CN108753963A - A kind of detection fecal cast-off cell methylation state of DNA is used to analyze the kit of colorectal cancer - Google Patents
A kind of detection fecal cast-off cell methylation state of DNA is used to analyze the kit of colorectal cancer Download PDFInfo
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Abstract
A kind of detection fecal cast-off cell methylation state of DNA is used to analyze the kit of colorectal cancer, kit and its application of the detection with the relevant PDX1 gene methylations of colorectal cancer in a kind of fecal cast-off cell from colorectal cancer are specially provided, the nucleic acid sequence that the present invention passes through design specific methylation primer detection PDX1 gene methylations, realize the screening to colorectal cancer and auxiliary diagnosis, the methylation level of PDX1 gene markers is analyzed by fluorescence quantitative PCR method, and according to the positive or negative result for the CT value interpretation samples reported.Can be detected in preclinical study absolute majority colorectal cancer and most cancer before adenoma；It can detect 90% Early cancer, adenoma (>=1 centimetre) before 64% cancer, specificity is 100%, and completely noninvasive, it is only necessary to which 4.5 grams of excrement, Silent cerebral infarction acceptance are high.
Description
Technical field
The invention belongs to molecular biology fields, are related to a kind of kit of methylation status of PTEN promoter method screening colorectal cancer,
With the presence or absence of knot colorectal cancer mutant DNA suitable for detection fecal cast-off cell.
Background technology
Colorectal cancer is one of most common malignant tumour of the mankind, and incidence occupies global malignant tumour third position.According to generation
The annual morbidity of boundary health organization statistics, whole world colorectal cancer is 1,200,000, year case fatality rate be about 600,000.Colorectal cancer
Incidence is gradually stablized in developed country.Developing country headed by China, with people life style and diet knot
The change of structure, the linear ascendant trend of incidence of colorectal cancer have statistical form to show that annual growth is about 4.2%, have become
China women third position, male the 4th malignant tumour.The survival rate of PATIENTS WITH LARGE BOWEL and neoplasm staging are closely related, early stage
5 years survival rates of Postoperative carcinoma are more than 90%, but when most of patients is medical has been in progressive stage.Since 93% colorectal cancer is derived from
Adenoma, early stage develop to cancer from adenoma and need 3~17 years mostly without specific symptom, this just provides the time for early screening
Basis, effective early screening can find precancerous lesion in time, take corresponding remedy measures, improve survival.
According to the suggestion that the World Health Organization proposes, it is suitble to the disease for carrying out screening that should meet 5 conditions：(1) this disease
It is high in this regional incidence；(2) there are suitable screening means；(3) it can effectively be treated after screening；(4) pass through screening
The death rate can be reduced with treatment；(5) screening means susceptibility and specificity are high, and cost is reasonable.Screening for colorectal accords with completely
Requirements above is closed, is classified as the cancer for being appropriate for screening by the World Health Organization at present.The result of study in American-European continent shows, greatly
Intestinal cancer screening can effectively reduce the morbidity and mortality of colorectal cancer.Carry out general population screening for colorectal as relatively early in the U.S.
Country, the variation of incidence of colorectal and the death rate especially merits attention.US National cancer research institute system in 2010
Counting display, the ratio that U.S. 50 years old or more crowd received sigmoidoscope or total colonoscopy is 54%-75%, 50
Year old or more the inscribed sigmoidoscope or the ratio of total colonoscopy of having received of crowd 5 years be 45%-63%.2003-2007 terms
Between, entire United States newly diagnose 722 542 colorectal cancers, and incidence is reduced to 45.5/10 in 2007 from 52.3/10 ten thousand in 2003
Ten thousand (annual change rate is 3.4%).The history that China carries out screening for colorectal work is more early, Zhejiang Province Haining and Jiashan area
A large amount of screening for colorectal work is carried out, major contribution is made that for the exploration of China's screening for colorectal strategy.China is for the first time
Screening for colorectal was carried out in 1977 in Haining county of Zhejiang Province, and screening object is occupying for 30 years old or more for the natural small towns in 21, Haining county
The people detect 1985 pure polyps, 6 adenomatous polyps, 20 polyps with canceration, 34 class cancers, 14 carcinoma of the rectum and 7 altogether
Example colon cancer.The screening for colorectal for the first time in Jiashan area was carried out in 1989, target group be randomly select 10 of this county from
30 years old or more the resident in right villages and small towns, detects 34 colorectal cancers altogether.City and Jiashan are regional under Hangzhou during 2007-2009 years
Screening for colorectal work covering 4.3 ten thousand people of population, finds 52 Cases with Colorectal Cancer, wherein Early cancer 34 (accounting for 65.38%) altogether,
Find various adenomas 569.Jiashan area colorectal cancer Follow-up Data is shown, after screening is carried out 8 years, the colon of target group
Cancer and carcinoma of the rectum cumulative mortality reduce 14.7% and 31.7% respectively.Early cancer clinical symptoms unobvious, need to pass through
Screening could be found.Therefore, there is an urgent need to improve screening method existing or that exploitation is new, and more rationally effective screening is formulated
Pattern reduces the incidence and case fatality rate of colorectal cancer to improve the efficiency and Population's compliance of screening for colorectal.
