CN108103196A - It is a kind of to be used to detect gene marker of the cancer of the esophagus and application thereof and detection method - Google Patents
It is a kind of to be used to detect gene marker of the cancer of the esophagus and application thereof and detection method Download PDFInfo
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- CN108103196A CN108103196A CN201810109130.7A CN201810109130A CN108103196A CN 108103196 A CN108103196 A CN 108103196A CN 201810109130 A CN201810109130 A CN 201810109130A CN 108103196 A CN108103196 A CN 108103196A
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Abstract
The invention discloses a kind of for detecting gene marker of the cancer of the esophagus and application thereof and detection method, gene marker includes one or more from the following gene：Phospholipid inositol phospho-esterase c X domains include albumen 3, sulfatase 1, F Box and full asphalt mixture albumen 7, Tolloid albuminoids 1, the single-stranded action protein matter 3 of RNA combination templates, ADAMTS albuminoids 1, phosphodiesterase 10 A, LIM domains ligase 2,3 subtribe of gamma amino butyric acid A receptor albumen gamma and 155 family protein member A of sequence similarity.The purposes of the gene marker is to be used as the detection cancer of the esophagus.The present invention can use more extensive DNA sample source；Safety is noninvasive, also high to the detection acceptance even if Silent cerebral infarction；Accuracy is high, there is higher sensitivity and specificity to the early stage cancer of the esophagus, and easy to operate, user experience is good, easily carries out the dynamic monitoring of recurrence of Esophageal Carcinoma and transfer.
Description
Technical field
The invention belongs to the clinical molecular diagnosis technical fields of the cancer of the esophagus, and specifically the present invention relates to one kind to pass through high pass
Measure the 5-hydroxymethyl cytosine content of sequence detection cancer of the esophagus gene marker so as to detect method that the cancer of the esophagus whether there is and
Kit.
Background technology
The cancer of the esophagus is increasingly taken seriously as one of highest cancer of China's Cancer Mortality.The cancer of the esophagus
Pathogenic factors have very much, it is to cause all kinds of chronic stimulations damaged, Yi Jihuan to oesophagus that generally acknowledged at present having, which drinks and smoke,
The main reason for border factor is Chinese esophageal squamous cell carcinoma morbidity.Investigation finds, like eating scald food, excess is drunk, low receipts people, low constitution
Index, the past esophageal lesion, be late for having dinner, the peppery food of eating and Family history of cancer etc. be increase cancer of the esophagus risk because
Element.
China's cancer of the esophagus is always based on esophageal squamous cell carcinoma, and the incidence of adenocarcinoma of esophagus, which had no, in recent years rises appreciably, oesophagus
Squamous carcinoma has become one of ten big characteristic tumours that the Ministry of Public Health of China determines.It is shown according to the update of World Health Organization's announcement,
2008 6,700,000,000 populations of the annual whole world newly send out the cancer of the esophagus 48.2 ten thousand, and incidence is 7/,100,000, occupies whole malignant tumours the 9th；
Dead 40.7 ten thousand, the death rate 5.8/10 ten thousand occupies the 8th.The 13.4 hundred million population cancer of the esophagus of China's Mainland newly sends out 25.9 ten thousand, morbidity
Rate is 16.7/10 ten thousand, occupies all kinds of malignant tumours the 5th in the whole nation；Dead 21.1 ten thousand, the death rate is 13.4/10 ten thousand, occupies the 4th.
It is counted by gender, China male esophageal cancer patient 17.6 ten thousand, incidence is 22.9/10 ten thousand；Dead 14.4 ten thousand, the death rate 18.7/
100000；Female patient 8.3 ten thousand, incidence are 10.5/10 ten thousand, dead 6.7 ten thousand, and the death rate 8.2/10 ten thousand, male's incidence occupies each
Class malignant tumour the 4th, women occupies the 7th, and death rate men and women occupies the 4th.Its prognosis and the TNM stage of patient are close
Correlation, 5 years survival rates of I phase patient with esophageal carcinoma are 50%~80%, and 5 years survival rates of III, IV phase patient be only 5%~
15%, therefore early diagnose and accurately just become the key factor for improving patient with esophageal carcinoma survival rate by stages.
