CN107385050A - For detecting the gene marker, kit and cancer of pancreas detection method of cancer of pancreas - Google Patents
For detecting the gene marker, kit and cancer of pancreas detection method of cancer of pancreas Download PDFInfo
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Abstract
The present invention relates to the gene marker for detecting cancer of pancreas, kit and cancer of pancreas detection method.Gene marker includes one or more following gene：Maltose, the member C of c-type lectin family 4, sorting protein 7, the homologous frame 2 of interstitial, FAT atypia cadherin 1, flavine include monooxygenase 3, cystic fibrosis transmembrane conductance regulator, phosphatide phosphatase GAP-associated protein GAP 3, α albumin and the chains of collagen V-type α 2；The content of 5 hydroxymethyl cytosines in cancer of pancreas gene marker is detected by high-flux sequence, so as to judge that cancer of pancreas whether there is.Present invention detection cancer of pancreas has that noninvasive, the DNA wide material sources of safety, accuracy are high, easy to operate, the good advantage of Consumer's Experience.The present invention can be combined with other clinical indices, provided for cancer of pancreas examination, diagnosis, treatment and prognosis and more accurately judged.
Description
Technical field
The present invention relates to the field of the clinical molecular diagnosis of cancer of pancreas.In particular it relates to for detecting cancer of pancreas
Gene marker, kit and cancer of pancreas detection method.
Background technology
Cancer of pancreas（PCA）It is that a kind of grade malignancy is very high, diagnoses and treat all highly difficult malignant tumor of digestive tract, about
90% is the duct adenocarcinoma originating from glandular tube epithelium.This sick incidence of disease male is 1.5~2 higher than the ratio between women, men and women:1, male
Patient is common far beyond premenopausal women, and the incidence of disease of postmenopausal women and male are similar.The cause of disease of cancer of pancreas still not ten at present
Distinguish one from the other.Its occur with smoking, drink, higher fatty acid and high-protein diet, excessive coffee for drinking, environmental pollution and inherent cause have
Close；Survey report in recent years finds the incidence of disease of cancer of pancreas in diabetic population apparently higher than general population；Also someone pays attention to
There is certain relation in the morbidity to chronic pancreatitis patient and cancer of pancreas, it is found that the ratio of cancer of pancreas occurs for chronic pancreatitis patient
It is obvious to increase.In recent years, the incidence of disease of cancer of pancreas is significantly raised, and since the thirties, American and Britain, the Deng states PCA incidences of disease add 2 ~
4 times.The incidences of disease of the Shanghai City PCA in malignant tumour is risen to the 8th in 1993 by the 14th in 1974.PCA falls ill
The increased speed of rate is only second to cancer of pancreas, about increases by 1.5% within every 10 years.
Although the development of abdominal surgery is maked rapid progress in recent years.Many belly illness, including tumour, it diagnoses, controlled
The level for the treatment of is greatly carried with the progress of preclinical medicine, the raising of Imaging Technology and the application of molecular diagnostic techniques
It is high.But the progress in terms of the diagnosis of cancer of pancreas and treatment is unsatisfactory.PCA onsets are hidden, and symptom lacks specificity, normal companion
There are early stage diffusion and transfer phenomena, be one of worst malignant tumour of prognosis.85% Patients with Pancreatic Cancer has belonged to late period when medical,
Only 20% or so operable is treated.Annual PCA is dead and incidence rate is 0.99, and the five year survival rate for making a definite diagnosis patient is 1.3%,
Only 4.1 months Mass median life cycle, it is referred to as " the obstinate fort of 21st century medical science ".
The detection of cancer of pancreas at present mainly passes through iconography, tissue biopsy, Serologic detection etc..However, iconography easily by
Operator's experience influences, and depends on equipment, somewhat expensive, especially in the case where medical resource is limited, its accuracy rate
It is difficult to ensure that, it is difficult to applied extensively with routine, and CT and ultrasonic wave are difficult to pancreatic neoplasm of the diagnosis less than 2cm.Organize biopsy
It is the goldstandard for clinically making a definite diagnosis cancer of pancreas at present, but organizes biopsy significant limitations to be present, such as the difficulty for sampling of performing the operation, or
The some cancer location inconvenience of person are punctured, and puncture also brings along certain clinical risk in itself, puncture examination more repeatedly
Great pain can be brought to patient.It is to carcinomebryonic antigen that Serologic detection is most widely used at present（CEA）Detection, but CEA pairs
The sensitivity and specificity of Early pancreatic carcinoma are not high, often just raised after tumour shifts.
