CN108660217A - A kind of detection peripheral blood cells methylation state of DNA is used to analyze the kit of liver cancer - Google Patents
A kind of detection peripheral blood cells methylation state of DNA is used to analyze the kit of liver cancer Download PDFInfo
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Abstract
The invention belongs to molecular biology fields, and in particular to a kind of detection peripheral blood cells methylation state of DNA is used to analyze the kit of liver cancer.Present invention firstly discovers that PTCH10 can be used as the marker that methylates for detecting liver cancer.The primer sequence that detection PTCH10 methylates is as follows：Forward primer sequence is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2；The fluorescence probe sequence is SEQ ID NO.3, and probe 5 ' holds flag F AM, 3 ' end label MGB.The present invention is with the marker that methylates that PTCH10 is for detecting liver cancer, and the primer and probe, sensitivity is strong and specificity is high, substantially increases the sensitivity of methylation in detection peripheral blood sample, high sensitivity of the present invention is up to 85%, and specifically up to 100%；Far above gene SLIT2 and DAPK.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of detection peripheral blood cells methylation state of DNA is used for
Analyze the kit of liver cancer.
Background technology
Hepatocellular carcinoma (HCC) is a kind of common malignant tumour, and early diagnosis, acquisition radical excision are that patient is long-term
Unique hope of existence.Due to lacking the tumor markers of specificity, late period is belonged to when most of patients is made a definite diagnosis, has lost radical-ability
The chance of excision.Early stage that tumor suppressor gene DNA promoter methylations are proved to be in tumour, take place frequently event, and different tumours or
There are greatest differences for the different times of same tumour.HCC patient's methylation of tumor suppressor spectrum is confirmed in various degree, and shows
Show good application prospect.But most of researchs are limited only to the research of postoperative tissue specimen, are influenced by specimen sampling to suffering from
The early diagnosis meaning of person is limited.In recent years the study found that there is free Tumour DNA in blood of cancer patients slurry, and
And there is gene alteration identical with DNA of tumor cell, therefore it is minimally invasive to detect the early stage that tumor patient peripheral circulation DNA is tumour
Diagnosis provides new means.
King equality people has found that SLIT2 and DAPK genes its methyl rate in HCC cancerous tissues is respectively 70.5% He
79.4%, methyl rate is respectively 44.1% and 50.0% in corresponding periphery blood plasma, and blood plasma SLIT2 and DAPK methylate inspection
The sensitivity of survey is respectively 62.5% and 63.0%, and specificity is 100%, and negative predictive value is respectively 52.6% He
41.2%, positive predictive value is 100%, still there is higher SLIT2 and DAPK gene methylations in result above prompt blood plasma
Recall rate, tissue and two gene methylations in blood plasma have good consistency, and (Wang Ping, Geng little Ping, Zhu Lixin wait liver cell livers
The general outer basis of the detection of periphery plasma dna gene methylation and meaning [J] China and clinical journals, 2012,19 (7) in cancer:727-
732.), but the sensitivity of above-mentioned two genes blood plasma DNA methylation assay is not high.
Therefore, a kind of marker for the high sensitivity that methylates about liver cancer peripheral blood, detection and treatment to liver cancer are screened
Have great importance.
Invention content
Present invention is primarily intended to provide a kind of examination for detecting peripheral blood cells methylation state of DNA and being used to analyze liver cancer
Agent box.Present invention firstly discovers that PTCH10 methylate it is related to liver cancer generation, and pass through detect peripheral blood in PTCH10 methyl
Change degree can be used for the detect and diagnose of liver cancer.
To achieve the goals above, the present invention uses following technical scheme：
One of the object of the invention, provides a kind of marker that methylates for liver cancer detection, and the marker that methylates is
PTCH10。
The two of the object of the invention provide the application of marker in preparing liver cancer detection reagent described above that methylates.
The three of the object of the invention provide one group of primer and probe for detecting the above-described marker that methylates, institute
It is as follows to state primer sequence：Forward primer sequence is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2；Institute
It is SEQ ID NO.3 to state fluorescence probe sequence, and probe 5 ' holds flag F AM, 3 ' end label MGB.
The four of the object of the invention provide above-described primer and probe and are preparing answering for the kit for liver cancer detection
With.