The detection method of the cancer of the esophagus mainly passes through iconography, tissue biopsy, Serologic detection etc. at present.However, image
Easily by operator's experience influenced, and depends on equipment, somewhat expensive, especially in the case where medical resource is limited,
Its accuracy rate is difficult to ensure that, it is difficult to be applied extensively with routine.With universal and people at highest risk the follow-up in situation of all-level hospitals and
Periodic review it is perfect, endoscopy progressively become cancer of the esophagus examination prefered method, but conventional endoscopic check when early stage eat
Pipe cancer is still easily failed to pinpoint a disease in diagnosis.Tissue biopsy is clinically to make a definite diagnosis the goldstandard of the cancer of the esophagus at present, but there is very big for tissue biopsy
Limitation, such as the difficulty for sampling of performing the operation or some cancer locations are not easy to be punctured, and puncture is also brought along in itself
Certain clinical risk, great pain but will be brought to patient by puncturing examination repeatedly.For clinical cancer of the esophagus blood serum tumor mark
Will object is squamous cell carcinoma antigen (SCC-Ag), carcinomebryonic antigen (CEA), cytokeratin 19 fragment (CYFRA 21-1) and P53
Antibody etc..But these blood serum designated objects are not high for the sensitivity and specificity of the early stage cancer of the esophagus, often occur to turn in tumour
It can just be raised after shifting.
Tumor markers is noninvasive since it is with simplicity, and substantially, can if can have breakthrough in accuracy
The early diagnostic rate of the cancer of the esophagus can be greatly improved, it is final to realize the purpose for reducing patient with esophageal carcinoma case fatality rate.It is but existing at present
Lack simple and effective in technology, be especially adapted for use in the diagnostic method of mass survey, so finding new cancer of the esophagus early diagnosis
Marker is significant.
The content of the invention
The present invention in order to overcome the shortcomings of the prior art, provide it is a kind of for detect the gene marker of the cancer of the esophagus and
Its purposes and detection method.
The present invention is achieved by the following technical solutions：A kind of gene marker for being used to detect the cancer of the esophagus, the gene
Marker includes one or more from the following gene：Phospholipid inositol phospho-esterase c X domains include albumen 3, sulfatase 1, F-
Box and the single-stranded action protein matter 3 of full asphalt mixture albumen 7, Tolloid albuminoids 1, RNA combination templates, ADAMTS albuminoids
1st, phosphodiesterase 10 A, LIM domains ligase 2, gamma -3 subtribe of aminobutyric acid type A receptor albumen gamma and sequence similarity 155
Family protein member A.The purposes of the gene marker is to be used as the detection cancer of the esophagus.
The method of the present invention detection cancer of the esophagus specifically includes following steps：（a）It measures in normal specimens and Samples subjects
The content of 5-hmC in gene marker；（b）By the use of the 5-hmC contents of gene marker in normal specimens as reference, by subject
The 5-hmC content standards of corresponding gene marker in sample；（c）To step（b）In normalised gene marker
5-hmC contents carry out mathematical, and obtain scoring P；（d）Testing result is obtained according to scoring P, scoring P ＞ 0.5 show this by
Examination person's sample suffers from the cancer of the esophagus.Step（a）It is to measure gene marker overall length or the content of the 5-hmC in its segment.
Wherein sample be in normal person or subject's body fluid dissociate DNA fragmentation or from organelle, cell
And the complete genome group DNA in tissue, body fluid is blood, urine, sweat, sputum, excrement, cerebrospinal fluid, ascites, hydrothorax, courage
Juice or oesophagus liquid.
The invention also discloses a kind of for detecting the kit of the cancer of the esophagus, which includes measuring genetic marker
The reagent and specification of the 5-hmC contents of object, wherein 5-hmC contents refer to the 5-hmC in gene marker overall length or its segment
Content.
Applicant to normal specimens and cancer of the esophagus sample by carrying out high-flux sequence, and to the 5- hydroxyls on wherein each gene
Methylcystein（5-hmC）Content is analyzed, it has surprisingly been found that multiple great information can be used for detect the cancer of the esophagus
Gene marker.
Therefore, the first aspect of the invention is related to the gene marker for detecting the cancer of the esophagus, the gene marker bag
Include one or more from the following gene：Phospholipid inositol phospho-esterase c X domains include albumen 3（PLCXD3）, sulfatase 1
（SULF1）, F-Box and full asphalt mixture albumen 7（FBXL7）, Tolloid albuminoids 1（TLL1）, RNA combination templates it is single-stranded
Action protein matter 3（RBMS3）, ADAMTS albuminoids 1（ADAMTSL1）, phosphodiesterase 10 A（PDE10A）, LIM domains ligase 2
（LDB2）, gamma -3 subtribe of aminobutyric acid type A receptor albumen gamma（GABRG3）With 155 family protein member A of sequence similarity
（FAM155A）.
