CN104762301B - A kind of kit and method for detecting liver cancer risk genes TSPYL5 methylation levels - Google Patents
A kind of kit and method for detecting liver cancer risk genes TSPYL5 methylation levels Download PDFInfo
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Abstract
The invention discloses one kind to detect liver cancer risk genesTSPYL5The kit and method of methylation level, belong to biological technical field.DetectionTSPYL5The horizontal kit of gene methylation includes the primer pair B for the primer pair A of MSRE qPCR methods or for BSP methods, and these primer sequences are as shown in SEQ ID NO.1 22.Present invention findsTSPYL5The relevance that gene methylation occurs with liver cancer, and identifyTSPYL5Methylation sites.The present invention combines specific primer using specific methylation sensitive restriction enzyme, establishes the detection of quantitative fluorescent PCRTSPYL5Methylation method.Detected by kit of the present invention based on MSRE qPCR and methodTSPYL5Methylation level, for its high sensitivity up to 83.9%, specificity is up to 93.25%.
Description
Technical field
The present invention relates to biological technical field, and liver cancer risk genes are detected more particularly to one kindTSPYL5Methylation level
Kit and method.
Background technology
DNA methylation is one of main contents of epigenetics research, and research is that gene base sequence does not change
The reversible hereditary change occurred in the case of change, it is covalent modification mode after a kind of DNA molecular replicates.In eucaryote
In, DNA methylation refers in dnmt rna（DNMTs）In the presence of, it is methyl donor by methyl using S-adenosylmethionine
The process being transferred in the particular bases in DNA molecular, it is occurred mainly on cytimidine in CpG dinucleotides.In gene
In group, the region of CpG dinucleotides distribution Relatively centralized is referred to as CpG islands（CpG islands, CGIs）, size 100~
1000bp, it is predominantly located at promoter region and the First Exon area of gene；The abnormal methylation on CpG islands can directly result in correlation
The expression silencing of gene.The specific methylation patterns of DNA are for maintaining Genome stability and the correct spatial and temporal expression of gene to have
Significant, the abnormal change of methylation patterns will directly participate in the human diseases even generation of cancer.
Methylation sensitive restriction enzyme combination PCR method（Methylation-sensitive
Restriction enzyme digestion and PCR, MSRE-PCR）It is to be based on methylation sensitive restriction enzyme
The characteristic that methylation sites are not cut, by DNA digestion to enter performing PCR amplification after different size of fragment again, produced according to electrophoresis
Thing variance analysis methylation state.Concrete principle is referring to Fig. 1, to the restricted of specific DNA fragment methylated DNA fragments sensitivity
After restriction endonuclease carries out digestion, enter performing PCR by amplification starting point of sequence on the outside of methylation sites to be measured.If the DNA fragmentation in the presence of
Methylate, it will have amplified production appearance；If without methylating, any fragment amplification is not had and is occurred.When unwise with methylating
When the endonuclease digestion product of sense is as pcr template, there should not be fragment to expand no matter whether the position methylates all.
Bisulfite cloning and sequencing（Bisulfite sequencing PCR, BSP）Method, the DNA of extraction is through Asia
After disulfate modification, the cytosine deamination not methylated is transformed into urea pyrimidine, and the cytimidine to methylate is kept not
Become.Thymidine is completely converted into by PCR urea pyrimidines.Glue purification is cut after PCR primer agarose electrophoresis, is subsequently attached to
PMD18-T carriers, Escherichia coli are converted, single positive colony extraction plasmid is chosen after being incubated overnight and is sequenced, obtains each DNA
The distribution map of molecule specific region methylation sites, so as to obtain the methylation state in each CpG sites in the region.This method is
The method of the very high detection methylation state of a kind of reliability and accuracy, each CpG site in the fragment that can have a definite purpose
Methylation state.On significant key CpG sites are found, there is the advantages of other method is incomparable.But the party
Method expends time, fund and energy, and the clone of more than 10, which is at least sequenced, could obtain authentic data, it is necessary to substantial amounts of gram
The sequencing of grand and plasmid extraction, process are relatively complicated, expensive.So it is difficult to realize high-throughout automatic detection in clinic, but
Can be as the control methods of DNA methylation assay new method.