Screening for colorectal method is broadly divided into excrement detection at present and colon structure detects two major classes.Excrement detects
Fecal occult blood detect (fecal occult blood test, FOBT) and faeces DNA detection (stool DNA testing,
sDNA)；Colon structure detection includes sigmoidoscopy (flexible sigmoidoscopy, FSIG), colonoscopy
Look into (colonoscopy, CS), double contrast barium enema (double contrast barium enema, DCBE) and CT knots
Intestines are imaged (CT colonoscopy, CTC).Fecal occult blood detection FOBT is a kind of simple, quick screening for colorectal method, is used
In detecting the micro hemoglobin in excrement.FOBT detections are easy, cheap, noninvasive, can be used for extensive screening research, but its shadow
The factor of sound is more, need to can just detect positive findings in the case of ulceration bleeding after tumour is formed, and polyp or cancerous tissue
Bleeding is in often intermittence, it is impossible to include that therefore, specificity is not high, is susceptible to and fails to pinpoint a disease in diagnosis by arbitrary stool sample.Second shape
Colonoscopy (FSIG) is the method for directly checking lower distal colon with endoscopic technic, and operation is simpler, INTESTINAL CLEANSING requirement
It is relatively low, without calm in art, biopsy and treatment can be carried out to lesion, be a kind of relatively inexpensive, easy and safe screening
Method；However the check point of FSIG is only limitted to distal end large intestine (using splenic flexure as boundary), if after FSIG has found distal end large bowel neoplasm
Just carry out colonoscopy, it will 72.0% proximal end large bowel neoplasm is caused to fail to pinpoint a disease in diagnosis.Colonoscopy (CS) is current colorectal cancer
The goldstandard of Clinics and Practices can completely observe entire colon, and can carry out tissue biopsy and excision discovery
Polyp.Although CS has many advantages, such as that good effect, accuracy are high, also there is certain limitation：(1) CS is a kind of invasive
The risk higher of complication occurs for inspection method, may cause perforation of colon (0.3%) or even death (1/5000)；(2) it consumes
Duration needs sufficient INTESTINAL CLEANSING and calm processing before inspection；(3) expensive, if necessary to organize biopsy and cut off
It was found that polyp then spend more；(4) Sigmoidoscope is 5% to the rate of missed diagnosis of colorectal cancer.CT Colonographies (CTC) belong to non-intruding
Property inspection method, can with the thickness of 1-2mm carry out tomoscan, and then on two and three dimensions image check enteron aisle lesion.CTC
Have the advantages that quick, accurate and complication is low etc., to the screening efficiency of larger polyp and colorectal cancer close to CS.But as one kind
Conventional screening method, there are clearly disadvantageous by CTC：In order to obtain satisfactory image, still need to discharge in CTC checking processes
2-4 rads X-rays increase the risk for suffering from cancer to patient abdomen；Air or carbon dioxide would generally be injected into enteric cavity to expand
Enteron aisle is opened, patient has certain discomfort when checking；In addition, the inspection fee of great number, no matter it all can band to personal or society
Carry out heavier financial burden.
Excrement cast-off cells and its genetic test (sDNA) are the forefront technologies of current screening for colorectal, are a kind of non-intrudings
Property screening method, compared with normal colorectal mucosa, colorectal cancer cells update accretion rate obviously speed, cast-off cells increase therewith
Add, by fecal cast-off cell DNA quantitative analyses, can effectively screening go out part colorectal cancer.The method generally includes the inspection of two classes
It surveys：One kind is the detection of fecal cast-off cell：The intestinal epithelial cell to fall off in excrement is exactly carried out to conventional pathology
It learns and checks to find out tumour cell therein, to reach the diagnosis of clear intestinal canal tumour.Second class is fecal cast-off cell
The detection of DNA：Because some researches show that mutator and tumor tissues itself in the tumour cell to fall off in patient's excrement
Mutator has very high consistency.If the genoid, enteron aisle can be detected from the tumour cell to fall off in excrement
The diagnosis of tumour can also define, and the method also has very high specificity, positive findings that can make a definite diagnosis the presence of lesion substantially；
Have no need to change the eating habit of screening crowd；The use etc. of drug need not be limited.