Preferably, gene marker include at least two, at least three, at least four, at least five, at least six, extremely
Seven, at least eight, at least nine or at least ten are selected from following gene less：PLCXD3、SULF1、FBXL7、TLL1、
RBMS3, ADAMTSL1, PDE10A, LDB2, GABRG3 and FAM155A.It is optimal, gene marker include PLCXD3, SULF1,
FBXL7, TLL1, RBMS3, ADAMTSL1, PDE10A, LDB2, GABRG3 and FAM155A.The invention further relates to said gene marks
Purposes of the will object in the cancer of the esophagus is detected.
The second aspect of the invention is related to the method for detecting the cancer of the esophagus, comprises the following steps：（a）Measure normal sample
The content of the 5-hmC of gene marker of the invention in product and Samples subjects；（b）With the 5- of gene marker in normal specimens
HmC contents are as reference, by the 5-hmC content standards of corresponding gene marker in Samples subjects；（c）To normalized
The 5-hmC contents of gene marker carry out mathematical, and scored；（d）Testing result is obtained according to scoring.
In one embodiment, sample be subject or normal person's body fluid middle reaches from DNA fragmentation or from cell
Complete genome group DNA in device, cell and tissue.Wherein body fluid is blood, urine, sweat, sputum, excrement, cerebrospinal fluid, abdomen
Water, hydrothorax, bile or oesophagus liquid etc..
In one embodiment, the 5-hmC contents of gene marker of the invention can be by those skilled in the art
Any method known is measured, for example, including but not limited to glucosylation method, restriction enzyme enzyme process, chemical labeling method, with
The real-time PCR sequencing PCR of the precipitation method, unimolecule associated with high-flux sequence method（SMRT）, oxidation bisulfite PCR sequencing PCR（OxBS-
Seq）Deng.
The principle of glucosylation method is using T4 bacteriophages β-glucosyl transferase (β-GT), in glucose donor substrate
In the presence of uridine nucleoside diphosphate glucose (UDP-Glu), by suction pressure to hydroxy position, so as to generate β-glucose
Base -5-hydroxymethyl cytosine (5-ghmC).It is quantified simultaneously using isotope labelled substrates, on the basis of glucosylation method
Further develop restriction enzyme enzyme process and chemical labeling method.
The principle of restriction enzyme enzyme process is：Glucosylation reacts the digestion characteristic for changing some restriction enzymes.
It methylates the restriction endonuclease MspI of dependence and HpaII can recognize that same sequence (CCGG), but they are to the shape that methylates
The sensibility of state is different:MspI is identified and is cut 5-methylcytosine（5-mC）And 5-hmC, but 5-ghmC cannot be cut;
HpaII only cuts unmodified site completely, and any modification (5-mC, 5-hmC, 5-ghmC) on cytimidine hinders to cut
It cuts.If CpG contains 5-hmC in site, then can detect band after glycosylation, enzymolysis, do not glycosylate and do not have in control reaction
There is band；QPCR can be used simultaneously and carry out quantitative analysis.In addition, other restriction enzymes, which similarly exist, hinders 5-ghmC
The situation of digestion can be applied to 5-hmC detections (such as：GmrSD, MspJI, PvuRts1I, TaqI etc.).
The principle of chemical labeling method is：Glucose on enzyme reaction substrate is chemically modified and is transformed into UDP-6-N3-
6-N3-glucose is transferred to hydroxymethyl position, generates N3-5ghmC by glucose.Then existed by click chemistry method
Molecular biosciences element is added on each 5-hmC, it, can with reference to next-generation high throughput DNA sequencing technologies or single-molecule sequencing technology
Analyze distribution situations of the 5-hmC in genomic DNA.
The precipitation method are again specifically to capture it from genomic DNA after 5-hmC is modified with particular form, and
Carry out sequencing analysis.Oxidation bisulfite PCR sequencing PCR is the first side for carrying out quantitative sequencing to 5-hmC with single base resolution ratio
5-hmC is carried out KRuO4 oxidation processes by method first, generates 5- formyl cytimidines（5fC）, then surveyed using bisulfite
Sequence.In the process, 5-hmC initial oxidations are 5fC, and then deamination forms U.In general, simultaneously using a variety of detection methods to 5-
HmC carries out quantitative detection.