The proposed methylation sensitive restriction enzyme combination quantitative fluorescent PCR that is based on of this kit（MSRE-qPCR）
Method, the subsequent detection of MSRE-PCR methods is changed to quantitative fluorescent PCR, can quantify detection the specific base of minim DNA sample
Because of the methylation level in site.This method is easy to use, has well without carrying out bisulf iotate-treated, and to paraffin section
Compatibility.
The content of the invention
Based on this, it is an object of the invention to provide the good detection liver cancer risk genes of a kind of high sensitivity, specificityTSPYL5The kit of methylation level.
The purpose of the present invention is achieved through the following technical solutions：
One kind is used to detectTSPYL5The method of the quantitative fluorescent PCR of gene methylation, comprises the following steps：1. extraction is treated
Survey sample genomic dna；2. digestion is carried out to genomic DNA using methylation sensitive restriction enzyme；3. use primer
Fluorescent quantitative PCR is carried out to digestion products to A；4. the Ct values of PCR primer are analyzed；5. judged according to analysis result
In sampleTSPYL5The methylation level of gene.
Wherein, step 3. in primer pair A be preferably a pair in following four pairs of primers（Primer pair amplifies are represented in bracket
The length of fragment）：
AF1：5’-CCCCGCGAGCGCATATCAGAG -3’（176bp）,
AR1：5’-GCAACCGCCGACGTCACGAAC-3’；
AF2：5’-ATATCAGAGAAACTCGCCGAG-3’（150bp）,
AR2：5’-CACGAACGTACAACTGTACCG-3’；
AF3：5’-CAGAGAAACTCGCCGAGACCTA-3’（231bp）,
AR3：5’-TTCAAAGACACGCTGTGACCCT-3’；
AF4：5’-AGCGCATATCAGAGAAACT-3’（140bp）,
AR4：5’-GTACCGTCGCGAGAGGACGTGA-3’.
Above-mentioned primer designs to obtain by the following method：Obtained from UCSC databasesTSPYL5CpG island sequence, passes through
The restriction enzyme site in the sequence is analyzed, using online primer-design software（http://simgene.com/Primer3）primer
The designs of premier 3.0 include the PCR primer of 2-6 restriction enzyme site.Screen and verify by repetition test, obtained amplification effect
Rate is high, specificity is good, can detect sampleTSPYL5The horizontal primer of gene methylation.
One kind is based on methylation sensitive restriction enzyme combination fluorescence quantitative PCR detectionTSPYL5Gene methylation water
Flat kit A, include above-mentioned primer pair A.
Described kit A also includes methylation sensitive restriction enzyme.
Described kit A is also comprising methylate negative control and positive control.Preferably, negative control includes normal person
Peripheral blood DNA, the unmethylated human genome DNA's control of commercialization；Positive control includes commercialized exhaustive methylation
Human genome DNA, the liver cancer cell lines DNA of certified target fragment exhaustive methylation.
Described kit A also SYBR Green fluorescent dyes comprising quantitative fluorescent PCR etc..
Mentioned reagent box A application method, comprises the following steps：1. extract sample to be tested genomic DNA；2. use methyl
Change sensitive restriction restriction endonuclease and digestion is carried out to genomic DNA；3. fluorescent quantitation is carried out to digestion products using primer pair A
PCR is expanded；4. the analysis of Ct values is carried out to PCR primer.
The system of described digestion is preferably：DNA 300ng, Buffer（10×）5 μ L, methylation sensitive are restricted interior
Enzyme cutting 30U, ddH2O complements to 50 μ L.
The reaction system of described PCR amplifications is preferably：SYBR Mix（2×）10 μ L, sense primer（F）0.5 μ L, downstream
Primer（R）0.5 μ L, ddH2The μ L of O 7, the μ L of digestion products 2.
The reaction condition of described PCR amplifications is preferably：95℃ 5min；95 DEG C of 30s, 61 DEG C of 30s, 72 DEG C of 30s,
35-40 cycles。
Kit A of the present invention based on methylation sensitive restriction enzyme combination quantitative fluorescent PCR can be divided into inspection
Two parts of examining system and monitoring system.Detecting system includes above-mentioned primer pair A, can by it is therein any one combine.