Pancreas duodenum homeotic gene 1 (Pancreatic duodenal homeobox1, PDX1) encodes pancreas ten
The homologous different frame albumen 1 of two duodenum 12, belongs to a member of Pancreatic families transcription factor.Studies have reported that PDX1 gene promoters
Son, which methylates, can lower the expression of PDX1 genes in gastric cancer.But currently, colorectal cancer is ground with PDX1 gene methylation correlations
Study carefully less, the correlative study report in detection or screening colorectal cancer is carried out there are no the detection kit about PDX1 gene methylations
Road.
Invention content
To solve the above-mentioned problems, it is de- to provide a kind of noninvasive, quick, high sensitive detection colorectal cancer excrement of energy by the present invention
Fall the method and its kit of mutator in cell.Specifically, the purpose of the present invention is to provide one kind from colorectal cancer excrement
Kit and its application of the detection with the relevant PDX1 gene methylations of colorectal cancer in cast-off cells, by detecting PDX1 target genes
Nucleic acid sequence that segment methylates is, it can be achieved that early screening and auxiliary diagnosis to colorectal cancer, its early screening to colorectal cancer
It is more sensitive than traditional enteroscopy and special.
Technical solution is used by the present invention solves above-mentioned technical problem：
The present invention provides a kind of for detecting and the primer of the relevant PDX1 gene methylations specific amplification of colorectal cancer
And probe, it is SEQ ID NO that the forward direction of PDX1 gene methylation specific amplifications, which draws sequence,:1, reverse primer sequences are SEQ ID
NO:2, fluorescence probe sequence is SEQ ID NO:3, wherein fluorescence probe 5 ' holds flag F AM (fluorescent reporter group), 3 ' end labels
MGB (fluorescent quenching group)；
Above-mentioned primer and probe is preparing the application in detecting colorectal cancer kit reagent.
The present invention also provides a kind of for detecting and the reagent of the relevant PDX1 gene methylations specific amplification of colorectal cancer
Box, the kit include fluorescent quantitation reaction solution, and the fluorescent quantitation reaction solution is PDX1 gene methylation specific amplifications
Primer and probe, it is SEQ ID NO that the forward direction of the PDX1 gene methylations specific amplification, which draws sequence,:1, reverse primer sequence
It is classified as SEQ ID NO:2, fluorescence probe sequence is SEQ ID NO:3, wherein fluorescence probe 5 ' holds flag F AM (fluorescence report bases
Group), 3 ' end label MGB (fluorescent quenching group).
Mentioned reagent box further includes PCR Master Mix, interior target primer and probe.
Preferably, the PCR Master Mix include Taq polymerase, dNTP, PCR Buffer.
Preferably, the internal standard gene is mankind's house-keeping gene GAPDH；The interior target primer and probe, are for supervising
The extraction of each sample and the amplification situation of PCR reaction tubes are controlled, to avoid the appearance of false negative or part histamine result.
Kit provided by the invention further includes lysate, Proteinase K, rinsing liquid, bisulfite solution；It is mainly used for
Urine sediments DNA extractions and the conversion that methylates.
The present invention also provides mentioned reagent boxes to detect the application of colorectal cancer in fecal cast-off cell.
Steps are as follows for the kit test method：
(1) certain amount human bladder cancer sample and normal sample are collected；
(2) genomic DNA is extracted from the fecal cast-off cell sample collected in step (1)；
(3) genomic DNA that step (2) is extracted is subjected to the conversion that methylates, is urine by the Cytosines of methylate DNA
Pyrimidine；
(4) by step (3) methylate conversion after DNA carry out PCR amplification.
Preferably, PCR reaction solution described in step (4) is by 20 μ L PCR Master Mix and 3 μ L fluorescent quantitation reaction solutions
Composition.
Preferably, PCR step described in step (4) is specially：
1) above-mentioned PCR reaction solution is mixed, is sub-packed in eight unions of PCR by 23 μ L/ person-portion volumes of every pipe；
2) DNA sample after the conversion to be checked that methylates is added to by the amount of 2 μ L of every hole in eight unions of PCR dispensed,
Of short duration centrifugation；
3) PCR amplification：Eight unions of PCR are placed on to the corresponding position of instrument sample slot, the selection of instrument sense channel：
Reporter Dye1：FAM, Quencher Dye1：none；Reporter Dye2(GAPDH)：Cy5, Quencher Dye2：
none；Passive Reference：none.