In one embodiment of the invention, gene of the present invention is measured using chemical labeling method combination high-flux sequence
The 5-hmC contents of marker.In the specific embodiment, measure the present invention gene marker 5-hmC contents method
Specifically include following steps：By the DNA fragmentation from patient with esophageal carcinoma and the sample of normal person；By the DNA ends of fragmentation
Repair simultaneously blunt end；The DNA of blunt end with sequence measuring joints is connected, obtains connection product；By marking reaction to connection
5-hydroxymethyl cytosine in product is marked；The DNA fragmentation containing 5-hmC marks is enriched with, obtains enriched product；To enrichment
Product carries out PCR amplification, obtains sequencing library；High-flux sequence is carried out to sequencing library, obtains sequencing result；It is tied according to sequencing
Fruit determines contents of the 5-hmC on gene.
Mark reaction includes：i）Sugar with modification group is covalently attached to the hydroxyl first of 5-hmC using glycosyl transferase
On base, ii) the click chemistry substrate for being directly or indirectly connected with biotin is reacted with the 5-hmC with modification group.Wherein walk
Rapid i）With step ii）It can carry out, can also be carried out at the same time in a reaction in order.This labeling method reduces sequencing
Required sample size, and the biotin label on 5-hmC makes it show higher dynamic signal in sequencing, improves core
The accuracy of thuja acid identification.In this embodiment, glycosyl transferase includes but not limited to：T4 bacteriophages β-glucosyltransferase
（β-GT), T4 bacteriophage alpha-glucosyl transferases（α-GT）And its with the derivative of same or similar activity, analog or again
Group enzyme；Sugar with modification group includes but not limited to：Carbohydrate with nitrine modification（6-N3- glucose）Or with other changes
Learn modification（Such as carbonyl, sulfydryl, hydroxyl, carboxyl, carbon-to-carbon double bond, carbon-carbon triple bond, disulfide bond, amido, amide groups, diene）'s
Carbohydrate, wherein it is preferred that the carbohydrate with nitrine modification；For being indirectly connected with the chemical group bag of biotin and click chemistry substrate
It includes but is not limited to：Carbonyl, sulfydryl, hydroxyl, carboxyl, carbon-to-carbon double bond, carbon-carbon triple bond, disulfide bond, amido, amide groups, diene.
In this embodiment it is preferred to the DNA fragmentation containing 5-hmC marks is enriched with by solid phase material.Specifically, may be used
By solid phase compatible reaction or other specific binding reactions solid phase material will be incorporated in containing the DNA fragmentation that 5-hmC is marked
On, then unbonded DNA fragmentation is removed by repeatedly washing.Solid phase material includes but not limited to the silicon chip with surface modification
Or other chips, such as artificial macromolecule bead, magnetic ball, agarose bead etc..Cleaning solution used is in solid phase enrichment
Buffer solution well known to those skilled in the art includes but not limited to：Contain Tris-HCl, MOPS, HEPES（pH=6-10）、NaCl
Or surfactant such as Tween20（0.01%~5%）Buffer solution.In this embodiment it is preferred to it is carried out directly in solid phase
PCR amplification is so as to preparing sequencing library.If it is desired, after carrying out PCR amplification in solid phase, carried out after amplified production can be recycled
Second takes turns PCR amplification to prepare sequencing library.
Second wheel PCR amplification can be carried out with conventional method well known by persons skilled in the art, optionally, prepare sequencing text
It can further comprise one or more purification steps during storehouse.It is that those skilled in the art know or commercially available any pure
Change kit and be used equally for the present invention.Purification process includes but not limited to gel electrophoresis gel extraction, pellosil centrifugal column method, magnetic
Pearl method, ethyl alcohol or isopropanol precipitating method or its combination.Optionally, before high-flux sequence, quality inspection is carried out to sequencing library
It looks into.For example, clip size analysis is carried out to library and absolute quantitation is carried out to the concentration in library using qPCR methods.Pass through quality
The sequencing library of inspection can be used for high-flux sequence.Then by certain amount（1~96）It presses in library containing different barcode
Same concentrations mixing is simultaneously sequenced according to machine in machine method in the standards of two generation sequenators, obtains sequencing result.It is known in the art
Various two generations microarray datasets and its relevant reagent can be used for the present invention.
In one embodiment of the invention, preferably sequencing result and standard human's genome reference sequences are compared
It is right, the sequence wherein compared on gene marker of the present invention is picked out, that is, is selected than loci and gene expression characteristics（Such as histone
Decorating site, Binding site for transcription factor, gene extron include subregion and gene promoter etc.）The read of overlapping region
Quantity, it is horizontal to represent modifications of the 5-hmC on the gene, so as to measure contents of the 5-hmC on the gene marker.It is preferred that
Before being compared, sequencing result is removed into low quality sequencing site first, wherein weighing the factor of sequencing site quality includes
But it is not limited to：Base quality, reads mass, G/C content, repetitive sequence and Overrepresented sequence quantity etc..The step
Various comparison softwares and analysis method involved in rapid are known in the art.
