CN110157804A - For pulmonary cancer diagnosis, outcome prediction or the methylation sites of prognosis, detection primer and kit - Google Patents
For pulmonary cancer diagnosis, outcome prediction or the methylation sites of prognosis, detection primer and kit Download PDFInfo
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Abstract
The invention discloses for pulmonary cancer diagnosis, outcome prediction or the methylation sites of prognosis, detection primer and kit.The present invention provides 15 methylations for pulmonary cancer diagnosis, outcome prediction and for 10 methylation sites of lung cancer for prognosis, it, being capable of the Accurate Diagnosis early stage of lung cancer, prediction lung cancer curative effect or prognosis by the change of the methylation level of methylation sites in measurement subject's blood sample using the combination of one or more of methylation sites as marker.The present invention also provides use these methylation sites for specific detection primer designed by target and probe.Invention further provides what is constructed using these primer and probes to be used for pulmonary cancer diagnosis, prediction or the kit of prognosis.Pulmonary cancer diagnosis, prediction or prognosis are carried out using kit of the present invention, have many advantages, such as that high specificity, sensitivity are strong and accuracy is high.
Description
Technical field
The present invention relates to diagnosing tumor, outcome prediction or the gene markers of prognosis, more particularly, to pulmonary cancer diagnosis, treatment
Effect prediction or prognosis methylation sites and detection primer, the invention further relates to diagnose diagnosis, outcome prediction or
The kit of prognosis belongs to pulmonary cancer diagnosis, outcome prediction or prognosis field.
Background technique
One of the main reason for lung cancer (Lung Cancer, LUNC) is global cancer related mortality.With other many cancers
Similar, its prognosis of the patients with lung cancer of early treatment has compared with patients with terminal and significantly improves, this is partly due to part
Compared with systemic therapy, therapeutic effect is relatively high for treatment.Therefore, the early screening of cancer, which has, reduces mortality
Significant potentiality.
Exist under normal physiological conditions, in human blood circulation by the dissociative DNA (ce1l of apoptosis and the release of the cell of necrosis
Free DNA, cfDNA), free Circulating tumor DNA (circulating tumor also can be detected in the blood circulation of tumor patient
DNA, ctDNA), change since ctDNA carries the molecular genetics consistent with primary tumor tissue, and can be comprehensively anti-
The hereditary information and evolution process for reflecting patient's body tumour cell, can be used as ideal tumor markers, for examining for tumour
Disconnected, molecule parting, therapeutic evaluation and tracking and prognosis evaluation.
Genetic alteration performance multiplicity in tumour cell and there are apparent individuality, including mutation (Mutation),
It copies number variation (Copy-number variation), microsatellite instability (Microsatellite Instability), miscellaneous
Conjunction property missing (Loss of Heterozygosity) etc..These are abnormal not only widely different between different tumours, even if
Also the individual with height is heterogeneous (Individual heterogeneity) between the different patients of same tumour, so that
These are detected in ctDNA to change and interpretation of result complex.In contrast to this, phase can be detected in the ctDNA of kinds of tumors
As methylate change, and the methylome of same type tumour ctDNA then it is more stable with it is consistent.With methylation-specific
PCR, two generations sequencing (next-generation sequencing, NGS) and digital pcr (droplet digital PCR,
The rapid development of technologies such as ddPCR) so that high-throughput, accurate, relatively inexpensive DNA methylation spectrum and methylation sites detection
It is possibly realized, the detection of ctDNA methylation-site is also promoted to have become one of the hot spot of liquid Biopsy a few days ago, it can be with
Accurate sieve Check early stage or extreme early cancer, facilitate early diagnosis, the earlier evaluations, early treatment, early prevention of cancer.
But the limited success that the method for the developed effectively screening lung cancer based on blood obtains at present, wherein being permitted
Specific serum biomarker mostly relevant to lung cancer not yet enters clinical use.Currently, being examined for identifying for lung cancer
Disconnected, outcome prediction and the serum biomarkers of prognosis simultaneously develop corresponding kit there are still urgent needs.
Summary of the invention
An object of the present invention is to provide for pulmonary cancer diagnosis or predicts the methylation sites of recurrence of postoperative lung cancer；
The second object of the present invention is to provide the methylation sites of lung cancer outcome prediction or prognosis；
The third object of the present invention is to provide for pulmonary cancer diagnosis, outcome prediction or the specific primer of prognosis and probe.
The fourth object of the present invention provides pulmonary cancer diagnosis, outcome prediction or the kit of prognosis.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
Present invention firstly provides the methylation sites of diagnosis, prediction recurrence of postoperative lung cancer for the early stage of lung cancer, including
Any methylation sites or more than one any combination as described below: 1-295864,3-1138223,6-
1526223、6-265969、7-14998、7-862737、9-954757、10-1205149、11-646423、12-1222773、
13-510867、14-1019927、16-571473、17-732857、19-179828。
The present invention is found by experiment that, using the group cooperation of 1-295864,3-1138223,6-1526223 and 6-265969
For the diagnosis marker of the early stage of lung cancer, specificity with higher and sensitivity, therefore, present invention preferably employs 1-295864,
Diagnosis marker of the combination of 3-1138223,6-1526223 and 6-265969 as the early stage of lung cancer.
The early stage of lung cancer or prediction can be accurately diagnosed by the change of methylation sites described in measurement subject's blood sample
Recurrence of postoperative lung cancer.
The present invention further provides a kind of detection kit of lung cancer early diagnosis on this basis, comprising: for expanding
Increase the specific primer and probe of the methylation sites, conversion fluid, in conjunction with liquid, cleaning solution, refined solution, eluent, DNA extraction
Reagent, PCR amplification reagent.
As reference, the conversion fluid can be bisulfite ammonium salt solution；The combination liquid can be guanidine hydrochloride；Institute
The cleaning solution stated can be purified water；The refined solution can be isopropanol, ethyl alcohol or sodium hydroxide；The eluent can
To be TE solution.
The DNA extract reagent can any conventional DNA extract reagent, the PCR amplification reagent can be with
It is any conventional amplifing reagent, preferably ddPCR amplifing reagent.
Wherein, for expanding the primer sets (including probe) of methylation sites 1-295864 by SEQ ID No.1-SEQ ID
The composition of primer sequence described in No.4；For expanding the primer sets (including probe) of methylation sites 3-1138223 by SEQ ID
The composition of primer sequence described in No.5-SEQ ID No.8；For expanding the primer sets of methylation sites 6-1526223 by SEQ
The composition of primer sequence described in ID No.9-SEQ ID No.12；For expanding the primer sets (packet of methylation sites 6-265969
Including probe) primer sequence described in SEQ ID No.13-SEQ ID No.16 forms；For expanding the primer sets of 7-14998
(including probe) primer sequence described in SEQ ID No.17-SEQ ID No.20 forms；For expanding methylation sites 7-
862737 primer sets (including probe) primer sequence described in SEQ ID No.21-SEQ ID No.24 forms；For expanding
Increase primer sets (including probe) primer sequence as described in SEQ ID No.25-SEQ ID No.28 of methylation sites 9-954757
Column composition；For expanding the primer sets (including probe) of methylation sites 10-1205149 by SEQ ID No.29-SEQ ID
The composition of primer sequence described in No.32；For expanding the primer sets (including probe) of methylation sites 11-646423 by SEQ ID
The composition of primer sequence described in No.33-SEQ ID No.36；For expanding the primer sets (packet of methylation sites 12-1222773
Including probe) primer sequence described in SEQ ID No.37-SEQ ID No.40 forms；For expanding methylation sites 13-
510867 primer sets (including probe) primer sequence described in SEQ ID No.41-SEQ ID No.44 forms；For expanding
Increase primer sets (including probe) primer as described in SEQ ID No.45-SEQ ID No.48 of methylation sites 14-1019927
Sequence composition；For expanding the primer sets (including probe) of methylation sites 16-571473 by SEQ ID No.49-SEQ ID
The composition of primer sequence described in No.52；For expanding the primer sets (including probe) of methylation sites 17-732857 by SEQ ID
The composition of primer sequence described in No.53-SEQ ID No.56；For expand methylation sites 19-179828 primer sets (including
Probe) primer sequence described in SEQ ID No.57-SEQ ID No.60 forms；
Invention further provides the methylation sites for lung cancer for prognosis or prediction patients with lung cancer mortality risk, are selected from
Any one of following methylation sites or more than one any combination: cg26205771, cg08436738,
cg27252696、cg24917945、cg03550506、cg06903569、cg12865837、cg18440897、cg19255783、
cg20634573。
By the change of the methylation level of methylation sites described in measurement subject's blood sample, lung can be accurately predicted
Cancer prognosis or prediction patients with lung cancer mortality risk.