Preferably, PCR amplification fluorescence program described in step (4) is：
4) judgement of result：
In the detection process of sample, baseline is determined as：Take the fluorescence signal of 3~15 cycles；Threshold value is set：Make threshold
It is worth the peak that line is more than normal negative controls amplification curve (random noise line), and Ct values are Undet.According to being reported
The positive of CT values (fluorescence signal in each reaction tube reaches the recurring number undergone when the threshold value of setting) interpretation sample of announcement
Or negative findings.
A kind of detection fecal cast-off cell methylation state of DNA, which is used to analyze the kit principle of colorectal cancer, is：Pass through inspection
The Exfoliated tumor cells ingredient in excrement is surveyed, to judge whether subject is possible to the molecule with adenoma before colorectal cancer or cancer
Diagnostic techniques, the tumour cell ingredient most often detected are the human gene for being mutated or being methylated.Large bowel neoplasm is general
It is happened in Colon and rectum epithelial tissue, first constantly there is tumour cell to drop to intestines during growth to intestines Intracavity
Intracavitary and as excrement is discharged, therefore detect the tumour cell ingredient in excrement a kind of noninvasive large bowel neoplasm sieve can be provided
Look into diagnostic method.The mutation of oncogenesis-related genes is that tumour is distinctive, and the methylation level of gene promoter region is then
It is significantly improved than in normal cell in tumour cell, so the change of this gene can be used as tumor markers for sieving
Look into or diagnose large bowel neoplasm.The present invention carries out early screening using methylation status of PTEN promoter method to colorectal cancer, and detection is
Nucleic acid sequence that PDX1 target fragments methylate is, it can be achieved that early screening and auxiliary diagnosis to colorectal cancer.
For the present invention in the fluorogenic quantitative detection experiment that methylate, the quality of primer and probe design is amplification success or failure
Key factor, design difficulty is also bigger, the analysis verification (software) of designed primer Need Hierarchy Theory, and is needed
Experimental verification and screening, so, the above-mentioned primer and probe of the present invention is that the present invention is directed to and the relevant PDX1 genes piece of colorectal cancer
The methylated nucleic acid sequence of section designs and demonstrates many places primer and probe composite sequence simultaneously, through repeatedly Optimal Experimental repeatedly,
It is preferred that the primer probe sequence that one group of detection PDX1 genetic fragment methylates.
Design, the design of Methylation-specific primer different from common PCR primer design of Methylation-specific primer
Principle is：Template DNA by sulphite after handling, the sites CpG 5 ' in the islands gene promoter region CpG that methylate
The sites CpG 5 ' hold Cytosines for uracil in the islands CpG that end cytimidine is remained unchanged, and do not methylated, i.e. C → U,
For modification front and back sequence difference design methylated primers and probe.
The difficulty that the present invention is encountered in design Methylation-specific primer is the methylated DNA fragments CG content phases to be expanded
To higher, methylation-specific amplification difficulty is big, and the present invention needs theoretically to speculate its amplification efficiency in design primer, right
In amplification efficiency<30% primer will optimize, by adjusting the leader primers amplification that methylates repeatedly before and after amplification region
Starting point makes amplification efficiency increase, and reduces amplification difficulty and improves PCR yield, secondly, it is also contemplated that at least containing 1 in primer sequence
Preferably contain multiple sites CpG, can guarantee the specificity of primer in this way, while DNA first can be improved in a above sites CpG
The recall rate of base choline base, in addition it is also necessary to be consistent as far as possible with the design of primers principle of regular-PCR.In PCR reactions, draw
The length and G/C content of object directly affect the Tm values of primer, and the upstream and downstream primer designed must must ensure annealing temperature phase
Closely, if 2 pairs of primers are not in the uncertain annealing of identical CpG, PCR results cannot accurately reflect the case where sample DNA methylates.
It is close that the present invention discloses primer Tm, respectively 58.4 DEG C, 58.6 DEG C, ensure that amplification efficiency and recall rate in PCR reactions, energy
It is enough efficiently to detect PDX1 methyl states in fecal cast-off cell, so that the sensitivity of detection colorectal cancer is greatly improved.
Whether fluorescence probe and target gene specifically bind the specificity for also directly affecting DNA methylation detection.Probe
Length etc., affect the Tm values of probe, the probe of design is necessary to ensure that the Tm values of probe are higher by 5-10 than the Tm values of primer
DEG C or so, it can guarantee that probe is combined prior to primer with target fragment in annealing in this way, and 5 ' ends of probe cannot be G, because
Even if single G bases are connected with FAM fluorescent reporter groups, the fluorescence signal that FAM groups are sent out can also be quenched, to lead
Cause the appearance of false negative.In addition it is also necessary to avoid forming secondary structure between probe and primer.Due to the sample detected in an experiment
This methylation is different, and primer and probe of the invention is and to be measured by testing optimal conditions repeatedly and preferably going out
PDX1 genetic fragments can be specifically bound and binding ability is strong one group, it is ensured that the accuracy rate of colorectal cancer detection.
