CN102134610A - Method for detecting methylation state of corresponding gene promoter of severe hepatitis and kit - Google Patents
Method for detecting methylation state of corresponding gene promoter of severe hepatitis and kit Download PDFInfo
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Abstract
The invention relates to a method for detecting the methylation state of a corresponding gene promoter of severe hepatitis and a kit. The method comprises reagent converting unmethylated cytosine into uracil, 2 times Taq PCR Mastermix, and a methylated specific and unmethylated specific primer pair of a promoter, which acts on the glutathione-S-transferase P1 gene of a human body. The method comprises the following steps: modifying extracted PBMC DNA, and mixing the modified PBMC DNA with the methylated (M) specific and the unmethylated (U) specific primer pair of the promoter, the 2 times Taq PCR Mastermix and double distilled water to generate methylated specific polymerase chain reaction, thereby obtaining amplification products. The detection on the methylation state of the corresponding gene promoter of severe hepatitis is beneficial to clinicians in judging the conditions of patients with severe hepatitis and carrying out treatment.
Description
Affiliated technical field
The present invention relates to medical field, is a kind of hepatitis gravis related gene promoter methylation state information and application thereof.Specifically detect the method and the test kit of the gene promoter methylation state relevant with hepatitis gravis.
Background technology
Slow extra urgaent dispatch hepatitis gravis (ACHBLF) is on the basis of chronic hepatopathy, the serious hepatic injury that causes by multiple factor, cause the function generation serious hindrances such as synthetic, drainage, bio-transformation and detoxifcation of liver, occurring with jaundice, coagulation disorders, hepatogenic encephalopathy and ascites etc. serve as the one group of clinical syndrome that mainly shows.China is the district occurred frequently of hepatitis B, and its hepatitis B surface antigen(HBsAg) (HBsAg) carrying rate reaches 10%, and existing patient is about more than 3,000 ten thousand, wherein is chronic viral hepatitis B (CHB) more than 60%.These carrier and chronic hepatitis patient often have possibility that worsens or develop into hepatitis gravis, therefore, in China, hepatitis B virus (HBV) infection be cause the main diseases of slow extra urgaent dispatch hepatitis gravis because of.According to statistics, account for 81% of hepatitis gravis by the caused by hepatitis B virus hepatitis gravis.The slow extra urgaent dispatch hepatitis gravis that takes place on hepatitis b virus infected basis often is called the heavy hepatitis B of slow extra urgaent dispatch.In China, slow extra urgaent dispatch liver failure is the sickness rate height not only, and state of an illness complexity, and the state of an illness is dangerous, and development is rapid, and various complication often appear in prognosis mala, seriously jeopardize patient's life.So study the control of chb and prevent that chb is to heavy liver, liver cirrhosis even liver cancer transformation have crucial meaning slowly.
The pathogenesis of slow extra urgaent dispatch liver failure is very complicated, also fails far away up to now to illustrate.Diseases associated present and the diseases related research of HBV mainly contains HBV dependency hepatitis, liver cirrhosis and liver cancer.Increase along with gene promoter zone methylation and various tumour comprise the correlation research of liver cancer, the diseases associated that methyl changes into the diseases related research of HBV mainly contains HBV dependency hepatitis, liver cirrhosis and liver cancer.Increase along with gene promoter zone methylation and various tumour comprise the correlation research of liver cancer, methylating becomes the increasing epigenetics content of research.Have research to point out, HBV infects body and causes the generation of dependency hepatic diseases to methylate relevant with the mistake of some gene.
HBV is a kind of hepatovirus of having a liking for, and behind the infected liver cell, HBV-DNA can integrate with the host liver cell, thereby makes host's genome take place to change widely, comprises the fusion of disappearance, transposition, transcription product and genomic instability widely.Virus enters body and induces the active increase of host's methylated transferase (DNMT), but catalysis vDNA (DNA) promoter methylation.At virogene by the methylated while, the modification because the active methylated transferase that increases also can methylate to host's gene performance simultaneously, therefore the human body normal DNA modification that also may be methylated, thus make host's gene promoter area itself also take place to methylate.