Therefore, the mark of new pancreas carcinoma marker, especially early warning and monitoring and early diagnosis is found to improving early stage
The diagnosis of cancer of pancreas, realize that early intervention is treated, reduce pancreatic cancer mortality and have very important significance.
The content of the invention
The present invention to normal specimens and pancreatic cancer samples 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 cancer of pancreas
Gene marker.
Therefore, the first aspect of the invention is related to the gene marker for detecting cancer of pancreas, including one or two
Following gene above：Maltose（SI）, the member C of c-type lectin family 4（CLEC4C）, sorting protein 7（SNX7）,
The homologous frame 2 of matter（MEOX2）, FAT atypia cadherin 1（FAT1）, flavine include monooxygenase 3（FMO3）, cystic fibrosis across
Film Conductance Regulator（CFTR）, phosphatide phosphatase GAP-associated protein GAP 3（PLPPR3）, α albumin（AFM）With collagen V-type α 2
Chain（COL5A2）.Preferably, the gene marker include SI, CLEC4C, SNX7, MEOX2, FAT1, FMO3, CFTR,
PLPPR3, AFM and COL5A2.
The invention further relates to purposes of the said gene mark in cancer of pancreas is detected, pancreas is detected by high-flux sequence
The content of 5-hydroxymethyl cytosine in oncogene mark, so as to judge that cancer of pancreas whether there is.
The second aspect of the invention is related to the method for detecting cancer of pancreas, comprises the following steps：
a）Determine the content of the 5-hmC of gene marker of the present invention in normal specimens and Samples subjects；
b）The 5-hmC contents of the gene marker described in normal specimens are as reference, by corresponding gene in Samples subjects
The 5-hmC content standards of mark；
c）Mathematical is carried out to the 5-hmC contents of the normalised gene marker, and scored；
d）Testing result is obtained according to the scoring.
In one embodiment, the sample be subject or normal person's body fluid middle reaches from DNA fragmentation, or derive from
Complete genome group DNA in organelle, cell and tissue.Wherein, body fluid is blood, urine, sweat, sputum, excrement, brain ridge
Liquid, ascites, hydrothorax, bile, pancreas liquid etc..
In one embodiment, the 5-hmC contents of gene marker of the present invention can pass through people in the art
Any method known to member is measured, such as is included but is not limited to, glucosylation method, restriction enzyme enzyme process, chemical labeling
The precipitation method, the real-time PCR sequencing PCR of unimolecule associated with method and high-flux sequence method（SMRT）, oxidation bisulfite PCR sequencing PCR
（OxBS-Seq）Deng.The principle of glucosylation method is to use T4 bacteriophages β-glucosyl transferase (β-GT), is supplied in glucose
In the presence of body substrate uridine nucleoside diphosphate glucose (UDP-Glu), by suction pressure to hydroxy position, so as to generate β-Portugal
Grape glycosyl -5-hydroxymethyl cytosine (5-ghmC).It can be quantified simultaneously using isotope labelled substrates.In glucosylation method base
Restriction enzyme enzyme process and chemical labeling method are further developed on plinth.The principle of restriction enzyme enzyme process is：Glucosylation is anti-
The digestion characteristic of some restriction enzymes should be changed.Methylate the restriction endonuclease MspI of dependence and HpaII can recognize that
Same sequence (CCGG), but their sensitiveness to methylation state are different:MspI is identified and is cut 5- methyl born of the same parents
Pyrimidine（5-mC）And 5-hmC, but 5-ghmC can not be cut;HpaII only cuts unmodified site completely, on cytimidine
Any modification (5-mC, 5-hmC, 5-ghmC) hinder cutting.If CpG contains 5-hmC in site, then glycosylation, enzymolysis
Band can be detected afterwards, and not glycosylating does not have band in control reaction;QPCR can be used to carry out quantitative analysis simultaneously.Separately
Outside, other restriction enzymes similarly there is a situation where to hinder 5-ghmC digestions, 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 entered
Row chemical modification is transformed into UDP-6-N3-glucose, and 6-N3-glucose is transferred into hydroxymethyl position, generates N3-
5ghmC.Then, molecular biosciences element is added on each 5-hmC by click chemistry method, with reference to high flux of future generation
DNA sequencing technologies or single-molecule sequencing technology, distribution situations of the 5-hmC in genomic DNA can be analyzed.The precipitation method are by 5-
HmC again specifically captures it after being modified with particular form from genomic DNA, and carries out sequencing analysis.Oxidation weight
Sulphite PCR sequencing PCR is that the first method for carrying out quantitative sequencing to 5-hmC with single base resolution ratio first enters 5-hmC
Row KRuO4 oxidation processes, generate 5- formyl cytimidines（5fC）, then it is sequenced using bisulfite.In the process,
5-hmC initial oxidations are 5fC, and then deamination forms U.Generally, while using a variety of detection methods to 5-hmC quantitative detection is carried out.