Monitoring system then includes：1. negative control includes 2 kinds, the 1st kind be the region to be measured that has confirmed for it is non-methylate it is normal
Human gene group DNA's template；2nd kind is the commercialized non-human genome DNA's control that methylates.All feminine genders are right under normal circumstances
According to methylation level answer convergence 0, otherwise show sample-adding process have pollution or digestion it is incomplete.2. positive control is commercialization
Exhaustive methylation human genome DNA, the liver cancer cell lines DNA of certified target fragment exhaustive methylation；Normally
The methylation level of the lower positive control of reaction answers convergence 1, otherwise illustrates the failure of an experiment.
Another kind is used to detectTSPYL5The BSP of gene methylation method, comprises the following steps：1. extract sample to be tested
Genomic DNA；2. genomic DNA is modified using sodium hydrogensulfite；3. the DNA after modification is carried out using primer pair B
PCR is expanded；4. PCR primer cloning and sequencing analyzes the methylation status in each CG sites.
Wherein, step 3. in primer pair B include Outside primer（W）And inner primer（N）, wherein Outside primer is following
A pair of 3 centerings：
BWF1：5’-TAAGAGATAATTGGAGGA-3’（465bp）,
BWR1：5’-ACCTTTACCCCGATTTTTA-3’；
BWF2：5’-ACGTTCGAGTATTTTTTTTA-3’（498bp）,
BWR2：5’-GACCTTTACCCCAATTTTTA-3’；
BWF3：5’-AGATAATTGGAGGAGTTGAAGA-3’（526bp）,
BWR3：5’-TACTATAAAAAATCCGAATCGC-3’；
Inner primer is a pair in following 4 pairs of primers：
BNF1：5’-AATAGGTGATGGGGGATAGGT-3’（376bp）,
BNR1：5’-CCGCTCATAATAACGACGAAA-3’；
BNF2：5’-AGAAAATAGGTGATGGGGGA-3’（383bp）,
BNR2：5’-CGACCGCTCATAATAACGAC-3’；
BNF3：5’-TTAGAAAATAGGTGATGGGGGATAG-3’（373bp）,
BNR3：5’-ATAACGACGAAAACAACTTCAAAAA-3’；
BNF4：5’-AATAGGTGATGGGGGATAG-3’（378bp）,
BNR4：5’-GACCGCTCATAATAACGAC-3’.
Above-mentioned primer designs to obtain by the following method：Obtained from UCSC databasesTSPYL5CpG island sequence, is used for
The primer of BSP methods uses online methylated primers design software（http://www.urogene.org/methprimer/）
MethPrimer is designed.Screen and verify by repetition test, obtained that amplification efficiency is high, specificity is good, sample can be detectedTSPYL5The horizontal BSP primers of gene methylation.
A kind of PCR detections based on sodium hydrogensulfite modificationTSPYL5The horizontal kit B of gene methylation, comprising above-mentioned
Primer pair B.
Described kit B is also comprising methylate negative control and positive control.Preferably, negative control includes normal person
Peripheral blood DNA, the non-human genome DNA's control to methylate of commercialization；Positive control includes the commercialized mankind's base that methylates
Because of a group DNA, the liver cancer cell lines DNA of certified target fragment exhaustive methylation.
Described kit B also includes PCR reagent（DNTPs, archaeal dna polymerase, archaeal dna polymerase buffer etc.）And sulfurous
Reagent needed for sour hydrogen sodium modification（Sodium hydrogensulfite, quinhydrones, sodium hydroxide, ammonium acetate, glycogen etc.）.
Mentioned reagent box B application method, comprises the following steps：1. extract sample to be tested genomic DNA；2. use sulfurous
Sour hydrogen sodium is modified genomic DNA；3. enter performing PCR amplification to the DNA after modification using primer pair B（Successively on the outside of use
Primer and inner primer carry out Chao Shi PCR amplifications）；4. PCR primer cloning and sequencing analyzes the methylation status in each CG sites.
The reaction system of described PCR amplifications is preferably：Buffer（10×）5 μ L, dNTPs（10mM）1 μ L, MgCl2
（25mM）4 μ L, Taq（1U/μL）2 μ L, sense primer（F）1 μ L, anti-sense primer（R）1 μ L, ddH2The μ L of O 32, the μ L of DNA profiling 4.