Dna methylation is the most deep a kind of mechanism of research on the epigenetics, and it is a kind of chemically modified process of enzyme mediation.Dna methylation is meant the 5-adenosylmethionine as methyl donor, under the effect of dna methylation transferring enzyme, the 5th carbon atom of pyrimidine ring of cytidylic acid(CMP) is methylated, and forms methylated CpG island with guanine of its 3 ' end.The CpG island is that length is some zones of being rich in the CpG dinucleotides of 300～3000 base pairs (bp) in the genome, mainly is present in 5 ' zone of gene.Under the normal circumstances, the not methylation state on CpG island is that genetic transcription is necessary in the promoter region, the methylation state in most sites is invariable in the genome, have only the methylation state in minority site to change, when the methylation state of genomic dna changes, can cause body karyomit(e) instability or abnormal gene expression, can cause genetic transcription to be suppressed.
Therefore study host gene that HBV causes methylate for alleviate hepatocellular injury that HBV causes, to reduce chronic hepatitis significant to the generation that hepatitis gravis transformed, reduced HBV dependency liver cancer.For research HBV dependency hepatitis, liver cirrhosis, liver cancer provide new thinking.
Reduced glutathion (GSH) is a kind of important enzyme that can protect liver cell to avoid the various factors damage.Reduced glutathion has to be removed free radical, anti-oxidant, anti-inflammatory, detoxifcation, the metabolism of promotion cholic acid, protect cytolemma, improves liver function, alleviates tissue injury and promotes functions such as tissue repair.Reduced glutathion extensively is present in the normal cell, and is especially abundant at the liver cell intensive amount.It participates in tricarboxylic acid cycle and carbohydrate metabolism in the body, can activate multiple enzyme, thereby impel sugar, fat and proteinic metabolism, and directly influence hepatocellular metabolic process.Can be the important component part of human body antioxidant system to anti peroxidation of lipid, have the protection liver plasma membrane, promote enzyme activity of liver, remove oxyradical and promote effects such as liver complex functionality.The chronic viral hepatitis B liver cell is impaired to cause that reduced glutathion lacks, make liver cell to oxyradical and inside and outside other removing ability of source property goods and materials reduce, increase the weight of hepatocellular injury.Often protect liver synthetic clinically, detoxifcation, functions such as deactivation hormone by the supplemented with exogenous reduced glutathion.Reduced glutathion brings into play that above-mentioned protection is hepatocellular does the katalysis that the time spent will depend on glutathione sulfydryl transferase (GST), and glutathione sulfydryl transferase belongs to metabolism and separates toxenzyme system, the reaction between main catalysis gsh and the Electron Affinities material.Glutathione sulfydryl transferase is a kind of dimer isozyme, have strong katalysis, energy catalytic substrate molecule, the yoke of electrophilic center and reduced glutathion closes, promoting its metabolism, inactivation, is one of the most important II phase of bio-transformation metabolic enzyme in the body.The glutathione sulfydryl transferase superfamily mainly is divided into α, μ, 4 subfamilies of π, θ by its chromosomal localization and sequence homology.The glutathione sulfydryl transferase subfamily participates in the metabolism of different chemical material respectively, but the substrate range of choice can be overlapped to each other.Wherein the effect of GST μ, π, θ is particularly outstanding.Glutathione S-transferase P1 (GSTP1) belongs to GST-π subfamily.GSTP1 genetic expression can have influence on the katalysis to gsh when reducing, and the expression reduction that the CpG island of GSTP1 gene promoter area methylates and can make GSTP1.The DNA promoter methylation may be thereby that gene such as the GSTP1 that the important mechanism of causing a disease of virus may make the protection liver avoid damaging expresses reduction in the virus induction cell, makes the GSH function impaired.Wang etc. discover prompting, and methylating of GSTP1 gene promoter area is not only just to occur in the liver cancer stage in the liver cancer tissue, but constantly take place under the effect at long term evolution that hepatic diseases takes place and HBV.