In one embodiment of the invention, the 5-hmC contents for measuring gene marker refer to measure the genetic marker
5-hmC contents in object overall length or 5-hmC contents or its combination for measuring a certain segment on the gene marker.According to this hair
It is bright, on each gene marker is measured after 5-hmC contents, by the use of the 5-hmC contents of gene marker in normal specimens as joining
According to by the 5-hmC content standards of corresponding gene marker in Samples subjects.Such as：Normal specimens and Samples subjects
The 5-hmC contents of middle same gene marker are respectively X and Y, then in Samples subjects the gene marker standardization 5-hmC
Content is Y/X.
According to the present invention, after data normalization, mathematical is carried out to the standardization 5-hmC contents of each gene marker
To be scored, so as to obtain testing result according to scoring.As used in the present invention, " mathematical " refers to biological sample in future
Gene marker 5-hmC contents any computational methods associated with esophagus cancer diagnosis result or machine learning method.This
Field those of ordinary skill understands, different computational methods may be selected or instrument is used to provide the mathematical of the present invention, such as
Elastomeric network regularization, decision tree, generalized linear model, logistic regression, highest score are to, neutral net, linear and secondary sentence
Other formula analysis (LQA and QDA), naive Bayesian, random forest and support vector machines.
In one embodiment of the invention, mathematical is carried out to the standardization 5-hmC contents of each gene marker
And it obtains scoring and is as follows：The standardization 5-hmC contents of each gene marker are multiplied by weighting coefficient, obtain the base
Because of the predictive factor t of marker；The predictive factor t of each gene marker is added, obtains total predictive factor T；To always predict because
Sub- T obtains scoring P by Logistic conversions；If P>0.5, then the Samples subjects are with the cancer of the esophagus；It, should be by if P≤0.5
Examination person's sample is normal.The weighting coefficient of the present invention refers to the factor that 5-hmC contents may be influenced in consideration（Such as subject
Domain, age, are less than, smoking history, history of drinking history, family history etc. gender）In the case of, by well known by persons skilled in the art each
The coefficient that kind advanced statistical analysis method obtains.
Third aspect of the present invention further relates to carry out the kit of cancer of the esophagus detection using said gene marker, including
For measuring the reagent and specification of the 5-hmC contents of said gene marker.For measuring the 5-hmC contents of gene marker
Reagent be known to the skilled in the art, such as T4 bacteriophages β-glucosyl transferase and isotope marks（For glucose
Base method）, restriction enzyme（For restriction enzyme enzyme process）, glycosyl transferase and biotin（For chemical labeling method）、
PCR and sequencing agents useful for same etc..
The beneficial effects of the invention are as follows：The present invention relates to gene markers for detecting the cancer of the esophagus and application thereof, also relate to
And the method being detected using the gene marker to the cancer of the esophagus.Compared with prior art, the present invention is used to detect the cancer of the esophagus
Method be based on the 5-hmC contents on gene marker, therefore more extensive DNA sample source can be used.The present invention
For detect the method for the cancer of the esophagus also have the advantages that it is following：（1）Safety is noninvasive, and also the detection is connect even if Silent cerebral infarction
It is high by degree；（2）DNA is derived from a wealth of sources, and there is no the check frequencies in iconography；（3）Accuracy is high, has to the early stage cancer of the esophagus higher
Sensitivity and specificity, be suitable for the early screening of the cancer of the esophagus；（4）Easy to operate, user experience is good, easily carries out oesophagus
Cancer recurrence and the dynamic monitoring of transfer.Gene marker in the present invention can be combined with other clinical indices, be the cancer of the esophagus
Examination, diagnosis, treatment and prognosis offer more accurately judge.
Description of the drawings
Fig. 1 is the result figure that cancer of the esophagus sample and normal healthy controls are distinguished with cancer of the esophagus gene marker of the present invention.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail, so that those skilled in the art can be more
Good understanding is of the invention and can be practiced.It should be appreciated by those skilled in the art attached drawing of the invention and embodiment are only
It is solely for the purpose of illustration, any restrictions, in the case of no contradiction, the implementation in the application can not be formed to the present invention
Feature in example and embodiment can be mutually combined.