In one embodiment of the invention, the base of the present invention is determined using chemical labeling method combination high-flux sequence
Because of the 5-hmC contents of mark.In the specific embodiment, the 5-hmC contents of the gene marker of the present invention are determined
Method comprises the following steps：By the DNA fragmentation from Pancreas cancer patients and the sample of normal person；By the DNA of the fragmentation
End is repaired and blunt end；The DNA of blunt end is connected with sequence measuring joints, obtains connection product；By marking reaction pair
5-hydroxymethyl cytosine in connection product is marked；The DNA fragmentation containing 5-hydroxymethyl cytosine mark is enriched with, is obtained rich
Collect product；Enter performing PCR amplification to enriched product, obtain sequencing library；High-flux sequence is carried out to sequencing library, obtains sequencing knot
Fruit；Content of the 5-hydroxymethyl cytosine on gene is determined according to sequencing result.Wherein, mark reaction includes：i）Utilize glycosyl
Sugar with modification group is covalently attached on the methylol of 5-hydroxymethyl cytosine by transferase, and ii) will be direct or indirect
The click chemistry substrate and the 5-hydroxymethyl cytosine with modification group for being connected with biotin react.Wherein, step i）And step
ii）It can in order carry out, can also be carried out simultaneously in a reaction.This labeling method reduces the sample needed for sequencing
Amount, and the biotin label on 5-hydroxymethyl cytosine makes it show higher dynamic signal in sequencing, improves core
The accuracy of thuja acid identification.In this embodiment, the glycosyl transferase includes but is not limited to：T4 bacteriophages β-glucityl
Transferase（β-GT), T4 bacteriophage alpha-glucosyl transferases（α-GT）And its with same or similar active derivative, similar
Thing or recombinase；The sugar with modification group includes but is not limited to：Carbohydrate with nitrine modification（Such as 6-N3- grapes
Sugar）Or with other chemical modifications（Such as carbonyl, sulfydryl, hydroxyl, carboxyl, carbon-to-carbon double bond, carbon-to-carbon triple bond, disulfide bond, amine
Base, amide groups, diene etc.）Carbohydrate, wherein it is preferred that with nitrine modification carbohydrate；It is described to be used to be indirectly connected with biotin and point
The chemical group for hitting chemical substrate includes but is not limited to：Carbonyl, sulfydryl, hydroxyl, carboxyl, carbon-to-carbon double bond, carbon-to-carbon triple bond, two sulphur
Key, amido, amide groups, diene.In this embodiment it is preferred to the DNA pieces containing 5-hmC marks are enriched with by solid phase material
Section.Specifically, will can be marked by solid phase compatible reaction or other specific binding reactions containing 5-hydroxymethyl cytosine
DNA fragmentation is incorporated on solid phase material, then removes uncombined DNA fragmentation by repeatedly washing.Solid phase material is included but not
It is limited to silicon chip or other chips with surface modification, such as artificial macromolecule bead (preferably a diameter of 1nm-100um), magnetic
(preferably a diameter of 1nm-100um) such as bead (preferably a diameter of 1nm-100um), agarose beads.Used in solid phase enrichment
Cleaning solution is buffer solution well known to those skilled in the art, is included but is not limited to：Contain Tris-HCl, MOPS, HEPES（pH=
6.0-10.0, concentration is between 1mM to 1M）、NaCl（0-2M）Or surfactant such as Tween20（0.01%-5%）Buffering
Liquid.In this embodiment it is preferred to directly expanded in the enterprising performing PCR of solid phase so as to prepare sequencing library.If it is desired, in solid phase
After enterprising performing PCR amplification, the second wheel PCR amplifications are carried out to prepare sequencing library after amplified production can be reclaimed.Second wheel
PCR amplifications can be carried out with conventional method well known by persons skilled in the art.Optionally, can enter during sequencing library is prepared