Described Chao Shi PCR expand outside, the reaction condition of inner side is both preferably：95 DEG C, 5min；95 DEG C, 30s, 68 DEG C-
56 DEG C, 2cycles/2 DEG C, 45s, 72 DEG C, 45s, annealing temperature carries out 25cycles when being down to 56 DEG C；72 DEG C, 10min.
Present invention findsTSPYL5The relevance that gene methylation occurs with liver cancer, and identifyTSPYL5Methylate
Site, with reference to methylation sensitive restriction enzyme and specific primer, establish the detection method of quantitative fluorescent PCR.And
And then this conclusion is verified using BSP method.
It is of the present inventionTSPYL5Up to 80.37%, specificity is up to the high sensitivity of gene methylation detection kit
93.25%。
This method is substantially increased detection efficiency, is reduced detection using fluorescence quantifying PCR method detection DNA methylation
Cost.Testing result is simple, intuitively, and high flux, it once can detect many samples.
Brief description of the drawings
Fig. 1 is MSRE-PCR schematic diagrams；In figure, CH3 represents to methylate, and enz represents methylation sensitive restriction enzyme
Enzyme；Without methylating, DNA is cut off left figure DNA, can not obtain PCR primer；Right figure DNA methylates, and DNA keeps complete, can be with
Obtain PCR primer.
Fig. 2 is the amplification curve (a) of negative control and melting curve (b) figure in embodiment 1, wherein grey and black line point
Do not represent enzyme-added with being not added with enzyme system, each two multiple holes of sample；RFU represents the fluorescent value of amplified reaction；- d (RFU)/dt is represented
The negative one subderivative that fluorescence signal changes.
Fig. 3 is the positives control amplification curve (a) of embodiment 1 and melting curve (b) figure, wherein grey and black line point
Do not represent enzyme-added with being not added with enzyme system, each two multiple holes of sample；RFU represents the fluorescent value of amplified reaction；- d (RFU)/dt is represented
The negative one subderivative that fluorescence signal changes.
Fig. 4 is embodiment 1 using in MSRE-qPCR detection liver cancer and cancer beside organismTSPYL5The horizontal scatterplot of gene methylation
Figure.
Fig. 5 is in embodiment 1TSPYL5Gene methylation is horizontal to distinguish liver cancer and cancer beside organism's ROC curve figure.
Fig. 6 is embodiment 2 using in BSP clone sequencings detection liver cancer and cancer beside organismTSPYL5Gene methylation is horizontal
Case figure.
Fig. 7 is liver cancer and cancer beside organism in embodiment 2TSPYL5Each CpG sites of gene methylate bar chart.
Embodiment
In order to be more clearly understood that the technology contents of the present invention, described in detail especially exemplified by following examples.It should be understood that this
A little embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.
The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook etc.
People, molecular cloning：Laboratory manual（New York: Cold Spring Harbor Laboratory Press, 1989）In
Described condition, or according to the condition proposed by manufacturer.Used various conventional chemical reagent, are city in embodiment
Sell product.Unless otherwise defined, the technical field of all technologies used in the present invention and scientific terminology with belonging to the present invention
The implication that is generally understood that of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality
The purpose of example is applied, is not used in the limitation present invention.
Embodiment 1TSPYL5Gene methylation fluorescent PCR detects 163 pairs of liver cancer and cancer beside organism's sample
Paraffin sample and frozen tissue sample genomic dna use the kit of Qiagen companies（QIAamp DNA
FFPE Tissue kit；QIAamp DNA Mini Kit）Extraction.The concentration and purity of DNA sample use NanoDrop-
2000c is detected.
300ng DNA samples carry out digestion using methylation sensitive restriction enzyme（HinP1I）, digestion system is：
DNA 300ng, Buffer（10×）5 μ L, methylation sensitive restriction enzyme 30U, ddH2O complements to 50 μ L, and this is
Enzyme-added group.Not enzyme-added group of processing removes and uses ddH2O is substituted outside restriction enzyme, and remaining is all identical with enzyme-added group.It is negative
Control（It is commercialized the non-human genome DNA's control to methylate）, positive control（The human gene of commercialized exhaustive methylation
Group DNA）Also same processing is done.