The five of the object of the invention provide a kind of kit, including above-described primer and probe, further include detection
The forward primer and probe of GAPDH, PCR Master Mix, lysate, Proteinase K, rinsing liquid, bisulfite solution and
ddH2O。
The six of the object of the invention provide the application method of kit described above, and steps are as follows：
(1) in sample genomic DNA extraction：Clinical samples are taken, lysate, Proteinase K, rinsing liquid and ddH2O are utilized
Extraction, obtains Genomic DNA solution；
(2) DNA methylation is modified：The Genomic DNA solution that step (1) extraction obtains is taken to be carried out with bisulfite solution
Modification obtains the DNA after modifying that methylates；
(3) DNA after modifying step (2) carries out PCR amplification.
Preferably, step (1) described sample is peripheral blood cells.Preferably, PCR reaction solution is：PCR Master
Mix20 μ L, primer and each 1 μ L of probe, 3 μ L of fluorescent quantitation reaction solution, 26 μ L of total amount/person-portion；PCR reaction conditions are：
The seven of the object of the invention provide application of the above-described kit in preparing liver cancer detection reagent.
The eight of the object of the invention provide kit described above and are preparing the application in detecting PTCH10 methylating reagents.
There is free Tumour DNA in peripheral blood, by detecting the methylation of gene in peripheral blood come diagnosing liver cancer,
The pain of liver cancer patient diagnosis can not only be mitigated, and can realize the early stage to liver cancer, quick diagnosis, early found conducive to patient
Early treatment.But in peripheral blood different genes methylation differ surely really reflection tissue true methylation status.This
Inventor has been surprisingly found that in probing into the relationship that gene methylation occurs with liver cancer, in Peripheral Blood of Patients with Hepatocellular Carcinoma, PTCH10 first
Base degree is higher, and methylates further across PTCH10 in verification experimental verification peripheral blood and methylate with PTCH10 in liver cancer tissue
Situation is consistent, and is the marker that methylates detected for liver cancer, detection sensitivity and known liver cancer mark with PTCH10
Object gene SLIT2 is compared with DAPK, and sensitivity greatly improves.
Compared with prior art, the present invention has the advantage that：
(1) present invention is using peripheral blood in patients as sample, by PTCH10 methylations in detection peripheral blood, it can be achieved that right
The diagnosis of liver cancer；Compared with being detection sample with tissue samples, the present invention is minimally invasive diagnosis, can greatly reduce patient pain's journey
Degree reduces patient and diagnoses expense, and quick diagnosis may be implemented.
(2) present invention is the marker that methylates detected for liver cancer with PTCH10, and the primer and probe, sensitive
Degree is strong and specificity is high, substantially increases the sensitivity of methylation in detection peripheral blood sample, and high sensitivity of the present invention reaches
85%, specifically up to 100%；Far above gene SLIT2 and DAPK.
Specific implementation mode
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific implementation mode, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or combination thereof.
In order to enable those skilled in the art can clearly understand technical scheme of the present invention, below with reference to tool
The embodiment of the body technical solution that the present invention will be described in detail.
Embodiment 1PTCH10 and its primer and probe
It can be used for detecting liver cancer present invention firstly discovers that PTCH10 methylates.
It is as follows for detecting the primer sequence that PTCH10 methylates：
Forward primer sequence is：5 '-TGTAAGTATCGTGTATGTGTAAACGTGTTC-3 ', such as SEQ ID NO：1 sequence
It is shown；
Reverse primer sequences are：5 '-CCGTACACACGAAAAACCCG-3 ', such as SEQ ID NO：Shown in 2 sequences；, fluorescence
Probe sequence is：5 '-TTCGTAGCGGTGTATATT-3 ', such as SEQ ID NO：Shown in 3 sequences；5 ' end labels of fluorescence probe
FAM (fluorescent reporter group), 3 ' end label MGB (fluorescent quenching group).
Other primers and probe used are as follows in screening process of the present invention：
First group of primer and probe：
Forward primer sequence as shown in SEQ ID NO.4 (5 '-CGCGTTGTTTTTGCGTTTTC-3 '),
Reverse primer sequences as shown in SEQ ID NO.5 (5 '-CAAAACTTAACTCTATATAAACCCCAACG-3 '),
Fluorescence probe sequence is as shown in SEQ ID NO.6 (5 '-AAGCGCGTTTTTCGGAATTCGGG-3 '), and fluorescence is visited
5 ' end flag F AM (fluorescent reporter group) of needle, 3 ' end label MGB (fluorescent quenching group).