The present invention is found by experiment that three kinds of methylation sites are combined the raising lung cancer for capableing of highly significant below
Prognosis or the accuracy for predicting patients with lung cancer mortality risk: (1) cg26205771, cg08436738 and cg27252696；(2)
Cg24917945, cg11252953, cg03550506 and cg06903569；(3) cg12865837, cg18440897,
Cg19255783 and cg20634573.
Invention further provides the kits for lung cancer for prognosis or prediction patients with lung cancer mortality risk, comprising: uses
In the specific primer and probe that expand the methylation sites, conversion fluid, in conjunction with liquid, cleaning solution, refined solution, eluent, DNA
Extract reagent, PCR amplification reagent.
Wherein, for expanding primer sets primer as described in SEQ ID No.61-SEQ ID No.64 of cg26205771
Sequence composition；For expanding the primer sets (including probe) of cg08436738 as described in SEQ ID No.65-SEQ ID No.68
Primer sequence composition；For expanding the primer sets (including probe) of cg27252696 by SEQ ID No.69-SEQ ID
The composition of primer sequence described in No.72；For expanding the primer sets (including probe) of cg24917945 by SEQ ID No.73-
The composition of primer sequence described in SEQ ID No.76；For expanding the primer sets (including probe) of cg03550506 by SEQ ID
The composition of primer sequence described in No.77-SEQ ID No.80；For expand the primer sets (including probe) of cg06903569 by
The composition of primer sequence described in SEQ ID No.81-SEQ ID No.84；For expand cg12865837 primer sets (including visit
Needle) primer sequence described in SEQ ID No.85-SEQ ID No.88 forms；For expanding the primer sets of cg18440897
(including probe) primer sequence described in SEQ ID No.89-SEQ ID No.92 forms；For expanding cg19255783's
Primer sets (including probe) primer sequence described in SEQ ID No.93-SEQ ID No.96 forms；For expanding
The primer sets (including probe) of cg20634573 primer sequence described in SEQ ID No.97-SEQ ID No.100 forms.
As reference, the conversion fluid can be bisulfite ammonium salt solution；The combination liquid can be guanidine hydrochloride；Institute
The cleaning solution stated can be purified water；The refined solution can be isopropanol, ethyl alcohol or sodium hydroxide；The eluent can
To be TE solution.
The DNA extract reagent can any conventional DNA extract reagent, the PCR amplification reagent can be with
It is any conventional amplifing reagent, preferably ddPCR amplifing reagent.
" reagent " used in the present invention can be used for any type of preparation of detection methylation level, including
But be not limited to organic reagent, inorganic reagent, probe, primer, chip etc..
In the case where knowing to be used for pulmonary cancer diagnosis, outcome prediction or the methylation sites of prognosis as disclosed herein, this
Field technical staff can use routine techniques design, synthesis correspondent probe or primer to detect the methyl of these methylation sites
Change horizontal.When needed, it is also designed to the form of chip.
Methylation sites provided by the invention and corresponding product or kit can be based on the Blood diagnosis lungs of patient
Cancer, outcome prediction or prediction prognosis.Diagnosis or method of prognosis pair compared to tumor biopsy, using methylation sites of the invention
Patient's is invasive small；And since any period during treatment blood can be obtained, allow tumour correlation point
The real-time monitoring of son variation；In addition, it can detecte by the way that imaging means are unobvious or uncertain tumour.In addition, utilizing this
The diagnosis of the methylation sites of invention or method of prognosis improve the accuracy of patients with lung cancer diagnosis and prognosis prediction.
Detailed description of the invention
Fig. 1 is shown to suffer from for diagnosing, the treatment curative effect of prediction lung cancer, prediction Lung Cancer Recurrence, or prediction lung cancer
The schematic diagram of the detection method of the methylation sites of the prognosis and mortality risk of person；Respectively indicate up and down: by t examine (A) or
LASSO (B) filters out the specific methylation site suitable for pulmonary cancer diagnosis, prediction or prognosis evaluation respectively, then according to ddPCR
Technology detects methyl rate to establish the workflow schematic diagram of diagnosis and prognosis evaluation.
Fig. 2 shows the accuracy of diagnosis prediction Model Diagnosis lung cancer of the invention.A. cfDNA is used in training group
The ROC curve and diagnosis prediction model of methyl rate analysis, relevant area under the curve (AUC)；B. compared with Normal group,
The methyl rate block diagram of lung cancer (LUNC) patient, the figure shows the data for coming self-training group, and wherein score comes from multicategory classification
Device；C. using below the relevant curve of cfDNA methylation subject analyte's curve (ROC) and methyl rate in verifying group
Product (AUC)；D. the block diagram of the methyl rate for classifying to normal, LUNC or HCC patient, the figure shows from verifying group
The data of group.
Fig. 3 has the cfDNA methyl rate and the removed patient of tumour and Normal group of the patient of tumor load
CfDNA methyl rate.
The cfDNA methyl rate comparative analysis by stages of Fig. 4 tumour difference.
Fig. 5 patients after surgery is compared with operation consent cfDNA methyl rate comparative analysis.
Comparative analysis of the patient compared with cfDNA methyl rate after treatment before Fig. 6 is treated.
Fig. 7 shows the LUNC survival expectancy based on cfDNA methylation analysis.A. according to the methyl rate in verifying group
Assessment, the overall survival curve of the LUNC patient with low or high mortality risk.B. verifying has I/II phase and III/ in group
The survival curve of the LUNC patient of IV phase.C. it assesses and is verified in group with methyl rate by the assessment of methyl rate, by stages
The ROC of 12 months survival rates of LUNC stage forecast.
Specific embodiment
Further describe the present invention below in conjunction with specific embodiment, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that can modify without departing from the spirit and scope of the invention to details and form of the invention or
Replacement, but these modifications and replacement are fallen within the protection scope of the present invention.