Advantageous effect：
1, present invention firstly discloses can be used for detecting with the kits of the relevant PDX1 gene methylations of colorectal cancer and its
Using the detection kit includes methylation-specific amplification forward primer, reverse primer and its fluorescence of PDX1 genes
Probe, PDX1 gene methylation levels lead to PATIENTS WITH LARGE BOWEL PDX1 gene low expressions, have more so as to cause colorectal cancer cells
Strong invasive ability, therefore, PDX1 gene methylation situations can serve as the molecular biosciences of colorectal cancer grade malignancy and prognostic evaluation
Learn label.Diagnostic kit based on detecting colorectal cancer PDX1 gene methylation levels can be conveniently and efficiently in molecular water
The flat upper detection realized to colorectal cancer, to colorectal cancer grade malignancy and Index for diagnosis, detection efficiency is high, with strong points, has standard
The advantages of really reliable, flexible quick and economy, is conducive to the early detection and treatment in time of colorectal cancer.
2, compared with traditional colonoscopy screening colorectal cancer, and conventional occult blood test compares, this kit is adopted
It is had the advantage that with methylation status of PTEN promoter method by detecting fecal cast-off cell DNA screening colorectal cancers：
1) completely noninvasive, it is only necessary to which that 4.5 grams of excrement, Silent cerebral infarction acceptance are high；
2) accuracy is high, can be detected in preclinical study absolute majority colorectal cancer and most cancer before gland
Tumor；It can detect 90% Early cancer, adenoma (>=1 centimetre) before 64% cancer, specificity is 95%；
3) tumour of left and right sides colon can be found simultaneously, reduce check frequency and failed to pinpoint a disease in diagnosis；
4) not only it can be found that colorectal cancer, it has also been discovered that adenoma before cancer, for before extracing cancer adenoma prevent large intestine
Cancer provides possibility；
5) easy to operate, after obtaining detection kit, user can complete the collection work of sample, later express delivery at home
It sends and is detected by professional.
3, this method uses fluorescent quantitation (Methylight), this technology to grow up in MSP technologies,
It is quantified using fluorescent dye in MSP amplification procedures, the application of sonde method makes the technology have higher accuracy, Gao Min
Perception and higher flux, and without in operations such as PCR rear electrophoresis, hybridization, reducing pollution and operating error.
Description of the drawings
Fig. 1 is that the FQ-PCR of 44,11,15,8,40,56 and 20 sample positive samples schemes (channels FAM)；
Fig. 2 is that the FQ-PCR of 25,18,50,7,12,23,1 and No. 28 sample negative samples schemes (channels Cy5).
Specific implementation mode
Feature of present invention and other correlated characteristics are described in further detail by the following examples, in order to the same industry
The understanding of technical staff：
Kit of the present invention is used to detect the primer and probe of colorectal cancer fecal cast-off cell PDX1 gene methylation levels
It is that Primer Premier are used according to mankind's whole genome sequence disclosed in US National Biotechnology Information center (NCBI)
5 designs, primer are synthesized by TAKARA (precious bioengineering Co., Ltd), and probe is synthesized by Life Technologies.
Embodiment 1 is used to detect the primer and probe design and its reagent of colorectal cancer fecal cast-off cell methylation level
Box.
The present invention is directed to the nucleic acid sequence that the relevant PDX1 target fragments of colorectal cancer methylate while designing and demonstrating
Multipair primer and probe composite sequence, through repeatedly Optimal Experimental repeatedly, preferred following (table 1) one group of detection PDX1 genetic fragment
The primer probe sequence to methylate.
The primer and probe sequence that 1 PDX1 genetic fragments of table methylate
The present invention provides a kind of kit of detection colorectal cancer fecal cast-off cell methylation level, including fluorescent quantitation is anti-
It is to further comprise detection internal reference-people except the primed probe of above-mentioned PDX1 target genes piece to answer liquid, the fluorescent quantitation reaction solution
The primer and probe sequence of class house-keeping gene (GAPDH), the amplification feelings of extraction and PCR reaction tubes for monitoring each sample
Condition；In addition, kit of the present invention further include have PCR Mix (including Taq polymerase, dNTP, PCR Buffer etc.), lysate,
Proteinase K, rinsing liquid, bisulfite solution.