One step includes one or more purification steps.Any purification kit that those skilled in the art know or commercially available is available
In the present invention.Purification process includes but is not limited to：Gel electrophoresis gel extraction, pellosil centrifugal column method, paramagnetic particle method, ethanol or different
The propyl alcohol precipitation method or its combination.Optionally, before high-flux sequence, quality examination is carried out to sequencing library.For example, to library
Carry out clip size analysis and absolute quantitation is carried out to the concentration in library using qPCR methods.Pass through the sequencing library of quality examination
Available for high-flux sequence.Then by certain amount（1-96）Library containing different barcode is mixed simultaneously by same concentrations
According to machine is sequenced in machine method in the standard of two generation sequenators, sequencing result is obtained.Various two generations sequencings known in the art are flat
Platform and its reagent of correlation 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 characteristicses（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 determine 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
The various comparison softwares and analysis method being related in rapid are known in the art.
In one embodiment of the invention, the 5-hmC contents for determining gene marker refer to determine the genetic marker
5-hmC contents in thing total length or 5-hmC contents or its combination for determining a certain fragment on the gene marker.
According to the present invention, on each gene marker is determined after 5-hmC contents, the genetic marker described in normal specimens
The 5-hmC contents of thing are as reference, by the 5-hmC content standards of corresponding gene marker in Samples subjects.Citing and
The 5-hmC contents of same gene mark are respectively X and Y in speech, normal specimens and Samples subjects, then are somebody's turn to do in Samples subjects
The standardization 5-hmC contents of gene marker are Y/X.
According to the present invention, after data normalization, the standardization 5-hmC contents to each gene marker carry out mathematical
To be scored, so as to obtain testing result according to the scoring.As used herein, " mathematical " refers to from biological sample
The 5-hmC contents of the gene marker of the product any computational methods associated with diagnosis of pancreatic cancer result or machine learning method.
Those of ordinary skill in the art understand, different computational methods may be selected or instrument is used to provide mathematical of the invention, example
If elastomeric network regularization, decision tree, generalized linear model, logistic regression, highest score are to, neutral net, linear and secondary
Discriminant analysis (LQA and QDA), naive Bayesian, random forest and SVMs.
In one embodiment of the invention, the standardization 5-hmC contents to each gene marker carry out mathematical
And obtain comprising the following steps that for scoring：The standardization 5-hmC contents of each gene marker are multiplied by weight coefficient, obtain the base
Because of the predictive factor t of mark；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 suffer from cancer of pancreas；, should be by if P≤0.5
Examination person's sample is normal.Weight coefficient as described herein refers to the factor that 5-hmC contents may be influenceed in consideration（Such as subject
Region, age, sex, it is less than, smoking history, history of drinking history, family history etc.）In the case of, by well known by persons skilled in the art
The coefficient that various advanced statistical analysis methods obtain.
Third aspect of the present invention further relates to carry out the kit of cancer of pancreas detection using said gene mark, and it includes
For the reagent and specification of the 5-hmC contents for determining said gene mark.For determining the 5-hmC contents of gene marker
Reagent be well known by persons 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..