Embodiment 1 is for diagnosing, the treatment curative effect of prediction lung cancer, prediction Lung Cancer Recurrence or predicts patients with lung cancer
The screening of prognosis and mortality risk
The present embodiment obtains for diagnosing by analysis lung cancer and normal blood sample sample, predicts that the treatment of lung cancer is treated
Effect, prediction Lung Cancer Recurrence, or predict the prognosis of patients with lung cancer and the methylation sites of mortality risk, specifically include following step
It is rapid:
The first step uses efficient dissociative DNA extracts kit (Guangzhou You Ze Bioisystech Co., Ltd；Article No.:
YZ000008 ctDNA) is extracted in blood sample；
Second step carries out sulphite conversion to the ctDNA of extraction, makes not methylate in the ctDNA
Cytosine deamination is transformed into uracil, and the cytimidine to methylate remains unchanged, and obtains sulphite conversion ctDNA；
Third step measures the change of the methylation level of the sulphite conversion ctDNA with the method for deep sequencing；
4th step, the design and synthesis of probe；Firstly, to TCGA (cancer and oncogene map) and NCBI GSE data
The 450K gene methylation chip data of cancerous lung tissue sample and normal human blood sample sample in library is analyzed, and methylation is found out
The gene of horizontal notable difference；Then, with the method measurement upper one of deep sequencing in the tissue of same patient and blood sample sample
The change of the methylation level of the gene filtered out is walked, and filters out while can organize to detected and methylate in blood sample
Level changes consistent gene；Change the probe of consistent gene with the methylation level that ppDesigner software design is elected
And it is synthesized, such as using nucleic acid hybridization technique, carry out probe synthesis with individual oligonucleotides.
5th step converts ctDNA to the sulphite by the probe and captures；Specifically: by 10ng sulfurous
Hydrochlorate conversion DNA and the probe of above-mentioned synthesis are mixed in 20ul reaction buffer by 1:20000 molar ratio and form reaction solution, instead
It answers on liquid plus 50ul mineral oil is to prevent liquid evaporation.It is annealed and is denaturalized to reaction solution, PCR amplification simultaneously gives each blood sample sample
Originally bar code is enclosed.Library is established in PCR reaction, and retains effective capture, removes the capture of blank.With fitting for Illumina company
Built library is purified with PCR method with primer and concentration, using Miseq the and Hiseq2500 system of Illumina company come into
Row sequencing, obtains sequencing result.
Since the original capture experimental result (i.e. sequencing result) of sequencing is in efficient probe and inefficient probe
The significant difference that the result of reading has carries out relative efficiency calculating, mixing to original sequencing result to improve such case
It is mixed when probe according to using the molar ratio after adjusting.
The analysis of 6th step data and the foundation of model
The blood sample sample of lung cancer blood sample sample and normal control is divided into training set in the ratio of 2:1 at random and verifying collects,
The method of LASSO and random forest is selected to carry out genescreen for the test result in the 5th step.
In LASSO method, using data set 500 times of double sampling method non-repeated sampling 75%, existing frequency is selected
Methylated genes more than 450 times totally 30；
In the method for random forest, criterion is minimized the error using OOB, by under setting variable every time duplicate score
The principle of drop 0.3 carries out the elimination of stochastic variable, finally filters out 24 methylation sites；
The methylation sites of above two method overlapping have 15, are respectively as follows: 1-295864,3-1138223,6-
1526223、6-265969、7-14998、7-862737、9-954757、10-1205149、11-646423、12-1222773、
13-510867,14-1019927,16-571473,17-732857,19-179828 (explanation: " 1-295864 " i.e. No. 1 chromosome
In the 295864th nucleotide, also the rest may be inferred in other 14 sites；These sites and neighbouring nucleic acid sequence pass through UCSC number
It is obtained according to library inquiry).
Methylation diagnostic marker of the methylation sites being overlapped using this 15 as lung cancer cfDNA, and collect in verifying
The middle detection and verifying that methyl rate is carried out with ddPCR technology；Methyl rate is methylation probe positive copy number in all sun
Property copy number in percentage, analysis, which obtains, can be used as the methyl rate threshold value of pulmonary cancer diagnosis.Specific screening mode referring to
The process of Fig. 1.
The methyl rate of each sample is detected and verified by ddPCR technology, and analysis obtains and screens to obtain 15 methyl
Change site as the early stage of lung cancer methyl rate threshold value diagnosed and the diagnosis for establishing diagnostic model applied to the early stage of lung cancer, is specifically shown in
Test example 1.
Equally, lung cancer blood sample sample is divided into training set and verifying collects, point counting is analysed for convenience: being first passed through to each base
Because establishing a single argument COX proportional hazard model as covariant, the gene of (p < 0.05) related to patient's prognosis is filtered out
Carry out next step analysis.
Then, using LASSO-COX multinomial logistic regression, finishing screen selects 10 methylation sites for predicting lung cancer
Treatment curative effect, prediction Lung Cancer Recurrence or predict patients with lung cancer prognosis and mortality risk marker (as shown in table 1), this
10 methylation sites are as follows: cg26205771, cg08436738, cg27252696, cg24917945, cg03550506,
cg06903569,cg12865837,cg18440897,cg19255783,cg20634573；
The DNA methylation data in 485,000 sites that the Infinium 450K methylation array that the experiment is related to generates
It is all obtained from (Gregory Hannum et al.Genome-wide methylation profiles disclosed in TCGA and document
reveal quantitative views of human aging rates.Molecular Cell.49,359-367,
January 24,2013) (GSE40279) data set for generating, wherein analyzing the DNA methylation spectrum of HCC and blood.
The IDAT formatted file for generating methylation data, it includes the rate values of each scanning magnetic bead.It uses
These data file transitions are score, referred to as Beta value by the minfi packet of Bioconductor., visited by using molecular inversion
The targeting bisulfite sequencing of needle obtains the methylation value of Chinese group and carries out subsequent analysis.
In the detection of lung cancer methylation sites, 10 methylations being screened by LASSO-COX multinomial logistic regression
Site (table 2).
1 10 methylation sites of table
| Title | Site |
| cg26205771 | 8:53851188 |
| cg08436738 | 2:235528864 |
| cg27252696 | 6:100912939 |
| cg24917945 | 10:120514926 |
| cg03550506 | 22:32149786 |
| cg06903569 | 6:28304139 |
| cg12865837 | 6:100911525 |
| cg18440897 | 4:46391413 |
| cg19255783 | 4:84035952 |
| cg20634573 | 2:96781406 |
10 methylation sites that table 2 is screened by LASSO-COX multinomial logistic regression
Finally, the methyl rate of each sample is detected and verified by ddPCR technology, analysis obtains and screens to obtain 12
Methylation sites are as the methyl rate threshold value assessed as lung cancer for prognosis and establish prediction model applied to the pre- of lung cancer for prognosis
It surveys, specific method and result are shown in test example 4.
Test example 1 is established the model of early stage of lung cancer diagnosis using 15 methylation sites screened and is examined applied to lung cancer
Disconnected prediction
One, the foundation of predictive diagnosis model
By logistic regression method, by screened 15 methylation sites (1-295864,3-1138223,6-
1526223、6-265969、7-14998、7-862737、9-954757、10-1205149、11-646423、12-1222773、
13-510867,14-1019927,16-571473,17-732857,19-179828) construct a diagnosis prediction model.