Embodiment 1：The screening of cancer of the esophagus gene marker：
（1）Extract plasma dna：Extract 10 ng blood plasma respectively from the sample from 20 patient with esophageal carcinoma and 20 normal persons
DNA.This step is carried out using any method for being suitable for extracting plasma dna well-known to those skilled in the art and reagent.
（2）Plasma dna is subjected to blunt end, outstanding A and is connected with sequence measuring joints：
It is prepared according to Kapa Hyper Perp Kit specifications containing 50uL plasma dnas, 7uL End Repair ＆ A-
The reaction mixture of Tailing Buffer and 3uL End Repair ＆ A-Tailing Enzyme mix（Total volume is 60
uL）, in 20 DEG C of warm bath 30min, then in 65 DEG C of warm bath 30min.Following coupled reaction is configured in 1.5mL low adsorption EP pipes to mix
Close object：5uL Nuclease free water, 30uL Ligation Buffer and 10 uL DNA Ligase.To 45uL
The sequence measuring joints of 5uL are added in coupled reaction mixture, mixing heats 20min in 20 DEG C, is then held in 4 DEG C.It uses
AmpureXP beads purify reaction product, contain Tris-HCl with 20uL（10mM, pH=8.0）And EDTA（0.1mM）
Buffer solution carry out elution and obtain final DNA connection samples.
（3）Mark 5-hmC：
Prepare the mark reaction mixture that total volume is 26 uL：The uridine diphosphate glucose of nitrine modification（That is UDP-N3-
Glu, final concentration of 50uM）、β-GT（Final concentration of 1uM）、Mg2+（Final concentration of 25mM）、HEPES（PH=8.0, it is final concentration of
50mM）With the 20uL DNA from above-mentioned steps.By mixed liquor when 37 DEG C of warm bath 1 are small.Mixed liquor is taken out, uses AmpureXP
Beads is purified, and obtains the 20uL DNA of purifying.
Then the diphenyl cyclooctyne that 1uL is connected with biotin is added in the 20uL DNA of above-mentioned purifying（DBCO-
Biotin）, when 37 DEG C of reactions 2 are small, then purified with AmpureXP beads, obtain the marked product of purifying.
（4）Solid phase is enriched with the DNA fragmentation containing markd 5-hydroxymethyl cytosine：
First, magnetic bead is prepared according to the following steps：Take out 0.5uL C1 streptadvin beads（life technology）And
Add in 100uL buffer solutions（5mM Tris, pH=7.5,1M NaCl, 0.02% Tween20）Vortex mixed 30s, then uses 100uL
Cleaning solution（5mM Tris pH=7.5,1M NaCl, 0.02% Tween20）It washs magnetic bead 3 times, is eventually adding 25 uL and combines and delay
Fliud flushing（10mM Tris, pH=7.5,2M NaCl, 0.04% Tween20 or other surfaces activating agent）, and be uniformly mixed.
Then, the marked product for the purifying that above-mentioned steps obtain is added in magnetic bead mixed liquor, and in impeller
Mixing 15min makes it fully combine.
Finally, with 100uL cleaning solutions（5mM Tris, pH=7.5,1M NaCl, 0.02% Tween20）Wash magnetic bead 3 times,
Supernatant is removed in centrifugation, adds in the water that 23.75uL is free of nuclease.
（5）PCR amplification：
2 X PCR master mix and the 1.25uL PCR primers of 25uL are added in into the final system of above-mentioned steps（Total volume
For 50uL）, it is expanded according to the temperature and condition of following PCR reaction cycles, amplified production is pure with AmpureXP beads
Change, obtain final sequencing library.
（6）High-flux sequence is carried out after carrying out quality inspection to sequencing library：The sequencing library of acquisition is carried out by qPCR dense
Degree measures, and DNA fragmentation size content in library is determined with Agilent2100.By by the sequencing library of quality inspection with
Same concentrations mix, and are sequenced with Illumina Hiseq 4000.
（7）Determine the 5-hmC contents and weighting coefficient of each gene marker：The sequencing result of acquisition is subjected to preliminary Quality Control
Assessment, after removing low quality sequencing site, the read for being up to sequencing quality standard utilizes Bowtie2 instruments and human standard base
Because a group reference sequences are compared.Then read quantity is counted with true using featureCounts and HtSeq-Count instruments
The 5-hmC contents of fixed each gene marker.Simultaneously using high-flux sequence as a result, the factor that will likely influence 5-hmC contents is made
For covariate, the weighting coefficient of each gene marker is obtained by logistic regression and elastomeric network regularization, as a result such as 1 institute of table
Show.