Studies show that both at home and abroad, there is the supermethylation of glutathione sulfydryl transferase (GSTP1) gene promoter in the HBV dependency liver cancer, but whether in chronic viral hepatitis B and hepatitis gravis patient, whether also there be progress and the prognosis that methylates and whether influenced the state of an illness, research is not also arranged.About the literature research of methylated the past of GSTP1 promotor main relevant with the generation of tumour, such as liver cancer, lung cancer and prostate cancer etc.Have and discovered in the liver cirrhosis patient blood plasma and have methylating of GSTP1 promoter region.Based on above research, it is relevant with slow urgent liver failure that the inventor has proposed methylating of GSTP1 promotor first.Consider that GSTP1 is a kind of enzyme of anti-oxidative damage, after being suppressed by promoter zone methylation GSTP1 so, may promotes oxidn stress be to the damage of liver, and oxidative stress is relevant with slow extra urgaent dispatch liver failure.It is a secular process that this genetics changes, and it may be present in hepatitis in each process of liver cirrhosis liver cancer, therefore methylatedly may play a role the pathogenic process from chb to heavy liver unusually.
Summary of the invention
The object of the present invention is to provide the test kit of the slow extra urgaent dispatch liver failure of the heavy hepatitis B of the suitable detection of a kind of convenience related gene promoter methylation state, and simple and feasible detection method, its detected result provides scientific basis for the clinical diagnosis and treatment of hepatitis gravis.
The present invention realizes like this, be provided with in this test kit: make the non-cytosine(Cyt) that methylates be converted into the reagent of uridylic, 2 * Taq PCR MasterMix, and right at the primer of the promotor GpG island methylation-specific of human glutathione sulfurtransferase P1 (GSTP1) gene and non-methylation-specific.
Use for convenience, can also be provided with the single nuclear DNA extraction of peripheral blood reagent in this test kit, like this, just can make the whole process that detects by the hepatitis gravis related gene promoter methylation state of taking a blood sample of a test kit.
For the ease of contrast, can also be provided with positive control and/or negative control in this test kit.
Methylate (M) specificity and non-methylating (U) Auele Specific Primer of the promotor described in this test kit to being selected from down group are:
In this test kit, the promoter methylation Auele Specific Primer length that is provided with is 15-40bp.
The reagent that makes the non-cytosine(Cyt) that methylates be converted into uridylic that is provided with in this test kit is hydrosulphite, '-biphenyl diphenol and sodium hydroxide.
The toughener, optimization agent and the stablizer enzyme that contain methylation-specific polymerase chain reaction (PCR) heat-resisting polymerase, triphosphoric acid dezyribonucleoside, magnesium chloride, reaction buffer, methylation-specific polymerase chain reaction (PCR) reaction in 2 * Taq PCR MasterMix enzyme.
The single nuclear DNA extraction of peripheral blood of the present invention reagent is: Trisodium Citrate, sodium hydroxide, phenol, different sulphur hydracid guanidine, trichloromethane, sarcosyl, ethanol, sodium-acetate, Tutofusin tris (Tris), ethylenediamine tetraacetic acid (EDTA) (EDTA) and N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd (HEPES).
The method of detection hepatitis gravis genes involved GSTP1 promoter methylation state of the present invention is: the reagent that makes the non-cytosine(Cyt) that methylates be converted into uridylic is modified the peripheral blood mononuclear cell DNA that extracts; Methylate (M) specificity and non-methylating (U) Auele Specific Primer, 2 * Taq PCR MasterMix, the bi-distilled water of the peripheral blood mononuclear cell DNA after modifying and promotor are mixed, make it produce methylation-specific polymerase chain reaction (PCR), the amplified production length that back, methylation-specific polymerase chain reaction (PCR) is amplified is 90-100bp, detect each gene promoter methylation state at last, draw detected result.Comprehensive patient's gpt (ALT), serum total bilirubin (TBIL), mda (MDA) level, thrombogen mobility (PTA), serum hepatitis B virus hepatopathy model in quantitative, whole latter stage (MELD) scoring, prompting exists the patient of GSTP1 gene promoter methylation in the presence of immune factor, cause the infringement repeatedly of the liver function of chb, had bigger chance to develop into slow extra urgaent dispatch liver failure.