The specific method is as follows:
(1) site and primer probe sequence
1-295864
F:GAGGTGGAGGGATGG(SEQ ID No.1)
R:CTTCCCAAACAATTTACAC(SEQ ID No.2)
M:FAM/GAATGGGTCGGGATGGG/BHQ1(SEQ ID No.3)
NM:HEX/GAATGGGTTGGGATGGG/BHQ1(SEQ ID No.4)
3-1138223
F:TTTTATATTTTTTATATTTAAAT(SEQ ID No.5)
R:TTCATTCAAATAACAAACA(SEQ ID No.6)
M:FAM/AATTTTAATTGACAAATTGGTTATT/BHQ1(SEQ ID No.7)
NM:HEX/AATTTTAATTGATAAATTGGTTATT/BHQ1(SEQ ID No.8)
6-1526223
F:GGGTATAGGGTTTTGTATT(SEQ ID No.9)
R:TATTCTCCCAAAACAACA(SEQ ID No.10)
M:FAM/TTGTTTGTTTTCGGTAGAATATT/BHQ1(SEQ ID No.11)
NM:HEX/TTGTTTGTTTTTGGTAGAATATT/BHQ1(SEQ ID No.12)
6-265969
F:TTTTATTTTAATGATTGTATTT(SEQ ID No.13)
R:ACAAACCTAATAACACCTAA(SEQ ID No.14)
M:FAM/TTTATAGTTTTCAGTGGGTTATTG/BHQ1(SEQ ID No.15)
NM:HEX/TTTATAGTTTTTAGTGGGTTATTG/BHQ1(SEQ ID No.16)
7-14998
F:TTATTATATTATAAATTTGAGTT(SEQ ID No.17)
R:ATACAAACTATATCAAACATAA(SEQ ID No.18)
M:FAM/GTGTGGTAAATTACTATGTATGG/BHQ1(SEQ ID No.19)
NM:HEX/GTGTGGTAAATTATTATGTATGG/BHQ1(SEQ ID No.20)
7-862737
F:TAGAGAGTGGTGGAGATTG(SEQ ID No.21)
R:ACCACACACCCTACTCAC(SEQ ID No.22)
M:FAM/GAGGATTGTTAGCTTAGTGGTAA/BHQ1(SEQ ID No.23)
NM:HEX/GAGGATTGTTAGTTTAGTGGTAA/BHQ1(SEQ ID No.24)
9-954757
F:TGTGTTTTTTAGGTTTAAGT(SEQ ID No.25)
R:ACACCTATAATCCCAACTA(SEQ ID No.26)
M:FAM/ATTTTTTTGTTTCAGTTTTTTTAG/BHQ1(SEQ ID No.27)
NM:HEX/ATTTTTTTGTTTTAGTTTTTTTAG/BHQ1(SEQ ID No.28)
10-1205149
F:TAGTGTGGTTGTTAAGGTT(SEQ ID No.29)
R:ACCAACAACTAAATATTTCA(SEQ ID No.30)
M:FAM/TTGTATTATTTCAAAGTGGGTGA/BHQ1(SEQ ID No.31)
NM:HEX/TTGTATTATTTTAAAGTGGGTGA/BHQ1(SEQ ID No.32)
11-646423
F:AGGAAGGGTTTTGGTT(SEQ ID No.33)
R:TCCCATAAAAAATACAAAC(SEQ ID No.34)
M:FAM/GTTGAGATTCTGTGTGGGG/BHQ1(SEQ ID No.35)
NM:HEX/GTTGAGATTTTGTGTGGGG/BHQ1(SEQ ID No.36)
12-1222773
F:GTTATTTTTGTAGATTTGTT(SEQ ID No.37)
R:CTTTCCACCAAAATATT(SEQ ID No.38)
M:FAM/ATTTATGGTTTACTTTATAGTATAT/BHQ1(SEQ ID No.39)
NM:HEX/ATTTATGGTTTATTTTATAGTATAT/BHQ1(SEQ ID No.40)
13-510867
F:AGAGGTTATAGGTTTAGTAGAG(SEQ ID No.41)
R:TCTAAAAAAACCTCCTAAA(SEQ ID No.42)
M:FAM/TTGTTTTGGCGATGTTGGT/BHQ1(SEQ ID No.43)
NM:HEX/TTGTTTTGGTGATGTTGGT/BHQ1(SEQ ID No.44)
14-1019927
F:GTTTGAAATGTTGTAAAATAT(SEQ ID No.45)
R:CTATTCTTCCTACCTCATAA(SEQ ID No.46)
M:FAM/TTTATTTTAGTCGTTTTAGTTGAA/BHQ1(SEQ ID No.47)
NM:HEX/TTTATTTTAGTTGTTTTAGTTGAA/BHQ1(SEQ ID No.48)
16-571473
F:TTGGTGTGTTTTTTAGTTT(SEQ ID No.49)
R:TAAAATAACCACATCTAACTA(SEQ ID No.50)
M:FAM/TTATGTTGTTCGGGTTATTGT/BHQ1(SEQ ID No.51)
NM:HEX/TTATGTTGTTTGGGTTATTGT/BHQ1(SEQ ID No.52)
17-732857
F:GTTTTGTATGTATTAGGTATTT(SEQ ID No.53)
R:ACACACTAAAACCTATTAACA(SEQ ID No.54)
M:FAM/GTTATTTTTTTCTTAGGTTTTTAT/BHQ1(SEQ ID No.55)
NM:HEX/GTTATTTTTTTTTTAGGTTTTTAT/BHQ1(SEQ ID No.56)
19-179828
F:GGATTTTATTTTAGTTGTTAT(SEQ ID No.57)
R:TCAAACATAAATTTTAATAAA(SEQ ID No.58)
M:FAM/GAAATATGTTTGTCGATAATTTGA/BHQ1(SEQ ID No.59)
NM:HEX/GAAATATGTTTGTTGATAATTTGA/BHQ1(SEQ ID No.60)
(2) reagent and amplification condition used in PCR amplification
1, reagent used in PCR amplification
Reagent used in 3 PCR amplification of table
| Ingredient names | Reagent component |
| Conversion fluid | Bisulfite ammonium salt solution |
| In conjunction with liquid | Guanidine hydrochloride |
| Cleaning solution | Purified water |
| Refined solution | Isopropanol, ethyl alcohol, sodium hydroxide |
| Eluent | TE solution |
2, PCR amplification condition
1-295864
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
52 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
3-1138223
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
46 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
6-1526223
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
50 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
6-265969
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
50 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
7-14998
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
48 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
7-862737
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
55 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
9-954757
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
51 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
10-1205149
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
53 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
11-646423
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
50 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
12-1222773
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
48 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
13-510867
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
51 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
14-1019927
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
50 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
16-571473
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
50 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
17-732857
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
52 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
19-179828
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
47 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
The definitive result of the cfDNA methylation ratio of the cancer patient and normal patient of (three) 15 methylation sites
Measurement result is shown in Table 4.Specificity and sensitivity result are shown in Table 5.
The cancer patient of 4 15 methylation sites of table and the cfDNA methylation ratio of normal patient
M: methylation, nM: non-methylation, by the methylation ratio for calculating M/ (M+NM) M+nM.
The sensitivity of 5 15 methylation sites diagnosis early stages of lung cancer of table and specific outcome
According to the test result of table 5 as it can be seen that this 15 methylation sites are for diagnosing the early stage of lung cancer have it is higher sensitive
Degree and specificity；Wherein together by 1-295864,3-1138223,6-1526223 and 6-265969 this four Sites Combinations
Sensitivity and specificity with higher；Therefore, in the diagnosis for the early stage of lung cancer, 1-295864,3- are preferentially used
1138223, the combination in this four sites 6-1526223 and 6-265969 is as diagnosis marker.
(4) foundation of diagnosis prediction analysis
CfDNA can a point two ways tested and analyzed:
The first test and analyze: first detection sample in 1-295864,3-1138223,6-1526223 and 6-265969 this
The methyl rate of four methylation sites；If pattern detection as the result is shown this 4 sites methyl rate be respectively less than or be equal to
Normal control cfDNA methyl rate then shows to suffer from cancer risk lower；If there is the methyl rate in 2 or 2 or more sites is greater than
Or equal to cancer patient's cfDN methyl rate in table, then shows to have and biggish suffer from cancer risk.
Second of detection and analysis: if this 4 sites (1-295864,3-1138223,6-1526223 and 6-265969)
In the methyl rate in only 1 site be greater than or equal to corresponding cancer patient cfDN methyl rate in table 3, or have 1 or 1
The methyl rate in a above site then detects whole 12 between normal control and cancer patient's cfDNA methyl rate
The methyl rate of methylation sites simultaneously calculates average value.If the average value of methyl rate is less than or equal to 16.90%, show have
Have and lower suffers from cancer risk；If the average value of methyl rate is greater than or equal to 46.93%, show with higher to suffer from cancer wind
Danger；If the average value of methyl rate falls between, need to do further diagnosis with other clinical methods.