Table 1：The Average normalized 5-hmC contents and weighting coefficient of the cancer of the esophagus gene marker of the present invention
| Formal symbol | Average normalized 5-hmC contents | P value (FDR) | Weighting coefficient | Gene I/D | Gene Name |
| PLCXD3 | 1.16 | 1.63E-06 | 0.84 | 345557 | Phospholipid inositol phospho-esterase c X domains include albumen 3 |
| SULF1 | 1.18 | 3.72E-06 | 0.26 | 23213 | Sulfatase 1 |
| FBXL7 | 0.93 | 7.32E-06 | 0.45 | 23194 | F-Box and full asphalt mixture albumen 7 |
| TLL1 | 1.16 | 2.85E-06 | 0.88 | 7092 | Tolloid albuminoids 1 |
| RBMS3 | 1.17 | 1.61E-06 | 0.39 | 27303 | The single-stranded action protein matter 3 of RNA combination templates |
| ADAMTSL1 | 0.89 | 4.38E-05 | -0.20 | 92949 | ADAMTS albuminoids 1 |
| PDE10A | 1.13 | 6.09E-05 | 0.59 | 10846 | Phosphodiesterase 10 A |
| LDB2 | 1.19 | 1.68E-05 | -0.17 | 9079 | LIM domains ligase 2 |
| GABRG3 | 1.14 | 1.25E-05 | 0.63 | 2567 | Gamma -3 subtribe of aminobutyric acid type A receptor albumen gamma |
| FAM155A | 1.17 | 1.09E-05 | 0.35 | 728215 | 155 family protein member A of sequence similarity |
As above, Average normalized 5-hmC contents refer in cancer of the esophagus sample the average 5-hmC contents of the gene marker with it is normal
The ratio between average 5-hmC contents of same gene marker in sample.As it can be seen from table 1 the cancer of the esophagus genetic marker of the present invention
The 5-hmC contents of object in normal specimens and in cancer of the esophagus sample there are significant difference, and in addition to ADAMTSL1, LDB2,
The 5-hmC contents of remaining gene marker compared with normally dramatically increasing per capita.
Embodiment 2：The validity of cancer of the esophagus gene marker：
The present embodiment is used to verify the validity that the cancer of the esophagus gene marker of the present invention is used to detect the cancer of the esophagus.According to embodiment
1 method measures first 82 samples（41 cancer of the esophagus and 107 normal healthy controls）10 oesophagus oncogenes of the middle present invention
The 5-hmC contents of marker.The standardization 5-hmC contents of each gene marker are multiplied by the marker to correspond in embodiment 1
Weighting coefficient, obtain the predictive factor t of the gene marker, be afterwards added the predictive factor t of each gene marker, obtain
Then total predictive factor T is obtained scoring P by total predictive factor T according to the following formula by Logistic conversions：P=1/(1+e-T), if P>0.5, then the Samples subjects are with the cancer of the esophagus；If P≤0.5, which is normal.It is shown in Fig. 1
The method according to the invention distinguish this batch of sample as a result, as shown in Figure 1, the method for the present invention can reach 90% sensitivity
With 92% specificity.
Finally it should be noted that more than content is merely illustrative of the technical solution of the present invention rather than the present invention is protected
The limitation of scope, simply modification or the equivalent substitution that those of ordinary skill in the art carry out technical scheme,
All without departing from the spirit and scope of technical solution of the present invention.
Claims (6)
1. a kind of gene marker for being used to detect the cancer of the esophagus, it is characterised in that：The gene marker includes one or more
Selected from following gene：Phospholipid inositol phospho-esterase c X domains include albumen 3, sulfatase 1, F-Box and full asphalt mixture egg
White 7, the single-stranded action protein matter 3 of Tolloid albuminoids 1, RNA combination templates, ADAMTS albuminoids 1, phosphodiesterase 10 A, LIM
155 family protein member A of domain ligase 2, gamma -3 subtribe of aminobutyric acid type A receptor albumen gamma and sequence similarity.
2. the purposes of the gene marker according to claim 1 for being used to detect the cancer of the esophagus, it is characterised in that：The gene mark
The purposes of will object is to be used as the detection cancer of the esophagus.