The inventor is through extensive and deep research, methylation state to slow extra urgaent dispatch liver failures of many cases (ACHBLF) and chronic viral hepatitis B (CHB) peripheral blood of patients mononuclearcell DNA range gene promotor is analyzed, and carry out correlation analysis with patient's liver function, mainly detect gpt (ALT), serum total bilirubin (TBIL), mda (MDA) level, thrombogen mobility (PTA), serum hepatitis B virus hepatopathy model in quantitative, whole latter stage (MELD) scoring.There is substantial connection in the promoter methylation state of finder's glutathione S-transferase P1 (GSTP1) gene with the development of slow extra urgaent dispatch liver failure, has finished the present invention on this basis, with auxiliary diagnosis, guidance treatment and prompting prognosis.
The inventor proves, there is dependency (r=0.57 in the mda level with slow extra urgaent dispatch liver failure patient's whole hepatopathy model scoring in latter stage, P＜0.01), the parallel rising with the degree of being in a bad way of the degree of oxidative stress among the slow extra urgaent dispatch liver failure of this results suggest patient, oxidative stress has participated in the generation of slow extra urgaent dispatch liver failure.The oxidative stress that the free radical that oxidation produced of DPNH I (NADPH) causes may be that chb morbidity and progress are the reason of slow extra urgaent dispatch liver failure.According to the inventor's research, HBV not only can make the oxidative damage aggravation, also may reduce the expression of antioxidant system involved enzyme such as GSTP1 by methylated mode.The promoter zone methylation that can infer GSTP1 may participate in the lipid peroxidation injury among the slow extra urgaent dispatch liver failure patient, and aggravated oxidative stress to hepatocellular damage, have the patient's who helps the high blood plasma level of mda recovery by taking oxidation resistant medicine.
In a word, our results suggest GSTP1 promoter zone methylation can aggravate the damage of slow extra urgaent dispatch liver failure oxidative stress to liver, and the mda level in the blood plasma is relevant with the severity extent of slow extra urgaent dispatch liver failure.Help the clinician to identify to make progress among the chb patient to be the patient of slow extra urgaent dispatch liver failure for the analysis of the methylation state of slow extra urgaent dispatch liver failure (ACHBLF) and chronic viral hepatitis B (CHB) peripheral blood of patients mononuclearcell DNA range gene promotor.
Description of drawings
Accompanying drawing: the result who has shown glutathione S-transferase P1 (GSTP1) promoter methylation primer amplified.
Among the figure, the non-primer amplification band (U) that methylates of 1-normal group; 2-chronic viral hepatitis B (CHB) patient organizes the non-primer amplification band (U) that methylates; The slow extra urgaent dispatch liver failure of 3-(ACHBLF) patient organizes the primer amplification band (M) that methylates; The 4-positive controls primer amplification band (M) that methylates; The non-primer amplification band (U) that methylates of 5-positive controls.
Methylate with non-methylation-specific primer condition under: normal group and chronic viral hepatitis B (CHB) patient organizes the general visible non-primer amplification band (U) that methylates, and does not see the primer amplification band (U) that methylates for above-mentioned two groups.Slow extra urgaent dispatch liver failure (ACHBLF) patient organizes the primer amplification band (M) that as seen obviously methylates.Negative control group should not have any primer amplification band appearance in the test, positive controls methylates (M) and non-methylating (U) primer amplification band all should occur, positive control and negative control are as quality control of the present invention, if above-mentioned accordingly result does not appear in positive control and negative control, then represent experimentation or operate wrong.