When carrying out carcinoma of urinary bladder detection using mentioned reagent box, it is as follows：
1, sample is collected
The sample collection since Wuhu reaches, face December by 40 in January, 2017~2017 year of strong Laboratory of medical test collection
Patient stool sample (20 men, 20 female) and 20 normal person stool samples (10 men, 10 female) of the bed pathological diagnosis for colorectal cancer, often
Each 10g of example sample.
2, genomic DNA is extracted from the fecal cast-off cell sample collected in step (1), the specific steps are：
A, it cracks
1) 200mg solid-states fecal sample or about 200 μ L liquid fecal samples are weighed, are placed in 1.5mL centrifuge tubes, are added
1mL lysates 1, then lasting vortex oscillation mixing 1min, makes tube bottom excrement mix well, is then placed in 1.5mL centrifuge tubes
70 DEG C of water-bath 5min.
2) 1.5mL centrifuge tubes are taken out out of water-bath, 12,000rpm centrifugation 5min slowly draw 300 μ L supernatants extremely
In new 1.5mL centrifuge tubes.
3) 200 μ L lysates and 20 μ L Proteinase Ks, vortex mixing are sequentially added into new 1.5mL centrifuge tubes.
4) new 1.5mL centrifuge tubes are placed in 70 DEG C of water-bath 10min.
B, DNA is combined
1) 200 μ L isopropanols, vortex mixing are added.
2) previous step acquired solution is all added in an adsorption column and (adsorption column is inserted in collecting pipe), 12,
000rpm centrifuges 30s, abandons waste liquid.
C, DNA is washed
1) 600 μ L rinsing liquids are added into adsorption column, 12,000rpm centrifugation 30s abandon waste liquid.It can be primary with repetitive operation.
2) 12,000rpm centrifuges 2min, abandons waste liquid；Adsorption column is stored at room temperature 2min, with residual in the adsorption column that thoroughly volatilizees
The rinsing liquid stayed.
3) adsorption column is transferred in 1.5mL centrifuge tubes, 50 μ L eluents is vacantly added dropwise to adsorbed film centre position, room temperature is quiet
2min is set, solution is collected into centrifuge tube by 12,000rpm centrifugation 2min.
3, the genomic DNA that step (2) is extracted is subjected to the conversion that methylates, is urine by the Cytosines of methylate DNA
Pyrimidine, the specific steps are：
1) DNA that step (2) is extracted is fully transferred in 0.2mL centrifuge tubes, adds 150 μ L bisulfite solutions；
By flick centrifuge tube or pipettor softly blow and beat operation mixing after of short duration centrifugation.
2) centrifuge tube is positioned in temperature cycles thermode and is arranged according to the following conditions：
①98℃10min
②64℃1.5h
③4℃(≦20h)
3) 500 μ L combination liquid are added into adsorption column, then the mixed liquor of previous step are transferred in adsorption column, lid upper tube cap
And reverse mixing, 12,000rpm centrifugation 30sec abandon waste liquid.
4) 600 μ L rinsing liquids are added into adsorption column, 12,000rpm centrifugation 30sec abandon waste liquid.
5) 12,000rpm centrifuges 2min, abandons waste liquid；Adsorption column is stored at room temperature 2min, with residual in the adsorption column that thoroughly volatilizees
The rinsing liquid stayed.
6) adsorption column is transferred in 1.5mL centrifuge tubes, 30 μ L eluents is vacantly added dropwise to adsorbed film centre position, room temperature is quiet
2min is set, solution is collected into centrifuge tube after 12,000rpm centrifugation 2min.
4, by step (3) methylate conversion after DNA carry out PCR amplification.
1) PCR reaction solution is configured according to following table：
2) it after PCR reaction solution mixes well, is sub-packed in eight unions of PCR by 23 μ L volumes of every pipe, and turns after marking
Move to sample process area.
3) DNA sample after the conversion that methylates that step (3) obtains is added to by the amount of 2 μ L of every hole and has been dispensed
In eight unions of PCR, compressing pipe lid, and of short duration centrifugation, tube wall liquid is centrifuged to tube bottom.
4) eight unions of PCR are placed on to the corresponding position of instrument sample slot, and records and puts order.
Instrument sense channel selects：Reporter Dye1：FAM, Quencher Dye1：none；Reporter Dye2
(GAPDH)：Cy5, Quencher Dye2：none；Passive Reference：none.
The setting of corresponding detection hole：Before amplified reaction starts, sample to be checked and quality-control product are set as " Unknown ".