Two, it is verified in verifying group using the diagnosis prediction model established
It is 86.7% in the sensibility of verifying group using the diagnosis prediction Model Diagnosis lung cancer of above-mentioned foundation, specificity
For 91.0% (being shown in Table 6).And the result as shown in Fig. 2 (A-E) is as it can be seen that this model extraordinary training dataset and can tested
It demonstrate,proves and distinguishes lung cancer and Normal group in data set, wherein concentrating the AUC=of lung cancer group in training data as shown in Fig. 2-A
0.937, as shown in Fig. 2-C, concentrated in verify data, the AUC=0.911 of lung cancer.Diagnostic result confirms that the application is screened
15 methylation sites can be used for accurately distinguishing lung cancer and normal control.
Table 6 diagnoses column in the normal binary classification between lung cancer based on the diagnosis of cfDNA methyl rate in verifying group
Join table
The diagnostic model that test example 2 is established using 15 methylation sites screened is applied to sentencing for lung cancer therapy curative effect
Disconnected, prediction tumour recurrence
The diagnosis prediction model established using test example 1 calculates the methyl rate of each sample, using this methyl
Rate predict patients with lung cancer treatment curative effect and tumor recurrence result as shown in figures 3 to 6.
According to Fig. 3 as it can be seen that there is the cfDNA methyl rate of patient's (56.3% ± 13.8) of tumor load to be apparently higher than tumour
Removed patient's (11.2% ± 5.7) and Normal group (9.7% ± 4.3) (p < 0.01, Fig. 3).
According to Fig. 4 as it can be seen that the value of cfDNA methyl rate is related to neoplasm staging.The cfDNA first of early stage patient (I, II phase)
Base rate (36.3% ± 10.4) is apparently higher than Normal group (9.7% ± 4.3), and is significantly lower than advanced stage (III, V phase)
(65.7% ± 12.6) patient (p < 0.05, Fig. 4).
According to Fig. 5 as it can be seen that the cfDNA methyl rate (11.2% ± 5.7) of patients after surgery compared with operation consent (56.3% ±
13.8) it is substantially reduced, patient is postoperative tumour (46.3% ± 14.3) recurrence occurs, and cfDNA methyl rate can then increase (p once again
=0.002, Fig. 5).
According to Fig. 6 as it can be seen that patient (56.3% ± 9.8) cfDNA methyl rate is apparently higher than (14.3% after treatment before treating
± 8.1) effective patient, and patient (60.7% ± 11.5) cfDNA methyl rate of the invalid tumour progression for the treatment of can be further
It increases (p < 0.001, Fig. 6).
Above-mentioned test result illustrates the early stage that can be used for lung cancer according to the diagnostic model that this 15 methylation sites are established
Diagnosis, the judgement for the treatment of curative effect, prediction tumor recurrence.
Prediction experiment of the application methylation sites of test example 3 to patients with lung cancer prognosis
By being fitted multivariable Cox proportional hazard model, with 10 methylation sites screened
(cg26205771、cg08436738、cg27252696、cg24917945、cg03550506、cg06903569、
Cg12865837, cg18440897, cg19255783, cg20634573) a prognostic risk model is constructed, and calculate every
The Prognostic scoring system index (being appointed as cp-score) of a sample.
Choosing -0.24 is cutoff value, and patients with lung cancer is divided into low-risk group and high risk group.
According to prediction result as it can be seen that no matter in training set or verifying collection, the existence of low-risk group patient is significantly better than that
High risk group (as shown in Figure 7).Multivariate analysis shows that cp-score is significant related to mortality risk, and cp-score
Existence be an independent risk factor.By stages and the independent hazard factor of patients with lung cancer, combine cp-score and point
Phase can be further improved the predictive ability (Fig. 7) to patients with lung cancer prognosis.
Test example 4 establishes the prediction model of lung cancer for prognosis and using the prediction model to lung using 10 methylation sites
Cancer patient's prognosis carries out prediction experiment
One, the foundation of the prediction model of lung cancer for prognosis
Droplet type digital pcr technology has the advantages that such as following: absolute quantitation does not depend on Ct value, is not necessarily to standard curve；
Hypersensitivity, the detection suitable for rare sequence and rare mutation；Accurately quantitative result and splendid repeatability；It is applicable in multiple
Miscellaneous sample detection is not influenced vulnerable to PCR mortifier；Compatible dye method and sonde method, while meeting scientific research and clinical detection
It is required that.
Clinically, ddPCR technology is high for cfDNA detection consistency, can be used for the early screening of tumour, clinic point
Phase, personalized medicine guidance, pharmacodynamic assessment and resistance analysis, prognosis evaluation and relapse and metastasis monitoring.Document report ddPCR is used
It is detected in hepatocellular carcinoma methylation sites, wherein ddPCR combines liver tissue-specific DNA methylation site primer hepatic tissue source
The diagnosable intestinal cancer hepatic metastases of cfDNA molecular number, this shows to utilize ddPCR technology detection tissue specificity methylation Biomarkers
The method of the quantitative each tissue cfDNA molecular number of object has the application potential of diagnosing tumour transfer.In addition, being followed using ddPCR detection
Ring tumour ctDNA monitoring recurrence, can be than the sign of CT scan discovery recurrence in average early 10 months.
Filtered out lung cancer methylation sites are utilized, by the design of biology routine techniques, synthesis correspondent probe and are drawn
Object detects the change of methylation level by ddPCR technology in subject's blood samples, to carry out noninvasive, early diagnosis lung
Cancer.
It is as follows using droplet type digital quantitative round pcr quantitative detecting method in this test example:
Firstly, obtaining tumour and corresponding blood samples from the patient for the tumor resection that undergos surgery；
Secondly, sample is freezed and is stored in -80 DEG C until using；
Finally, therefrom separation DNA and RNA uses AllPrep DNA/RNA Mini kit and cfDNA to extract examination respectively
Agent box (Qiagen, Valencia, CA) carries out sample extraction.
Basic principle:
It is allowed to be assigned to different droplets by preparing reaction system dilution separation individual molecule, and is individually expanded by PCR；
Detect the fluorescence signal value of droplet respectively by fluorescence probe to analyze every kind of product.Pass through above two step in subject's blood sample
The cf DNA obtained after processing carries out subsequent ddPCR experiment, and its step are as follows:
Workflow:
First step ddPCR reaction system is prepared: by DNA/RNA sample, primer and probe/EvaGreen dyestuff and ddPCR
Premixed liquid mixes (20ul)；
DdPCR reaction system: being added in the aperture of drop generator chip by the preparation of second step droplet first, droplet hair
Raw device each run at most can produce the droplet of 8*20000 Water-In-Oil, and target nucleic acid and background nucleic are randomly distributed in micro-
In drop；
Third step droplet PCR: droplet is transferred in 96 hole PCR reaction plates, and is sealed and carried out droplet PCR；
The analysis of 4th step droplet: after PCR reaction, 96 hole PCR reaction plates is transferred on droplet analyzer, each sample is analyzed
The fluorescence signal of the droplet of product, software quantify DNA/RNA concentration in conjunction with Poisson distribution principle according to the ratio of negative droplet
(copies/ul)。
It is as follows in relation to primer and probe sequence, the specific reaction condition of PCR amplification and used reagent component:
1, site and primer probe sequence:
cg26205771
cg08436738
cg27252696
cg24917945
cg03550506
cg06903569
cg12865837
cg18440897
cg19255783
cg20634573
2. reagent component
Reagent used in 6 PCR amplification of table
| Ingredient names | Reagent component |
| Conversion fluid | Bisulfite ammonium salt solution |
| In conjunction with liquid | Guanidine hydrochloride |
| Cleaning solution | Purified water |
| Refined solution | Isopropanol, ethyl alcohol, sodium hydroxide |
| Eluent | TE solution |
3, the actual conditions of PCR amplification
cg26205771
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
53 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg08436738
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
53 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg01604601
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
51 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg27252696
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
47 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg24917945
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
51 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg11252953
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
51 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg03550506
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
50.5 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg06903569
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
48 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg12865837
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
52 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg18440897
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
49 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg19255783
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
49 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
cg20634573
PCR amplification condition
First stage: 95 DEG C, 10min, 1 circulations；
Second stage: 94 DEG C, 30s,
51 DEG C, 1min, 45 circulations；
Phase III: 98 DEG C, 10min, 1 circulations；
Fourth stage: 12 DEG C, 30min.