Show by above result displayed: 1. in slow extra urgaent dispatch liver failure (ACHBLF) patient, serum mda (MDA) level will be apparently higher than chronic viral hepatitis B (CHB) patient (P＜0.01), slow extra urgaent dispatch liver failure (ACHBLF) patient that methylates organizes the mda level apparently higher than the non-group that methylates (P＜0.01), slow extra urgaent dispatch liver failure (ACHBLF) patient's mda level and whole hepatopathy model in latter stage (MELD) scoring relevant (P＜0.01).Mda level in the group that methylates among the slow extra urgaent dispatch liver failure patient will be apparently higher than the blood plasma level (P＜0.01) of the non-group that methylates.2. slow extra urgaent dispatch liver failure (ACHBLF) patient GSTP1 gene promoter methylation situation shows, have 31.43% to find to methylate among the slow extra urgaent dispatch liver failure patient, have 8.57% to find to methylate among chronic viral hepatitis B (CHB) patient, difference between the two has statistical significance (χ
2=5.71, P=0.02).The group that methylates has significant difference (P＜0.01) with group slow extra urgaent dispatch liver failure (ACHBLF) the patient TBIL level that do not methylate.Methylating of GSTP1 gene promoter can increase slow extra urgaent dispatch liver failure patient liver oxidative stress damage, and oxidative stress is relevant with slow extra urgaent dispatch liver failure.
There is close dependency in above analysis revealed glutathione S-transferase P1 (GSTP1) gene promoter methylation state with slow extra urgaent dispatch liver failure.Comprehensive patient's gpt (ALT), serum total bilirubin (TBIL), mda (MDA) level, thrombogen mobility (PTA), serum hepatitis B virus hepatopathy model in quantitative, whole latter stage (MELD) scoring, prompting exists the patient of GSTP1 gene promoter methylation in the presence of immune factor, caused the infringement repeatedly of the liver function of chb. there is bigger chance to develop into slow extra urgaent dispatch liver failure.
Embodiment
Be provided with in this test kit: make the non-cytosine(Cyt) that methylates be converted into the reagent of uridylic, 2 * Taq PCRMasterMix, and right at the primer of the promoter methylation specificity of human glutathione sulfurtransferase P1 (GSTP1) gene and non-methylation-specific.
Use for convenience and contrast, can also be provided with the single nuclear DNA extraction of peripheral blood reagent, positive control and/or negative control in this test kit.Like this, not only can just can do the whole process that detects by the slow extra urgaent dispatch liver failure related gene promoter methylation state of taking a blood sample with a test kit.And, can also carry out the positive control and/or the negative control of test result easily, conveniently draw test result.
Methylate (M) specificity and non-methylating (U) Auele Specific Primer of promotor of the present invention to being selected from down group are:
In this test kit, the promoter methylation Auele Specific Primer length that is provided with is 15-40bp.
The reagent that makes the non-cytosine(Cyt) that methylates be converted into uridylic of the present invention is: hydrosulphite, '-biphenyl diphenol and sodium hydroxide.
The toughener, optimization agent and the stablizer enzyme that contain methylation-specific polymerase chain reaction (PCR) heat-resisting polymerase, triphosphoric acid dezyribonucleoside, magnesium chloride, reaction buffer, methylation-specific polymerase chain reaction (PCR) reaction in 2 * Taq PCR MasterMix enzyme.
The single nuclear DNA extraction of peripheral blood of the present invention reagent is: Trisodium Citrate, sodium hydroxide, phenol, different sulphur hydracid guanidine, trichloromethane, sarcosyl, ethanol, sodium-acetate, Tutofusin tris (Tris), ethylenediamine tetraacetic acid (EDTA) (EDTA) and N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd (HEPES).
The method of detection hepatitis gravis genes involved GSTP1 promoter methylation state of the present invention following (wherein, the experimental technique of unreceipted actual conditions is according to the experiment condition and the method for routine, or the condition of advising according to manufacturer):
The extraction of DNA: after extracting blood from slow extra urgaent dispatch liver failure (ACHBLF) patient, can extract patient's DNA, also can extract DNA with additive method according to the following step.
(1) prepares reagent: ethanol, Trisodium Citrate, sodium hydroxide, phenol, different sulphur hydracid guanidine, trichloromethane, sarcosyl, sodium-acetate, Tutofusin tris (Tris), ethylenediamine tetraacetic acid (EDTA) (EDTA) and N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd (HEPES).