It is reacted by the following conditions setting PCR
4) judgement of result：
In the detection process of sample, baseline is determined as：Take the fluorescence signal of 3~15 cycles；Threshold value is set：Make threshold
It is worth the peak that line is more than normal negative controls amplification curve (random noise line), and Ct values are Undet.According to being reported
The positive of CT values (fluorescence signal in each reaction tube reaches the recurring number undergone when the threshold value of setting) interpretation sample of announcement
Or negative findings.
Conclusion：(1) 40 channel positive sample FAM, which corresponds to detection hole, amplification curve, and testing result is the positive, with disease
Reason diagnostic result is consistent, and 44,11,15,8,40,56 and 20 sample fluorescence quantitative PCR detection amplifications are as shown in Figure 1；20
Negative sample only has the channels reference gene Cy5 compareed to have amplification curve, and testing result is feminine gender, with pathological diagnosis result one
It causes, 25,18,50,7,12,23,1 and No. 28 sample fluorescence quantitative PCR detection amplifications are as shown in Fig. 2, show this kit
Testing result have high specificity and reliability.The CT values of the detailed testing result of each sample of the present invention are shown in Table 1.
Each sample PDX1 genes are high in colorectal cancer sample it can be seen from the fluorescence quantitative PCR detection result of sample
Degree amplification, and expanded in normal sample seldom, it is de- thus to prove that kit of the present invention can fast and accurately detect colorectal cancer excrement
Fall mutator and normal gene in cell.It is therefore seen that kit of the present invention is not only fast and efficiently, and its result
Interpretation is very clear, intuitive, as a result reliably, specifically.
The CT values of 2 each sample fluorescence quantitative PCR detection result of table
(2) sensitivity and specificity are calculated according to following formula in the testing result of Comprehensive Criteria object：
Sensitivity=testing result is the colorectal cancer sample number of positive colorectal cancer sample number/total；
Specificity=testing result is the non-carcinoma of urinary bladder sample number of negative non-colorectal cancer sample number/total；
The results are shown in Table 2, in 40 bladder cancer patients, 20 normal person's urine specimens, is examined using primer of the present invention
The specificity for surveying PDX1 gene methylations is 100%, sensitivity 97.5%.
In order to verify specificity and the sensitivity of primer of the present invention, the present invention uses while other primers of design are opposed
According to.
Comparative example 1：
The PDX1 gene forward primer sequences of the present invention are SEQ ID NO：4, reverse primer sequences are SEQ ID NO：5,
Fluorescence probe sequence is SEQ ID NO:6, quantitative fluorescent PCR is carried out to the application sample, to verify this hair PDX1 gene primers
The special specificity of sensitivity.That difference lies in primers is different from embodiment 1, other same as Example 1.
By comparing discovery, the sensitivity of comparative example is 92.5%, and specificity is 95%, so the application primer is sensitive
Degree and specificity are higher than other primer sensitivity and specificity of Synchronization Design.
Finally illustrate, the foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, to the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, for those skilled in the art, still can be with
It modifies to the technical solution recorded in previous embodiment, or equivalent replacement is carried out to which part.It is all the present invention
Within spirit and principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not to the limit of the scope of the present invention
System, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art need not pay
Go out various modifications or changes that creative work can be made still within protection scope of the present invention.
SEQUENCE LISTING
<110>Anhui reaches strong medical science and technology Co., Ltd
<120>A kind of detection fecal cast-off cell methylation state of DNA is used to analyze the kit of colorectal cancer
<130> 2010
<160> 6
<170> PatentIn version 3.3
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<213>It is artificial synthesized
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<213>It is artificial synthesized
<400> 2
aaacaaaacc gaactatcct accg 24
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<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 3
ttggttcggt atatgcgttt 20
<210> 4
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 4
cgcgttggtt cggtatatgc 20
<210> 5
<211> 29
<212> DNA
<213>It is artificial synthesized
<400> 5
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<210> 6
<211> 21
<212> DNA
<213>It is artificial synthesized
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Claims (10)
1. a kind of exist for detecting with the primer and probe of the relevant PDX1 gene methylations specific amplification of colorectal cancer, feature
In it is SEQ ID NO that the forward direction of the PDX1 gene methylations specific amplification, which draws sequence,:1, reverse primer sequences are SEQ ID
NO:2, fluorescence probe sequence is SEQ ID NO:3, wherein fluorescence probe 5 ' holds flag F AM, 3 ' end label MGB.
2. primer and probe described in claim 1 is preparing the application in detecting colorectal cancer kit reagent.