PCR primer is designed for restriction enzyme site, using quantitative fluorescent PCR to being handled by digestion（Enzyme-added group）And non-digestion
（Not enzyme-added group）DNA sample, negative control and positive control expanded（Two multiple holes of each sample）.Amplification system is：
SYBR Mix（2×）10 μ L, sense primer（F）0.5 μ L, anti-sense primer（R）0.5 μ L, ddH2The 2 μ L of μ L, DNA of O 7；On wherein
Anti-sense primer is as follows：
Sense primer AF2：5 '-ATATCAGAGAAACTCGCCGAG-3 ',
Anti-sense primer AR2：5’-CACGAACGTACAACTGTACCG-3’；
Amplification condition is：95℃ 5min；95 DEG C of 30s, 61 DEG C of 30s, 72 DEG C of 30s, 35 cycles.
Using 100% × 2△ Cq (not enzyme-added group-enzyme-added group)Formula calculates the level to methylate, wherein △ Cq (not enzyme-added group-enzyme-added group)
It is expressed as the average value of two multiple holes Cq values of average value-not enzyme-added group of enzyme-added group of two multiple holes Cq values.
As a result the methylation level for finding negative control is 0.00%（Fig. 2）, the methylation level of positive control is
100.00%（Fig. 3）.In cancerous tissueTSPYL5Gene methylation level is significantly higher than cancer beside organism（P<0.0001, Fig. 4）, and
ROC curve, which analyzes it and can be very good to distinguish cancer and cancer beside organism, its TG-AUC, can reach 91.4%（88.0%-
94.8%）（Fig. 5）, when methylation level is cut off values with 7.015%, its sensitivity and specificity are respectively 80.37% He
93.25%。
The BSP clone sequencings of embodiment 2 are detected in liver cancer and cancer beside organismTSPYL5Gene methylation is horizontal
Paraffin sample and frozen tissue sample genomic dna use the kit of Qiagen companies（QIAamp DNA
FFPE Tissue kit；QIAamp DNA Mini Kit）Extraction.The concentration and purity of DNA sample use NanoDrop-
2000c is detected.
2 μ g DNA samples are modified using sodium hydrogensulfite, negative control（It is commercialized the non-human genome to methylate
DNA is compareed）, positive control（The human genome DNA of commercialized exhaustive methylation）Also same processing is done, detailed process is such as
Under：
（1）Take 2 μ g DNA to be placed in 2mL EP pipes, add appropriate autoclaving water and supply volume to 50 μ L；
（2）Add 5.5 μ L Fresh 3mol/L NaOH, 55 DEG C of water-bath 20min；
（3）Add the hydroquinone solution of Fresh（0.04mol/L）30 μ L and solution of sodium bisulfite（3.6 mol/L）
520 μ L, add 200 μ L paraffin oils, 55 DEG C of lucifuge water-baths 16 hours；
（4）Wizard DNA Clean-up System are purified（Carried out by operational manual）；
（5）3mol/L NaOH 4 μ L, 37 DEG C of water-bath 15min are added into above-mentioned DNA after purification；
（6）22 μ L 10M ammonium acetates are added, 4 μ L 20g/L glycogens, 250 μ L ice absolute ethyl alcohols, -20 DEG C is placed in and precipitated
Night；
（7）The DNA of precipitation is reclaimed, 70% ethanol is washed twice, is dissolved in after drying at room temperature in 50 μ L TE, is placed in -20 DEG C of guarantors
Deposit.
For the sequences Design PCR primer after conversion, using Chao Shi PCR（Outside primer, inner primer, sequence are used successively
Row are as follows）DNA after modification is expanded, primer sequence is specific as follows：
Outside primer：
Upstream BWF2：5 '-ACGTTCGAGTATTTTTTTTA-3 ',
Downstream BWR2：5’-GACCTTTACCCCAATTTTTA-3’；
Inner primer：
Upstream BNF2：5 '-AGAAAATAGGTGATGGGGGA-3 ',
Downstream BNR2：5’-CGACCGCTCATAATAACGAC-3’.