3. the method according to claim 1 using the genetic marker analyte detection cancer of the esophagus for detecting the cancer of the esophagus, feature exists
In：The method of the detection cancer of the esophagus specifically includes following steps：
（a）Measure the content of 5-hmC in gene marker in normal specimens and Samples subjects；
（b）By the use of the 5-hmC contents of gene marker in normal specimens as reference, by corresponding genetic marker in Samples subjects
The 5-hmC content standards of object；
（c）To step（b）In normalised gene marker 5-hmC contents carry out mathematical, and obtain scoring P；
（d）Testing result is obtained according to scoring P, scoring P ＞ 0.5 show that the Samples subjects suffer from the cancer of the esophagus.
4. the method according to claim 3 using the genetic marker analyte detection cancer of the esophagus for detecting the cancer of the esophagus, feature exists
In：The step（a）It is to measure gene marker overall length or the content of the 5-hmC in its segment.
5. the method according to claim 3 using the genetic marker analyte detection cancer of the esophagus for detecting the cancer of the esophagus, feature exists
In：Wherein sample be in normal person or subject's body fluid dissociate DNA fragmentation or from organelle, cell and
Complete genome group DNA in tissue.
6. the method according to claim 5 using the genetic marker analyte detection cancer of the esophagus for detecting the cancer of the esophagus, feature exists
In：The body fluid is blood, urine, sweat, sputum, excrement, cerebrospinal fluid, ascites, hydrothorax, bile or oesophagus liquid.
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| CN201810109130.7A CN108103196A (en) | 2018-02-05 | 2018-02-05 | It is a kind of to be used to detect gene marker of the cancer of the esophagus and application thereof and detection method |
| PCT/CN2019/072398 WO2019149093A1 (en) | 2018-02-05 | 2019-01-18 | Gene marker for detecting esophageal cancer, use thereof and detection method therefor |
| TW108103804A TW201934568A (en) | 2018-02-05 | 2019-01-31 | Gene marker for detecting esophageal cancer, use thereof and detection method therefor |
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| CN (1) | CN108103196A (en) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019149093A1 (en) * | 2018-02-05 | 2019-08-08 | 上海易毕恩生物技术有限公司 | Gene marker for detecting esophageal cancer, use thereof and detection method therefor |
| CN112180088A (en) * | 2020-04-30 | 2021-01-05 | 郑州大学第一附属医院 | Test paper strip for early screening of esophageal cancer |
| CN113406328A (en) * | 2021-06-17 | 2021-09-17 | 福建省立医院 | Application of CST1, CEA and SCC-Ag in preparation of esophageal squamous cell carcinoma early diagnosis kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DK3159406T3 (en) * | 2014-06-18 | 2021-01-11 | Toray Industries | Kit or device for detecting esophageal cancer and method for detecting |
| CN115927612A (en) * | 2015-03-27 | 2023-04-07 | 精密科学公司 | Detecting esophageal disorders |
| US20190042695A1 (en) * | 2016-02-10 | 2019-02-07 | Fukushima Medical University | Method for differentiating contraction of esophageal basaloid carcinoma |
| CN107385051A (en) * | 2017-08-04 | 2017-11-24 | 上海易毕恩生物技术有限公司 | For detecting liver tumour good pernicious gene marker, kit and detection method |
| CN107365845A (en) * | 2017-08-04 | 2017-11-21 | 上海易毕恩生物技术有限公司 | For detecting the gene marker, kit and lung cancer detection method of lung cancer |
| CN108103196A (en) * | 2018-02-05 | 2018-06-01 | 上海易毕恩生物技术有限公司 | It is a kind of to be used to detect gene marker of the cancer of the esophagus and application thereof and detection method |
-
2018
- 2018-02-05 CN CN201810109130.7A patent/CN108103196A/en active Pending
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019149093A1 (en) * | 2018-02-05 | 2019-08-08 | 上海易毕恩生物技术有限公司 | Gene marker for detecting esophageal cancer, use thereof and detection method therefor |
| CN112180088A (en) * | 2020-04-30 | 2021-01-05 | 郑州大学第一附属医院 | Test paper strip for early screening of esophageal cancer |
| CN112180088B (en) * | 2020-04-30 | 2022-05-06 | 郑州大学第一附属医院 | Test paper strip for early screening of esophageal cancer |
| CN113406328A (en) * | 2021-06-17 | 2021-09-17 | 福建省立医院 | Application of CST1, CEA and SCC-Ag in preparation of esophageal squamous cell carcinoma early diagnosis kit |
| CN113406328B (en) * | 2021-06-17 | 2022-04-29 | 福建省立医院 | Application of CST1, CEA and SCC-Ag in preparation of esophageal squamous cell carcinoma early diagnosis kit |
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| WO2019149093A1 (en) | 2019-08-08 |
| TW201934568A (en) | 2019-09-01 |
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