(2) preparation of TRIzol: with following reagent mix, and with distilled water (ddH
2O) be settled to 2000ml:500ml phenol, the different sulphur hydracid of 250g guanidine, the 0.75mol/L sodium citrate solution of 17.6ml (pH 〉=7), 10% the sarcosyl solution of 26.4ml, the 2mol/L sodium acetate soln of 50ml (pH 〉=4);
(3) centrifugal collecting cell, every (5-10) * 10
6Peripheral blood mononuclear cell adds the TRIzol of 1ml, inhales repeatedly and beats.Room temperature (15-30 ℃) was placed 5 minutes, and the nucleic acid-protein mixture is separated fully;
(4) TRIzol of every use 1ml adds the 0.2ml trichloromethane, thermal agitation 15 seconds, and room temperature was placed 3 minutes;
(5) under 2-8 ℃ of condition centrifugal 15 minutes with 10000 * g centrifugal force.Sample is divided into three layers: bottom is yellow organic phase, and the upper strata is colourless water and a middle layer;
(6) remove the upper strata water, with the DNA in ethanol sedimentation middle layer and the organic phase, the TRIzol of every use 1ml adds 0.3ml dehydrated alcohol mixing, and room temperature was placed 3 minutes, centrifugal 5 minutes of 2-8 ℃ of centrifugal force that is no more than 2000 * g;
(7) remove supernatant, with containing 10% alcoholic acid 0.1mol/L Trisodium Citrate washing DNA precipitation.Every TRIzol with 1ml adds the 0.1mol/L Trisodium Citrate (containing 10% ethanol) of 1ml, and room temperature was placed 30 minutes, and centrifugal 5 minutes of the centrifugal force of 2-8 ℃ of 2000 * g is abandoned supernatant, repeats once;
(8) wash DNA precipitation again with 75% ethanol, whenever add 75% ethanol of 1.5ml-2ml with the TRIzol of 1ml, room temperature was placed under 10-20 minute (putting upside down mixing frequently) 2-8 ℃ condition 2000 * g centrifugal 5 minutes, abandoned supernatant liquor;
(9) room temperature is placed and was dried DNA in 5-15 minute, with 8mmol/L dissolution of sodium hydroxide DNA.From 10
7Separated DNA is dissolved in the 8mmol/L sodium hydroxide of 300--600 μ l in the individual cell, and the concentration of DNA is generally 0.2-0.3 μ g/ μ l.Available Tris-EDTA solution and HEPES regulate about pH to 7.5 behind the dissolving DNA, obtain that the sample peripheral blood mononuclear cell extracts DNA.
Hepatitis gravis genes involved GSTP1 promoter methylation status detection:
1, make the non-cytosine(Cyt) that methylates be converted into the reagent modifying DNA of uridylic
(1) the sample peripheral blood mononuclear cell is extracted the DNA of 2 μ g, placed the EP pipe of 1.5ml to use distilled water to be diluted to 50 μ l;
(2) add the freshly prepared 3mol/L sodium hydroxide of 5.5 μ l, 42 ℃ of water-baths 30 minutes;
(3) '-biphenyl diphenol that adds 30 μ l concentration 10mmol/L is to the above-mentioned water-bath in the mixed solution;
(4) preparation 3.6mol/L sodium bisulfite, method is as follows: the 1.88g sodium bisulfite uses the distilled water dilution, and with 3mol/L sodium hydroxide volumetric soiutions to pH 5.0, final volume is 5ml, adds 520 μ l to the above-mentioned water-bath in the solution;
(5) EP pipe is outer wraps up in aluminium-foil paper, and lucifuge is softly put upside down mixing solution, adds 200 μ l paraffin oils, prevents moisture evaporation, restriction oxidation, 50 ℃ of lucifuge water-baths 16 hours;
(6) spectrophotometer method is surveyed DNA concentration and purity, is stored in-20 ℃.