Compared with prior art, the present invention detection cancer of pancreas method be based on the 5-hmC contents on gene marker, because
This can use more extensive DNA sample source.Therefore, the method for present invention detection cancer of pancreas has following advantage：
（1）Safety is noninvasive, also high to the detection acceptance even if Silent cerebral infarction；（2）DNA wide material sources, in the absence of in iconography
Check frequency；（3）Accuracy is high, has higher sensitivity and specificity to Early pancreatic carcinoma, is suitable for the early stage of cancer of pancreas
Examination；（4）Easy to operate, Consumer's Experience is good, easily carries out the dynamic monitoring of pancreas cancer recurrence and transfer.The gene mark of the present invention
Will thing can be combined with other clinical indices, provided for cancer of pancreas examination, diagnosis, treatment and prognosis and more accurately judged.
Brief description of the drawings
Fig. 1 is the curve map that differentiating pancreatic cancer sample of the present invention compares with healthy sample.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in detail, so that those skilled in the art are better understood from
The present invention, and can be practiced.
The screening of the cancer of pancreas gene marker of embodiment 1.
1）Extract plasma dna：
Extract 10 ng plasma dnas respectively from the sample from 20 Pancreas cancer patients and 20 normal persons.Using this area
Any method for being applied to extracting plasma dna and reagent known to technical staff carry out this step.
2）Plasma dna is subjected to blunt end, outstanding A and is connected with sequence measuring joints：
Prepared according to Kapa Hyper Perp Kit specifications containing 50uL plasma dnas, 7uL End Repair ＆ A-
Tailing Buffer and 3uL End Repair ＆ A-Tailing Enzyme mix reaction mixture（Cumulative volume is 60
uL）, in 20 DEG C of warm bath 30 minutes, then in 65 DEG C of warm bath 30 minutes.Following coupled reaction is configured in the low absorption EP pipes of 1.5mL
Mixture：5uL Nuclease free water, 30uL Ligation Buffer and 10 uL DNA Ligase.To
5uL sequence measuring joints are added in 45uL coupled reaction mixtures, mixing, is heated 20 minutes in 20 DEG C, is then held in 4 DEG C.Make
Reaction product is purified with AmpureXP beads, contains 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-hydroxymethyl cytosine：
Prepare the mark reaction mixture that cumulative 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 in 37 DEG C of warm bath 1 hour.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）, reacted 2 hours in 37 DEG C, 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 100uL buffer solutions（5mM Tris, pH=7.5,1M NaCl, 0.02% Tween20）, vortex mixed 30 seconds, Ran Houyong
100uL cleaning solutions（5mM Tris, pH=7.5,1M NaCl, 0.02% Tween20）Wash magnetic bead 3 times, be eventually adding 25
UL combination buffers（10mM Tris, pH=7.5,2M NaCl, 0.04% Tween20 or other surfaces activating agent）, and mix
Close uniform.
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
Secondary, supernatant is removed in centrifugation, adds the water that 23.75uL is free of nuclease.
5）PCR is expanded：
25uL 2 X PCR master mix and 1.25uL PCR primers are added into the final system of above-mentioned steps（Cumulative volume
For 50uL）, expanded according to the temperature and condition of following PCR reaction cycles：
Amplified production AmpureXP beads are purified, 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 subjected to concentration mensuration by qPCR, and DNA fragmentation size in library contained with Agilent2100
Amount is determined.It will be mixed with same concentrations by the sequencing library of quality inspection, be sequenced with Illumina Hiseq 4000.
7）Determine the 5-hmC contents and weight coefficient of each gene marker：
The sequencing result of acquisition is subjected to preliminary Quality Control assessment, after removing low quality sequencing site, is up to sequencing quality standard
Read using Bowtie2 instruments compared with human standard genome reference sequences.Then using featureCounts and
HtSeq-Count instruments count read quantity to determine the 5-hmC contents of each gene marker.Utilize high-flux sequence simultaneously
As a result, it would be possible to influence the factor of 5-hmC contents as covariate, each base is obtained by logistic regression and elastomeric network regularization
Because of the weight coefficient of mark.As a result it is as shown in table 1.