Second group of primer and probe：
Forward primer sequence be as shown in SEQ ID NO.7 (5 '-GTGTATATTCGGGTTTTTCGTGTGTAC-3 '),
Reverse primer sequences are, shown in SEQ ID NO.8 (5 '-AATCCTAAAATCAACCCGCGA-3 '),
Fluorescence probe sequence as shown in SEQ ID NO.9 (5 '-AATCCTAAAATCAACCCGCGA-3 '), fluorescence probe
5 ' end flag F AM (fluorescent reporter group), 3 ' end label MGB (fluorescent quenching group).
A kind of 2 liver cancer detection kit of embodiment
The kit includes PTCH10 forward primers, and sequence is the reverse primer as shown in SEQ ID NO.1, and sequence is
SEQ ID NO.2 and fluorescence probe, sequence are SEQ ID NO.3；The probe 5 ' holds flag F AM, 3 ' end label MGB；This
Outer further includes the forward primer and probe for detecting GAPDH, PCR Master Mix, lysate, Proteinase K, rinsing liquid, sulfurous acid
Hydrogen salt solution and ddH2O；Primer and probe known in the art can be used in the forward primer and probe for detecting GAPDH.
The application method of kit described in embodiment 3
The application method of 2 kit of embodiment, it is specific as follows：
(1) extraction of peripheral blood cells genomic DNA：Take peripheral blood cells, using lysate, Proteinase K, rinsing liquid and
ddH2O is extracted, and obtains Genomic DNA solution；
The specific steps are：
200 μ L peripheral blood samples are drawn, are placed in 1.5mL centrifuge tubes, 1mL lysates 1 are added, then lasting vortex oscillation
Mixing 1min, mixes well, and 1.5mL centrifuge tubes are then placed in 70 DEG C of water-bath 5min.
1.5mL centrifuge tubes are taken out out of water-bath, 12,000rpm centrifugation 5min slowly draw 300 μ L supernatants to new
1.5mL centrifuge tubes in.
200 μ L lysates 2 and 20 μ L Proteinase Ks, vortex mixing are sequentially added into new 1.5mL centrifuge tubes.
New 1.5mL centrifuge tubes are placed in 70 DEG C of water-bath 10min.
Add 200 μ L isopropanols, vortex mixing.
Previous step acquired solution is all added in an adsorption column and (adsorption column is inserted in collecting pipe), 12,000rpm
30sec is centrifuged, waste liquid is abandoned.
600 μ L rinsing liquids are added into adsorption column, 12,000rpm centrifugation 30sec abandon waste liquid.
Aforesaid operations are repeated, 12,000rpm centrifugation 2min abandon waste liquid；Adsorption column is stored at room temperature 2min, thoroughly to volatilize
Remaining rinsing liquid in adsorption column.
Adsorption column is transferred in 1.5mL centrifuge tubes, 50 μ L eluents are vacantly added dropwise to adsorbed film centre position, are stored at room temperature
2min, 12,000rpm centrifugation 2min, solution is collected into centrifuge tube.
(2) DNA methylation is modified：The Genomic DNA solution that step (1) extraction obtains is taken to be carried out with bisulfite solution
Modification obtains the DNA after modifying that methylates；
The DNA eluents that step (1) is extracted are fully transferred in 0.2mL centrifuge tubes, 150 μ L bisulfites of addition are molten
Liquid；By flick centrifuge tube or pipettor softly blow and beat operation mixing after of short duration centrifugation.
Centrifuge tube is positioned in temperature cycles thermode and is arranged according to the following conditions：
①98℃10min
②64℃1.5h
③4℃(≦20h)
500 μ L combination liquid are added into adsorption column, then the mixed liquor of previous step is transferred in adsorption column, lid upper tube cap is simultaneously
Reverse mixing, 12,000rpm centrifugation 30sec, abandons waste liquid.
600 μ L rinsing liquids are added into adsorption column, 12,000rpm centrifugation 30sec abandon waste liquid.