3. a kind of for detecting and the kit of the relevant PDX1 gene methylations specific amplification of colorectal cancer, which is characterized in that
Including fluorescent quantitation reaction solution, the fluorescent quantitation reaction solution expands for PDX1 gene methylations specificity described in claim 1
The primer and probe of increasing.
4. kit according to claim 3, which is characterized in that the kit further includes PCR Master Mix, interior
Mark the primer and probe of gene, lysate, Proteinase K, rinsing liquid, bisulfite solution.
5. kit according to claim 4, which is characterized in that in the PCR Master Mix containing Taq polymerase,
DNTP and PCR Buffer；The internal standard gene is mankind's house-keeping gene GAPDH.
6. application of any one of the claim 3-5 kits in detecting colorectal cancer reagent.
7. according to the application method of any kit of claim 3~5, which is characterized in that the kit detects large intestine
The step of cancer, is as follows：
(1) certain amount human large intestine cancer sample and normal sample are collected；
(2) genomic DNA is extracted from the fecal cast-off cell sample collected in step (1)；
(3) genomic DNA that step (2) is extracted is subjected to the conversion that methylates, is that urine is phonetic by the Cytosines of methylate DNA
Pyridine；
(4) by step (3) methylate conversion after DNA carry out PCR amplification.
8. the method according to the description of claim 7 is characterized in that sample described in step (1) is fecal cast-off cell.
9. the method according to the description of claim 7 is characterized in that PCR step described in step (4) is specially：
1) PCR reaction solution is mixed, is sub-packed in eight unions of PCR by 23 μ L/ person-portion volumes of every pipe；
2) DNA sample after the conversion to be checked that methylates is added to by the amount of 2 μ L of every hole in eight unions of PCR dispensed, is centrifuged；
3) PCR amplification is carried out, judges result.
10. according to the method described in claim 9, it is characterized in that, the PCR reaction solution of the PCR amplification is by 20 μ L PCR
Master Mix and 3 μ L fluorescent quantitation reaction solutions composition；
The PCR amplification fluorescence program is：
The first step is 25 DEG C, time 10min, 98 DEG C, time 3min of temperature, is recycled 1 time, fluorescence signal collection is no；Second step
For 95 DEG C, time 30s of temperature, phosphor collection is no, 66 DEG C, time 20s of temperature, and phosphor collection is no, is recycled 20 times；Third walks
For 95 DEG C, time 30s of temperature, phosphor collection is no, 62 DEG C, time 35s of temperature, and phosphor collection is yes, is recycled 25 times.
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| CN201810555890.0A CN108753963A (en) | 2018-06-01 | 2018-06-01 | A kind of detection fecal cast-off cell methylation state of DNA is used to analyze the kit of colorectal cancer |
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| CN109486955A (en) * | 2018-12-29 | 2019-03-19 | 厦门艾德生物医药科技股份有限公司 | It is a kind of for excrement Detection progress phase adenoma, the kit of colorectal cancer related gene methylation sites |
| CN112266964A (en) * | 2020-11-23 | 2021-01-26 | 广州齐凯生物科技有限公司 | Multi-site colorectal cancer methylation detection primer, probe and kit |
| CN113699237A (en) * | 2020-07-29 | 2021-11-26 | 上海吉凯医学检验所有限公司 | Kit for detecting DNA methylation markers of early colorectal cancer and adenoma |
| CN114085905A (en) * | 2020-08-24 | 2022-02-25 | 广州达健生物科技有限公司 | Composition for detecting colorectal cancer, kit and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486955A (en) * | 2018-12-29 | 2019-03-19 | 厦门艾德生物医药科技股份有限公司 | It is a kind of for excrement Detection progress phase adenoma, the kit of colorectal cancer related gene methylation sites |
| CN109486955B (en) * | 2018-12-29 | 2022-05-24 | 厦门厦维健康科技有限公司 | Kit for detecting methylation sites of genes related to advanced adenomas and colorectal cancers in excrement |
| CN113699237A (en) * | 2020-07-29 | 2021-11-26 | 上海吉凯医学检验所有限公司 | Kit for detecting DNA methylation markers of early colorectal cancer and adenoma |
| CN114085905A (en) * | 2020-08-24 | 2022-02-25 | 广州达健生物科技有限公司 | Composition for detecting colorectal cancer, kit and application thereof |
| CN114085905B (en) * | 2020-08-24 | 2024-02-23 | 广州达健生物科技有限公司 | Composition for detecting colorectal cancer, kit and application thereof |
| CN112266964A (en) * | 2020-11-23 | 2021-01-26 | 广州齐凯生物科技有限公司 | Multi-site colorectal cancer methylation detection primer, probe and kit |
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