Table 1：The Average normalized 5-hmC contents and weight coefficient of the cancer of pancreas gene marker of the present invention
| Formal symbol | Average normalized 5-hmC contents | P value (FDR) | Weight coefficient | Gene I/D | Gene Name |
| SI | 1.19 | 1.08E-06 | 0.24 | 6476 | Maltose |
| CLEC4C | 0.87 | 1.52E-06 | -2.44 | 170482 | The member C of c-type lectin family 4 |
| SNX7 | 1.18 | 1.54E-06 | 0.45 | 51375 | Sorting protein 7 |
| MEOX2 | 1.18 | 4.85E-06 | 0.84 | 4223 | The homologous frame 2 of interstitial |
| FAT1 | 1.17 | 7.81E-06 | 0.47 | 2195 | FAT atypia cadherin 1 |
| FMO3 | 1.17 | 1.08E-05 | 0.20 | 2328 | Flavine includes monooxygenase 3 |
| CFTR | 1.16 | 1.09E-05 | -0.19 | 1080 | Cystic fibrosis transmembrane conductance regulator |
| PLPPR3 | 0.88 | 1.98E-05 | -2.15 | 79948 | Phosphatide phosphatase GAP-associated protein GAP 3 |
| AFM | 1.15 | 2.25E-05 | 0.23 | 173 | α albumin |
| COL5A2 | 1.13 | 2.29E-05 | 0.67 | 1290 | The chains of collagen V-type α 2 |
As described above, Average normalized 5-hmC contents refer in pancreatic cancer samples the average 5-hmC contents of the gene marker with
The ratio between average 5-hmC contents of same gene mark in normal specimens.As it can be seen from table 1 the pancreas oncogene of the present invention
Significant difference be present in normal specimens and in pancreatic cancer samples in the 5-hmC contents of mark, and except CLEC4C, PLPPR3 it
Outside, the 5-hmC contents of remaining gene marker relative to normally dramatically increasing per capita.
The validity of the cancer of pancreas gene marker of embodiment 2.
The cancer of pancreas gene marker of the present embodiment checking present invention is used for the validity for detecting cancer of pancreas.
First 82 samples are determined according to the method for embodiment 1（41 cancers of pancreas and 41 normal healthy controls）The middle present invention
The 5-hmC contents of 10 described cancer of pancreas gene markers.
The standardization 5-hmC contents of each gene marker are multiplied by the mark corresponding weight coefficient in embodiment 1,
The predictive factor t of the gene marker is obtained, the predictive factor t of each gene marker is added afterwards, obtains total predictive factor
T, total predictive factor T is then obtained into scoring P according to below equation by Logistic conversions：
If P>0.5, then the Samples subjects suffer from cancer of pancreas；If P≤0.5, the Samples subjects are normal.
Fig. 1 shows that the method according to the invention distinguishes the result of this batch of sample.As shown in figure 1, the method energy of the present invention
Enough reach 95% sensitivity and 87% specificity.
Finally it should be noted that above content is merely illustrative of the technical solution of the present invention, rather than the present invention is protected
The limitation of scope, the simple modification or equivalent substitution that one of ordinary skill in the art is carried out to technical scheme,
All without departing from the spirit and scope of technical solution of the present invention.
Claims (10)
1. gene marker is used for the purposes for detecting cancer of pancreas, 5- hydroxyls in cancer of pancreas gene marker are detected by high-flux sequence
The content of methylcystein, so as to judge that cancer of pancreas whether there is, the gene marker is included below one or more
Gene：Maltose（SI）, the member C of c-type lectin family 4（CLEC4C）, sorting protein 7（SNX7）, the homologous frame 2 of interstitial
（MEOX2）, FAT atypia cadherin 1（FAT1）, flavine include monooxygenase 3（FMO3）, CF transmembrane conductance regulation
Albumen（CFTR）, phosphatide phosphatase GAP-associated protein GAP 3（PLPPR3）, α albumin（AFM）With the chains of collagen V-type α 2（COL5A2）.
2. purposes according to claim 1, it is characterised in that：The gene marker include SI, CLEC4C, SNX7,
MEOX2, FAT1, FMO3, CFTR, PLPPR3, AFM and COL5A2.