Diagnosis prediction analysis
CfDNA is carried out in three steps detection and analysis.
The cfDNA of first group of methylation sites is analyzed first: if the methylation value in 3 sites cfDNA is respectively less than or is equal to
Normal control cfDNA methyl rate is then prognosis low-risk；If there is the methyl rate in 2 or 2 or more sites is greater than or waits
Cancer patient's cfDN methyl rate in table, then be prognosis high risk；If the methyl rate in only 1 site is greater than or waits
Cancer patient's cfDN methyl rate in table, or have the methyl rate in 1 or 1 or more site between normal control and cancer
Between patient's cfDNA methyl rate, then need to calculate average value.If average value is lower than 13.83%, show that there is lower trouble
Cancer risk；If average value is greater than or equal to 13.83%, need to do second group of detection.
If the methylation value in second group of 4 site cfDNA is respectively less than or is equal to normal control cfDNA methyl rate, for
Prognosis low-risk；The cancer patient cfDN methylation being greater than or equal in table if there is the methyl rate in 1 or 1 or more site
Rate is then prognosis high risk；If there is the methyl rate in 1 or 1 or more site between normal control and cancer patient's cfDNA first
Between base rate, then need to calculate average value.If average value is lower than 12.20%, shows to have and lower suffer from cancer risk；If
Average value is greater than or equal to 12.20%, then needs to do the detection of third group.
If the methyl rate in 4 sites of cfDNA third group is respectively less than or is equal to normal control cfDNA methyl rate, can
Energy prognosis low-risk, needs to do further clinical monitoring；If if there is the methyl rate in 1 or 1 or more site is greater than or waits
In normal control cfDNA methyl rate, then need to calculate average value.If average value is lower than 12.93%, the low wind of possible prognosis
Danger, needs to do further clinical monitoring；If average value is higher than in 12.93%, for prognosis high risk.Measurement result is shown in Table 7.
The cancer patient cfDNA methylation ratio and normal control cfDNA methylation ratio of 7 methylation markers of table
M: methylation, nM: non-methylation, by the methylation ratio for calculating M/ (M+NM).
SEQUENCE LISTING
<110>Guangzhou You Ze Bioisystech Co., Ltd
<120>for pulmonary cancer diagnosis, outcome prediction or the methylation sites of prognosis, detection primer and kit
<130> BJ-4005-190204A
<160> 100
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> DNA
<213> Artifical sequence
<400> 1
gaggtggagg gatgg 15
<210> 2
<211> 19
<212> DNA
<213> Artifical sequence
<400> 2
cttcccaaac aatttacac 19
<210> 3
<211> 17
<212> DNA
<213> Artifical sequence
<400> 3
gaatgggtcg ggatggg 17
<210> 4
<211> 17
<212> DNA
<213> Artifical sequence
<400> 4
gaatgggttg ggatggg 17
<210> 5
<211> 23
<212> DNA
<213> Artifical sequence
<400> 5
ttttatattt tttatattta aat 23
<210> 6
<211> 19
<212> DNA
<213> Artifical sequence
<400> 6
ttcattcaaa taacaaaca 19
<210> 7
<211> 25
<212> DNA
<213> Artifical sequence
<400> 7
aattttaatt gacaaattgg ttatt 25
<210> 8
<211> 25
<212> DNA
<213> Artifical sequence
<400> 8
aattttaatt gataaattgg ttatt 25
<210> 9
<211> 19
<212> DNA
<213> Artifical sequence
<400> 9
gggtataggg ttttgtatt 19
<210> 10
<211> 18
<212> DNA
<213> Artifical sequence
<400> 10
tattctccca aaacaaca 18
<210> 11
<211> 23
<212> DNA
<213> Artifical sequence
<400> 11
ttgtttgttt tcggtagaat att 23
<210> 12
<211> 23
<212> DNA
<213> Artifical sequence
<400> 12
ttgtttgttt ttggtagaat att 23
<210> 13
<211> 22
<212> DNA
<213> Artifical sequence
<400> 13
ttttatttta atgattgtat tt 22
<210> 14
<211> 20
<212> DNA
<213> Artifical sequence
<400> 14
acaaacctaa taacacctaa 20
<210> 15
<211> 24
<212> DNA
<213> Artifical sequence
<400> 15
tttatagttt tcagtgggtt attg 24
<210> 16
<211> 24
<212> DNA
<213> Artifical sequence
<400> 16
tttatagttt ttagtgggtt attg 24
<210> 17
<211> 23
<212> DNA
<213> Artifical sequence
<400> 17
ttattatatt ataaatttga gtt 23
<210> 18
<211> 22
<212> DNA
<213> Artifical sequence
<400> 18
atacaaacta tatcaaacat aa 22
<210> 19
<211> 23
<212> DNA
<213> Artifical sequence
<400> 19
gtgtggtaaa ttactatgta tgg 23
<210> 20
<211> 23
<212> DNA
<213> Artifical sequence
<400> 20
gtgtggtaaa ttattatgta tgg 23
<210> 21
<211> 19
<212> DNA
<213> Artifical sequence
<400> 21
tagagagtgg tggagattg 19
<210> 22
<211> 18
<212> DNA
<213> Artifical sequence
<400> 22
accacacacc ctactcac 18
<210> 23
<211> 23
<212> DNA
<213> Artifical sequence
<400> 23
gaggattgtt agcttagtgg taa 23
<210> 24
<211> 23
<212> DNA
<213> Artifical sequence
<400> 24
gaggattgtt agtttagtgg taa 23
<210> 25
<211> 20
<212> DNA
<213> Artifical sequence
<400> 25
tgtgtttttt aggtttaagt 20
<210> 26
<211> 19
<212> DNA
<213> Artifical sequence
<400> 26
acacctataa tcccaacta 19
<210> 27
<211> 24
<212> DNA
<213> Artifical sequence
<400> 27
atttttttgt ttcagttttt ttag 24
<210> 28
<211> 24
<212> DNA
<213> Artifical sequence
<400> 28
atttttttgt tttagttttt ttag 24
<210> 29
<211> 19
<212> DNA
<213> Artifical sequence
<400> 29
tagtgtggtt gttaaggtt 19
<210> 30
<211> 20
<212> DNA
<213> Artifical sequence
<400> 30
accaacaact aaatatttca 20
<210> 31
<211> 23
<212> DNA
<213> Artifical sequence
<400> 31
ttgtattatt tcaaagtggg tga 23
<210> 32
<211> 23
<212> DNA
<213> Artifical sequence
<400> 32
ttgtattatt ttaaagtggg tga 23
<210> 33
<211> 16
<212> DNA
<213> Artifical sequence
<400> 33
aggaagggtt ttggtt 16
<210> 34
<211> 19
<212> DNA
<213> Artifical sequence
<400> 34
tcccataaaa aatacaaac 19
<210> 35
<211> 19
<212> DNA
<213> Artifical sequence
<400> 35
gttgagattc tgtgtgggg 19
<210> 36
<211> 19
<212> DNA
<213> Artifical sequence
<400> 36
gttgagattt tgtgtgggg 19
<210> 37
<211> 20
<212> DNA
<213> Artifical sequence
<400> 37
gttatttttg tagatttgtt 20
<210> 38
<211> 17
<212> DNA
<213> Artifical sequence
<400> 38
ctttccacca aaatatt 17
<210> 39
<211> 25
<212> DNA
<213> Artifical sequence
<400> 39
atttatggtt tactttatag tatat 25
<210> 40
<211> 25
<212> DNA