It is primary to repeat aforesaid operations, 12,000rpm centrifugation 2min abandon waste liquid；Adsorption column is stored at room temperature 2min, with thorough
Remaining rinsing liquid in volatilization adsorption column.
Adsorption column is transferred in 1.5mL centrifuge tubes, 30 μ L eluents are vacantly added dropwise to adsorbed film centre position, are stored at room temperature
Solution is collected into centrifuge tube after 2min, 12,000rpm centrifugation 2min.
(3) DNA after modifying step (2) carries out PCR amplification.
PCR reaction solution is configured according to following table：
Table 1
After PCR reaction solution mixes well, it is sub-packed in eight unions of PCR by 26 μ L volumes of every pipe, and shifted after marking
To sample process area.
By BisDNA to be checked (DNA after the conversion that methylates) sample, the PCR eight dispensed is added to by the amount of 2 μ L of every hole
In union, compressing pipe lid, and of short duration centrifugation, tube wall liquid is centrifuged to tube bottom.
Eight unions of PCR are placed on 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
Table 2
Test example 1
Strong Laboratory of medical test in May, 2017~2018 year Clinicopathologic Diagnosis in May is reached collected from Wuhu for liver cancer to suffer from
The peripheral blood of person (20 people), colorectal cancer patients (5 people) and normal person (20 people).
It is detected by 3 method of embodiment, the CT values of the detailed testing result of each sample are shown in Table 3.
The CT values of 3 each sample fluorescence quantitative PCR detection result of table
(2) according to table 3, sensitivity and specificity are calculated according to following formula：
Sensitivity=testing result is liver cancer sample number/liver cancer patient total number of samples of the positive；
Specificity=testing result is liver cancer sample number/normal person's total sample number of feminine gender；
The results are shown in Table 4.
4 kit of the present invention of table detects specificity and the sensitivity of liver cancer
| 45 pattern detection results | |
| Sensitivity | 85% |
| Specificity | 100% |
PTCH10 is highly expanded in Peripheral Blood of Patients with Hepatocellular Carcinoma sample it can be seen from table 3,4 testing result of table, and
PTCH10 methylation positives are consistent with liver cancer positive patient in peripheral blood.Detection sensitivity of the present invention is up to 85%, and specificity is
100%.From the above results, the present invention is using PTCH10 as target gene, and the primer and probe, and sensitivity is by force and special
Property it is high, substantially increase the sensitivity of methylation in detection peripheral blood sample, high sensitivity of the present invention is special high up to 85%
Up to 100%；Far above gene SLIT2 and DAPK.
Test example 2
It is detected with the sample described in test example 1 of the present invention, using first group in embodiment 1 and second group of primer and spy
Needle carries out the detection of liver cancer, and testing result is as shown in table 5 below.
5 first groups to second group primer specificity of table and sensitivity technique result
| First group of primer | Second group of primer | |
| Sensitivity | 75% | 80% |
| Specificity | 90% | 100% |
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.
Although the above-mentioned specific implementation mode to the present invention is described, it is not intended to limit the protection scope of the present invention, affiliated neck
Field technique personnel should be understood that based on the technical solutions of the present invention those skilled in the art need not pay creativeness
The various modifications or changes that can be made work 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 peripheral blood cells methylation state of DNA is used to analyze the kit of liver cancer
<130>
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence
<400> 1
tgtaagtatc gtgtatgtgt aaacgtgttc 30
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ccgtacacac gaaaaacccg 20
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
ttcgtagcgg tgtatatt 18
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
cgcgttgttt ttgcgttttc 20
<210> 5
<211> 29
<212> DNA
<213>Artificial sequence
<400> 5
caaaacttaa ctctatataa accccaacg 29
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence
<400> 6
aagcgcgttt ttcggaattc ggg 23
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence
<400> 7
gtgtatattc gggtttttcg tgtgtac 27
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence
<400> 8
aatcctaaaa tcaacccgcg a 21
<210> 9
<211> 23
<212> DNA
<213>Artificial sequence
<400> 9
tttcgttcgc gttcgtcgtc gtt 23
Claims (10)
1. a kind of marker that methylates for liver cancer detection, which is characterized in that the marker that methylates is PTCH10.
2. the application of marker in preparing liver cancer detection reagent as described in claim 1 that methylates.