A kind of 3. method for detecting cancer of pancreas, it is characterised in that comprise the following steps：
a）Determine the 5-hydroxymethyl cytosine of the gene marker in normal specimens and Samples subjects described in claim 1 and 2
Content；
b）The 5-hydroxymethyl cytosine content of the gene marker described in normal specimens, will be right in Samples subjects as reference
Same gene mark in the 5-hydroxymethyl cytosine content standard for the gene marker answered, normal specimens and Samples subjects
5-hydroxymethyl cytosine content be respectively X and Y, then the standardization 5- methylol born of the same parents of the gene marker are phonetic in Samples subjects
Pyridine content is Y/X；
c）To step b）In the 5-hydroxymethyl cytosine content of the normalised gene marker carry out mathematical, and obtain
Must be scored P；
d）According to the numerical values recited of the scoring P obtain Samples subjects whether suffer from cancer of pancreas testing result.
4. according to the method for claim 3, it is characterised in that step a）Comprise the following steps：Will come from Pancreas cancer patients and
The DNA fragmentation of the sample of normal person；Simultaneously blunt end is repaired into the DNA ends of the fragmentation；By the DNA of blunt end with
Sequence measuring joints connect, and obtain connection product；By marking reaction that the 5-hydroxymethyl cytosine in connection product is marked；It is rich
Collect the DNA fragmentation containing 5-hydroxymethyl cytosine mark, obtain enriched product；Enter performing PCR amplification to enriched product, be sequenced
Library；High-flux sequence is carried out to sequencing library, obtains sequencing result；Determine 5-hydroxymethyl cytosine in base according to sequencing result
Because of upper content.
5. according to the method for claim 4, it is characterised in that：The mark reaction includes：i）Will using glycosyl transferase
Sugar with modification group is covalently attached on the methylol of 5-hydroxymethyl cytosine, and ii) it will directly or indirectly be connected with biology
The click chemistry substrate of element reacts with the 5-hydroxymethyl cytosine with modification group.
6. according to the method described in claim 3,4 or 5, it is characterised in that：The step（a）It is the measure gene marker
The content of total length or the 5-hydroxymethyl cytosine in its fragment.
7. according to the method described in claim 3,4 or 5, it is characterised in that step c）Comprise the following steps：By each genetic marker
The standardization 5-hydroxymethyl cytosine content of thing is multiplied by weight coefficient, obtains the predictive factor t of the gene marker；By each gene
The predictive factor t of mark is added, and obtains total predictive factor T；Total predictive factor T is obtained into scoring P by Logistic conversions；
If P>0.5, then the Samples subjects suffer from cancer of pancreas；If P≤0.5, the Samples subjects are normal.
8. according to the method for claim 3, it is characterised in that：The sample is to come from normal person or subject's body fluid middle reaches
From DNA fragmentation, or from organelle, cell and tissue in complete genome group DNA.
9. according to the method for claim 8, it is characterised in that：The body fluid be blood, urine, sweat, sputum, excrement,
Cerebrospinal fluid, ascites, hydrothorax, bile or pancreas liquid.
A kind of 10. kit for being used to detect cancer of pancreas, it is characterised in that including：
a）For the reagent for the 5-hydroxymethyl cytosine content for determining gene marker described in claim 1；And b) specification；
The 5-hydroxymethyl cytosine content refers to the gene marker total length or the 5-hydroxymethyl cytosine in its fragment
Content.
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| CN201710662851.6A CN107385050A (en) | 2017-08-04 | 2017-08-04 | For detecting the gene marker, kit and cancer of pancreas detection method of cancer of pancreas |
| PCT/CN2017/119005 WO2019024404A1 (en) | 2017-08-04 | 2017-12-27 | Genetic marker for detecting pancreatic cancer, kit, and pancreatic cancer detection method |
| TW107122397A TWI680296B (en) | 2017-08-04 | 2018-06-28 | Gene marker, reagent set for detecting pancreatic cancer, and method for detecting pancreatic cancer |
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Also Published As
| Publication number | Publication date |
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| TWI680296B (en) | 2019-12-21 |
| WO2019024404A1 (en) | 2019-02-07 |
| TW201910774A (en) | 2019-03-16 |
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