<213> Artifical sequence
<400> 40
atttatggtt tattttatag tatat 25
<210> 41
<211> 22
<212> DNA
<213> Artifical sequence
<400> 41
agaggttata ggtttagtag ag 22
<210> 42
<211> 19
<212> DNA
<213> Artifical sequence
<400> 42
tctaaaaaaa cctcctaaa 19
<210> 43
<211> 19
<212> DNA
<213> Artifical sequence
<400> 43
ttgttttggc gatgttggt 19
<210> 44
<211> 19
<212> DNA
<213> Artifical sequence
<400> 44
ttgttttggt gatgttggt 19
<210> 45
<211> 21
<212> DNA
<213> Artifical sequence
<400> 45
gtttgaaatg ttgtaaaata t 21
<210> 46
<211> 20
<212> DNA
<213> Artifical sequence
<400> 46
ctattcttcc tacctcataa 20
<210> 47
<211> 24
<212> DNA
<213> Artifical sequence
<400> 47
tttattttag tcgttttagt tgaa 24
<210> 48
<211> 24
<212> DNA
<213> Artifical sequence
<400> 48
tttattttag ttgttttagt tgaa 24
<210> 49
<211> 19
<212> DNA
<213> Artifical sequence
<400> 49
ttggtgtgtt ttttagttt 19
<210> 50
<211> 21
<212> DNA
<213> Artifical sequence
<400> 50
taaaataacc acatctaact a 21
<210> 51
<211> 21
<212> DNA
<213> Artifical sequence
<400> 51
ttatgttgtt cgggttattg t 21
<210> 52
<211> 21
<212> DNA
<213> Artifical sequence
<400> 52
ttatgttgtt tgggttattg t 21
<210> 53
<211> 22
<212> DNA
<213> Artifical sequence
<400> 53
gttttgtatg tattaggtat tt 22
<210> 54
<211> 21
<212> DNA
<213> Artifical sequence
<400> 54
acacactaaa acctattaac a 21
<210> 55
<211> 24
<212> DNA
<213> Artifical sequence
<400> 55
gttatttttt tcttaggttt ttat 24
<210> 56
<211> 24
<212> DNA
<213> Artifical sequence
<400> 56
gttatttttt ttttaggttt ttat 24
<210> 57
<211> 21
<212> DNA
<213> Artifical sequence
<400> 57
ggattttatt ttagttgtta t 21
<210> 58
<211> 21
<212> DNA
<213> Artifical sequence
<400> 58
tcaaacataa attttaataa a 21
<210> 59
<211> 24
<212> DNA
<213> Artifical sequence
<400> 59
gaaatatgtt tgtcgataat ttga 24
<210> 60
<211> 24
<212> DNA
<213> Artifical sequence
<400> 60
gaaatatgtt tgttgataat ttga 24
<210> 61
<211> 23
<212> DNA
<213> Artifical sequence
<400> 61
gatygtagga tagtttttat tag 23
<210> 62
<211> 20
<212> DNA
<213> Artifical sequence
<400> 62
caacacactc tctacttatc 20
<210> 63
<211> 20
<212> DNA
<213> Artifical sequence
<400> 63
ggagaatcgg gtaagtgtgt 20
<210> 64
<211> 20
<212> DNA
<213> Artifical sequence
<400> 64
ggagaattgg gtaagtgtgt 20
<210> 65
<211> 16
<212> DNA
<213> Artifical sequence
<400> 65
gagatgggga tggatt 16
<210> 66
<211> 18
<212> DNA
<213> Artifical sequence
<400> 66
accaactaaa acttcctt 18
<210> 67
<211> 17
<212> DNA
<213> Artifical sequence
<400> 67
ttttggggcg ttagggg 17
<210> 68
<211> 17
<212> DNA
<213> Artifical sequence
<400> 68
ttttggggtg ttagggg 17
<210> 69
<211> 17
<212> DNA
<213> Artifical sequence
<400> 69
ggttaatagg gttgagt 17
<210> 70
<211> 19
<212> DNA
<213> Artifical sequence
<400> 70
aactcattaa aaatccaac 19
<210> 71
<211> 20
<212> DNA
<213> Artifical sequence
<400> 71
gataygagtc ggtttygagt 20
<210> 72
<211> 20
<212> DNA
<213> Artifical sequence
<400> 72
gataygagtt ggtttygagt 20
<210> 73
<211> 16
<212> DNA
<213> Artifical sequence
<400> 73
agtgattgga ggtgtt 16
<210> 74
<211> 17
<212> DNA
<213> Artifical sequence
<400> 74
crtcccaaac aaaaacc 17
<210> 75
<211> 20
<212> DNA
<213> Artifical sequence
<400> 75
ggggttttcg tatttttgag 20
<210> 76
<211> 21
<212> DNA
<213> Artifical sequence
<400> 76
ggggtttttg tatttttgag t 21
<210> 77
<211> 18
<212> DNA
<213> Artifical sequence
<400> 77
gattaggagg aaatggat 18
<210> 78
<211> 18
<212> DNA
<213> Artifical sequence
<400> 78
cttaaaccac ctcctaac 18
<210> 79
<211> 19
<212> DNA
<213> Artifical sequence
<400> 79
tttagtgttc gagggtagg 19
<210> 80
<211> 19
<212> DNA
<213> Artifical sequence
<400> 80
tttagtgttt gagggtagg 19
<210> 81
<211> 18
<212> DNA
<213> Artifical sequence
<400> 81
aggtattttt atggggag 18
<210> 82
<211> 15
<212> DNA
<213> Artifical sequence
<400> 82
ttcctaacac cttcc 15
<210> 83
<211> 22
<212> DNA
<213> Artifical sequence
<400> 83
gttagatggt tatcgagatt ta 22
<210> 84
<211> 22
<212> DNA
<213> Artifical sequence
<400> 84
gttagatggt tattgagatt ta 22
<210> 85
<211> 21
<212> DNA
<213> Artifical sequence
<400> 85
ttgatggtag taaaagatta g 21
<210> 86
<211> 19
<212> DNA
<213> Artifical sequence
<400> 86
ctttcctata ctacatccc 19
<210> 87
<211> 19
<212> DNA
<213> Artifical sequence
<400> 87
ggggtgaacg gaaaatatg 19
<210> 88
<211> 20
<212> DNA
<213> Artifical sequence
<400> 88
ggggtgaatg gaaaatatgt 20
<210> 89
<211> 18
<212> DNA
<213> Artifical sequence
<400> 89
ggttttgatt aagtgttg 18
<210> 90
<211> 21
<212> DNA
<213> Artifical sequence
<400> 90
cctacctaac taatatttac t 21
<210> 91
<211> 22
<212> DNA
<213> Artifical sequence
<400> 91
gttttggagt cgaggattat aa 22
<210> 92
<211> 22
<212> DNA
<213> Artifical sequence
<400> 92
gtttyggagt tgaggattat aa 22
<210> 93
<211> 20
<212> DNA
<213> Artifical sequence
<400> 93
gtagggtata gtatatagaa 20
<210> 94
<211> 19
<212> DNA
<213> Artifical sequence
<400> 94
ccacttctac taaattact 19
<210> 95
<211> 22
<212> DNA
<213> Artifical sequence
<400> 95
tagttttaga cgtttttatt gg 22
<210> 96
<211> 22
<212> DNA
<213> Artifical sequence
<400> 96
tagttttaga tgtttttatt gg 22
<210> 97
<211> 19
<212> DNA
<213> Artifical sequence
<400> 97
ggttaggatg tattaggtt 19
<210> 98
<211> 15
<212> DNA
<213> Artifical sequence
<400> 98
accaaaaccc ccaac 15
<210> 99
<211> 16
<212> DNA
<213> Artifical sequence
<400> 99
gtattggggg cgttyg 16
<210> 100
<211> 18
<212> DNA
<213> Artifical sequence
<400> 100
ttgtattggg ggtgttyg 18
Claims (10)
1. for lung cancer early diagnosis, the methylation sites of prediction recurrence of postoperative lung cancer, which is characterized in that selected from as described below
Any one of methylation sites or more than one combination are as diagnosis marker: 1-295864,3-1138223,6-
1526223、6-265969、7-14998、7-862737、9-954757、10-1205149、11-646423、12-1222773、
13-510867、14-1019927、16-571473、17-732857、19-179828。
2. described in accordance with the claim 1 for lung cancer early diagnosis, the methylation sites of prediction recurrence of postoperative lung cancer, feature
It is, the diagnostic markers using the combination of 1-295864,3-1138223,6-1526223 and 6-265969 as the early stage of lung cancer
Object.