3. one group of primer and probe for requiring the marker that methylates described in 1 for test right, which is characterized in that the primer
Sequence is as follows：Forward primer sequence is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2；The fluorescence
Probe sequence is SEQ ID NO.3, and probe 5 ' holds flag F AM, 3 ' end label MGB.
4. primer as described in claim 1 and probe are in the application for preparing the kit for liver cancer detection.
5. a kind of kit, which is characterized in that further include detecting GAPDH just including the primer and probe described in claim 3
To primer and probe, PCR Master Mix, lysate, Proteinase K, rinsing liquid, bisulfite solution and ddH2O。
6. the application method of kit as claimed in claim 5, which is characterized in that steps are as follows：
(1) in sample genomic DNA extraction：Clinical samples are taken, lysate, Proteinase K, rinsing liquid and ddH are utilized2O is extracted,
Obtain Genomic DNA solution；
(2) DNA methylation is modified：The Genomic DNA solution that step (1) extraction obtains is taken to be repaiied with bisulfite solution
Decorations obtain the DNA after modifying that methylates；
(3) DNA after modifying step (2) carries out PCR amplification.
7. the application method of kit according to claim 6, which is characterized in that step (1) described sample is that peripheral blood is thin
Born of the same parents.
8. the application method of kit according to claim 6, which is characterized in that PCR reaction solution is：PCR Master Mix
20 μ L, primer and each 1 μ L of probe, 3 μ L of fluorescent quantitation reaction solution, 26 μ L of total amount/person-portion；PCR reaction conditions are：
9. application of the kit according to claim 5 in preparing liver cancer detection reagent.
10. kit is preparing the application in detecting PTCH10 methylating reagents according to claim 5.
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| CN201810745130.6A CN108660217A (en) | 2018-07-09 | 2018-07-09 | A kind of detection peripheral blood cells methylation state of DNA is used to analyze the kit of liver cancer |
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| CN201810745130.6A CN108660217A (en) | 2018-07-09 | 2018-07-09 | A kind of detection peripheral blood cells methylation state of DNA is used to analyze the kit of liver cancer |
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Citations (4)
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|---|---|---|---|---|
| WO2013129397A1 (en) * | 2012-02-29 | 2013-09-06 | シスメックス株式会社 | Method for determining presence or absence of cancer cell derived from hepatocellular carcinoma, and determination marker and kit |
| CN104762301A (en) * | 2015-04-24 | 2015-07-08 | 武汉大学 | Kit and method for detecting methylation level of liver cancer risk gene TSPYL5 |
| CN108009400A (en) * | 2018-01-11 | 2018-05-08 | 至本医疗科技（上海）有限公司 | Full-length genome Tumor mutations load forecasting method, equipment and storage medium |
| CN108070655A (en) * | 2017-09-21 | 2018-05-25 | 北京旌准医疗科技有限公司 | For predicting the kit of risk of hepatic cancer and method |
-
2018
- 2018-07-09 CN CN201810745130.6A patent/CN108660217A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013129397A1 (en) * | 2012-02-29 | 2013-09-06 | シスメックス株式会社 | Method for determining presence or absence of cancer cell derived from hepatocellular carcinoma, and determination marker and kit |
| CN104762301A (en) * | 2015-04-24 | 2015-07-08 | 武汉大学 | Kit and method for detecting methylation level of liver cancer risk gene TSPYL5 |
| CN108070655A (en) * | 2017-09-21 | 2018-05-25 | 北京旌准医疗科技有限公司 | For predicting the kit of risk of hepatic cancer and method |
| CN108009400A (en) * | 2018-01-11 | 2018-05-08 | 至本医疗科技（上海）有限公司 | Full-length genome Tumor mutations load forecasting method, equipment and storage medium |
Non-Patent Citations (4)
| Title |
|---|
| GANG WU等: "Epigenetic high regulation of ATAD2 regulates the Hh pathway in human hepatocellular carcinoma", 《INTERNATIONAL JOURNAL OF ONCOLOGY》 * |
| J YING等: "Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple othercarcinomas with frequent methylation", 《ONCOGENE》 * |
| SHUAI GAO等: "DNA methylation in liver diseases", 《WORLD JOURNAL OF CLINICAL INFECTIOUS DISEASES》 * |
| 范颂: "HCC中PCDH10甲基化与HBV的相关性探讨", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
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