Interior outside PCR amplification conditions are：95 DEG C, 5min；95 DEG C, 30s, 68 DEG C -56 DEG C, 2cycles/2 DEG C, 45s, 72
DEG C, 45s, annealing temperature carries out 25cycles when being down to 56 DEG C；72 DEG C, 10min.
PCR primer is connected after cutting glue purification with pMD18-T carriers, then continues in DH5 α competence, in Amp (+) flat board
It is incubated overnight rear 10 positive colony extraction plasmid order-checkings of picking.Sequencing result is analyzed, using 100 ×（Methylate
The quantity of cytimidine quantity/all cytimidines）Calculate the methylation level of each site and each sample.
The present embodiment is by using BSP clone sequencings in the part liver cancer in embodiment 1 and cancer beside organismTSPYL5
Gene methylation level is detected.As a result cancerous tissue is foundTSPYL5The methylation level on gene promoter area CpG islands is high
In cancer beside organism（P<0.0001, Fig. 6）, and in cancerous tissueTSPYL5Each CpG sites methylates in gene amplification fragment
Level is above cancer beside organism（Fig. 7）.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope that this specification is recorded all is considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.
Claims (6)
1. one kind is horizontal based on methylation sensitive restriction enzyme combination fluorescence quantitative PCR detection TSPYL5 gene methylations
Kit, it is characterised in that：Include following primer pair：
AF2：5 '-ATATCAGAGAAACTCGCCGAG-3 ',
AR2：5’-CACGAACGTACAACTGTACCG-3’；
Described methylation sensitive restriction enzyme is HinP1I.
2. kit according to claim 1, it is characterised in that：Also include methylation sensitive restriction enzyme.
3. kit according to claim 1, it is characterised in that：Also include quantitative fluorescent PCR reagent.
4. according to the kit described in claim any one of 1-3, it is characterised in that：Also comprising methylate negative control and methyl
Change positive control.
5. kit according to claim 4, it is characterised in that：The described negative control that methylates includes Normal human peripheral
Blood DNA, the non-human genome DNA to methylate.
6. kit according to claim 4, it is characterised in that：Described methylation positive control includes the people to methylate
The liver cancer cell lines DNA that type genomic dna, target fragment methylate.
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| KR101848803B1 (en) | 2016-06-15 | 2018-04-17 | 한국원자력연구원 | Method to block the growth of cancer stem cells via inhibiting the phosphorylation of 120th threonine residue in TSPYL5 and Peptide inhibitor and the use thereof |
| CN116064795A (en) * | 2016-09-02 | 2023-05-05 | 梅约医学教育与研究基金会 | Methods and kits for determining methylation status of differentially methylated regions |
| CN106916893A (en) * | 2016-12-26 | 2017-07-04 | 中国科学院上海微系统与信息技术研究所 | A kind of gene methylation degree quantitative approach based on digital pcr chip |
| CN106811523B (en) * | 2017-01-17 | 2021-05-18 | 首都医科大学附属北京佑安医院 | Methylation gene for screening liver cancer |
| CN109207618B (en) * | 2017-06-30 | 2020-05-29 | 中国农业科学院植物保护研究所 | Method and kit for determining methylation rate of target sequence in biological material |
| CN108103163A (en) * | 2018-01-17 | 2018-06-01 | 湖南大地同年生物科技有限公司 | A kind of detection method of gene methylation |
| CN108660217A (en) * | 2018-07-09 | 2018-10-16 | 安徽达健医学科技有限公司 | A kind of detection peripheral blood cells methylation state of DNA is used to analyze the kit of liver cancer |
| CN110257511A (en) * | 2018-12-29 | 2019-09-20 | 武汉生命科技股份有限公司 | A method of for detecting the quantitative fluorescent PCR of TSPYL5 gene methylation |
| CN113897434A (en) * | 2021-11-02 | 2022-01-07 | 北京艾克伦医疗科技有限公司 | Method and kit for identifying liver cancer status |
| CN116200499B (en) * | 2023-03-23 | 2024-01-19 | 北京和瑞精湛医学检验实验室有限公司 | Gene combination for liver cancer detection, related reagent and application |
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