3. methylation sites described in claim 1 are in the preparation diagnosis early stage of lung cancer or the reagent for predicting recurrence of postoperative lung cancer
Purposes.
4. the PCR detection kit of recurrence of postoperative lung cancer is early diagnosed or predicted for lung cancer, comprising: amplification claim 1 institute
The specific primer and probe of methylation sites are stated, conversion fluid, in conjunction with liquid, cleaning solution, refined solution, eluent, DNA, which is extracted, to be tried
Agent, PCR amplification reagent.
5. PCR detection kit described in accordance with the claim 3, it is characterised in that: for expanding methyl described in claim 1
The primer sets primer sequence described in SEQ ID No.1-SEQ ID No.4 for changing site 1-295864 forms；It is weighed for expanding
Benefit require 1 described in methylation sites 3-1138223 primer sets primer sequence as described in SEQ ID No.5-SEQ ID No.8
Column composition；For expanding the primer sets of methylation sites 6-1526223 described in claim 1 by SEQ ID No.9-SEQ ID
The composition of primer sequence described in No.12；For expand the primer sets of methylation sites 6-265969 described in claim 1 by
The composition of primer sequence described in SEQ ID No.13-SEQ ID No.16；For expanding methylation sites described in claim 1
The primer sets of 7-14998 primer sequence described in SEQ ID No.17-SEQ ID No.20 forms；For expanding claim
The primer sequence group as described in SEQ ID No.21-SEQ ID No.24 of the primer sets of methylation sites 7-862737 described in 1
At；For expanding the primer sets of methylation sites 9-954757 described in claim 1 by SEQ ID No.25-SEQ ID
The composition of primer sequence described in No.28；For expand the primer sets of methylation sites 10-1205149 described in claim 1 by
The composition of primer sequence described in SEQ ID No.29-SEQ ID No.32；For expanding methylation sites described in claim 1
The primer sets of 11-646423 primer sequence described in SEQ ID No.33-SEQ ID No.36 forms；It is wanted for expanding right
The primer sequence as described in SEQ ID No.37-SEQ ID No.40 of the primer sets of methylation sites 12-1222773 described in asking 1
Column composition；For expanding the primer sets of methylation sites 13-510867 described in claim 1 by SEQ ID No.41-SEQ
The composition of primer sequence described in ID No.44；For expanding the primer of methylation sites 14-1019927 described in claim 1
Group primer sequence described in SEQ ID No.45-SEQ ID No.48 forms；For expanding methylation described in claim 1
The primer sets of site 16-571473 primer sequence described in SEQ ID No.49-SEQ ID No.52 forms；It is weighed for expanding
Benefit require 1 described in methylation sites 17-732857 primer sets primer as described in SEQ ID No.53-SEQ ID No.56
Sequence composition；For expanding the primer sets of methylation sites 19-179828 described in claim 1 by SEQ ID No.57-SEQ
The composition of primer sequence described in ID No.60.
6. for lung cancer for prognosis or the methylation sites of prediction patients with lung cancer mortality risk, which is characterized in that be selected from following methyl
Change any one of site or more than one any combination: cg26205771, cg08436738, cg27252696,
cg24917945、cg03550506、cg06903569、cg12865837、cg18440897、cg19255783、cg20634573。
7. methylation sites according to claim 6, which is characterized in that any one in following (1)-(3) combination
Kind is a variety of: (1) cg26205771, cg08436738 and cg27252696；(2) cg24917945, cg11252953,
Cg03550506 and cg06903569；(3) cg12865837, cg18440897, cg19255783 and cg20634573.
8. methylation sites described in claim 6 or 7 are preparing diagnosing, lung cancer for prognosis or prediction patients with lung cancer death wind
Purposes in the reagent of danger.
9. for pulmonary cancer diagnosis, prognosis or the detection kit for predicting patients with lung cancer mortality risk, comprising: wanted for expanding right
Ask the specific primer and probe of 6 or 7 methylation sites, conversion fluid, in conjunction with liquid, cleaning solution, refined solution, eluent, DNA
Extract reagent, PCR amplification reagent；Preferably, the conversion fluid is bisulfite ammonium salt solution；The combination liquid is hydrochloric acid
Guanidine；The cleaning solution is purified water；The refined solution is isopropanol, ethyl alcohol or sodium hydroxide；The eluent is TE
Solution；The PCR amplification reagent is ddPCR amplifing reagent.
10. detection kit according to claim 9, which is characterized in that for expanding methylation sites cg26205771
Primer sets primer sequence described in SEQ ID No.61-SEQ ID No.64 form；For expanding methylation sites
The primer sets of cg08436738 primer sequence described in SEQ ID No.65-SEQ ID No.68 forms；For expanding methyl
The primer sets primer sequence described in SEQ ID No.69-SEQ ID No.72 for changing site cg27252696 forms；For expanding
The primer sets primer sequence described in SEQ ID No.73-SEQ ID No.76 for increasing methylation sites cg24917945 forms；
For expanding primer sets primer sequence as described in SEQ ID No.77-SEQ ID No.80 of methylation sites cg03550506
Composition；Primer sets for expanding methylation sites cg06903569 are drawn as described in SEQ ID No.81-SEQ ID No.84
Object sequence composition；For expanding the primer sets of methylation sites cg12865837 by SEQ ID No.85-SEQ ID No.88 institute
The primer sequence composition stated；For expanding the primer sets of methylation sites cg18440897 by SEQ ID No.89-SEQ ID
The composition of primer sequence described in No.92；For expanding the primer sets of methylation sites cg19255783 by SEQ ID No.93-
The composition of primer sequence described in SEQ ID No.96；For expanding the primer sets of methylation sites cg20634573 by SEQ ID
The composition of primer sequence described in No.97-SEQ ID No.100.
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