permethylated human genome DNA, the feminine gender
Quality-control product is the bisulphite modified non-human genome DNA that methylates, and the no template control refers to be free of human gene
Group DNA reaction system.
4. the reagent of esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree is detected as claimed in claim 3
Box, it is characterised in that：The no template control is the reaction system that template DNA is replaced with sterilized water.
5. the reagent of esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree is detected as claimed in claim 1
Box, it is characterised in that：The purity of the primer reaches PAGE grades or HPLC grades.
6. the reagent of esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree is detected as claimed in claim 1
Box, it is characterised in that：In the kit, the final concentration composition of fluorescent PCR amplification reaction system is：1~10 × PCR is buffered
Liquid, 0.1~1mMdNTPs, 0.05~20ng/ μ l template DNAs, 0.01~0.10 Μ/μ l Taq archaeal dna polymerases, 1~5mM chlorine
Change magnesium, 1~2 × SYBR Green nucleic acid dyes, 0.1~1 μM of primer.
7. the reagent of esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree is detected as claimed in claim 6
Box, it is characterised in that：The final concentration of the fluorescent PCR amplification reaction system is constituted：1~2 × PCR buffer solutions, 0.4~
0.8mM dNTPs, 0.2~0.5 μM of primer, 0.1~10ng/ μ l template DNAs, 0.05~0.10 Μ/μ l Taq archaeal dna polymerases,
2~3mM magnesium chlorides, 1~1.5 × SYBR Green nucleic acid dyes.
8. the reagent of esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree is detected as claimed in claim 6
Box, it is characterised in that：Also include TrisCl, sodium chloride, potassium chloride and magnesium sulfate in the fluorescent PCR amplification reaction system.
9. the reagent of esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree is detected as claimed in claim 1
Box, it is characterised in that：In the kit, fluorescent PCR amplification condition is：Pre-degeneration：94~95 DEG C 30~60 seconds；Circulation：Become
Property 94~95 DEG C 5~10 seconds, annealing 50~65 DEG C 10~30 seconds, extension 68~72 DEG C 15~60 seconds, totally 35~50 circulation；It is molten
Solution curve：94~95 DEG C 5~10 seconds, 65 DEG C 60~90 seconds, be warming up to 94~95 DEG C with 0.1~0.5 DEG C/sec of speed.
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| Application Number | Priority Date | Filing Date | Title |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710422533.2A CN107177679A (en) | 2017-06-07 | 2017-06-07 | A kind of kit for detecting esophageal squamous cell carcinoma related gene TFPI2 and HSPB2 promoter methylation degree |
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Cited By (1)
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|---|---|---|---|---|
| CN109266749A (en) * | 2018-11-23 | 2019-01-25 | 皖南医学院 | A kind of primer and its detection method detecting RNASET2 gene promoter methylation |
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| CN109266749A (en) * | 2018-11-23 | 2019-01-25 | 皖南医学院 | A kind of primer and its detection method detecting RNASET2 gene promoter methylation |
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Application publication date: 20170919 |