2, methylation-specific polymerase chain reaction (PCR) detects each gene promoter methylation state
Right with methylation-specific or non-methylation-specific primer respectively, the DNA that handled is carried out methylation-specific polymerase chain reaction (PCR), wherein do not contain any DNA substrate composition in the negative control, positive control is that DNA substrate in the system is replaced with positive control dna (commercialization DNA positive control), and concentration is 10-20ng/ μ l.Reaction system such as following table:
The methylation status of PTEN promoter reaction system
Wherein, upstream primer and downstream primer refer to: methylate (M) specificity and non-methylating (U) Auele Specific Primer of promotor are right, that is: foregoing M-upstream primer, M-downstream primer; U-upstream primer, U-downstream primer.
Reaction conditions is as follows: be heated to 95 ℃, be incubated 5 minutes, totally 1 circulation; Be heated to 95 ℃, be incubated 30 seconds, under 55-60 ℃ temperature, annealed 45 seconds, 72 ℃ 30 seconds, 40 circulations; 72 ℃ of 1 circulations in 10 minutes; The amplified production length that back, methylation-specific polymerase chain reaction (PCR) is amplified is 90-100bp, and the 1%-1.5% agarose electrophoresis detects amplified production, obtains detected result.
Claims (8)
1. the test kit that detects of hepatitis gravis related gene promoter methylation state, it is characterized in that: be provided with in the box and make the non-cytosine(Cyt) that methylates be converted into the reagent of uridylic, 2 * Taq PCR MasterMix, and right at the primer of the promoter methylation specificity of human glutathione sulfurtransferase P1 gene and non-methylation-specific.
2. the test kit that hepatitis gravis related gene promoter methylation state according to claim 1 detects is characterized in that: also be provided with the single nuclear DNA extraction of peripheral blood reagent in the box.
3. hepatitis gravis related gene promoter methylation state detection kit according to claim 1 is characterized in that: also be provided with positive control and/or negative control in the box.
4. the test kit that hepatitis gravis related gene promoter methylation state according to claim 1 detects is characterized in that: methylate (M) specificity and non-methylating (U) Auele Specific Primer of promotor to being selected from down to organize is in the box:
5. the test kit that hepatitis gravis related gene promoter methylation state according to claim 1 detects, it is characterized in that: the length of the promoter methylation Auele Specific Primer that is provided with in the described box is 15-40bp.
6. the test kit that hepatitis gravis related gene promoter methylation state according to claim 1 detects, it is characterized in that: the reagent that makes the non-cytosine(Cyt) that methylates be converted into uridylic that is provided with in the box is hydrosulphite, '-biphenyl diphenol and sodium hydroxide.
7. the test kit that hepatitis gravis related gene promoter methylation state according to claim 2 detects, it is characterized in that: the single nuclear DNA extraction of the peripheral blood reagent in the described box is: Trisodium Citrate, sodium hydroxide, phenol, different sulphur hydracid guanidine, trichloromethane, sarcosyl, ethanol, sodium-acetate, Tutofusin tris (Tris), ethylenediamine tetraacetic acid (EDTA) (EDTA) and N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd (HEPES).
8. the detection method of hepatitis gravis related gene promoter methylation state is characterized in that: the reagent that makes the non-cytosine(Cyt) that methylates be converted into uridylic is modified the peripheral blood mononuclear cell DNA that extracts; Methylate (M) specificity and non-methylating (U) Auele Specific Primer, 2 * Taq PCR MasterMix, the bi-distilled water of the peripheral blood mononuclear cell DNA after modifying and promotor are mixed, make it produce the methylation-specific polymerase chain reaction, the amplified production length that is amplified behind the methylation-specific polymerase chain reaction is 90-100bp, detect each gene promoter methylation state at last, draw detected result.
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| CN103805699A (en) * | 2014-01-25 | 2014-05-21 | 山东大学齐鲁医院 | Quantitative detection method and reagent kit of GSTP1 (Glutathione S-Transferase P1) methylation for predicting hepatic failure prognosis |
| CN106755466A (en) * | 2017-01-12 | 2017-05-31 | 宁夏医科大学 | A kind of its method for building up of specific DNA methylome and application |
| CN112011619A (en) * | 2020-09-17 | 2020-12-01 | 山东大学深圳研究院 | Application of SFRP2 as marker in early diagnosis of hepatitis B virus-related hepatocellular carcinoma and detection kit |
| CN112795643A (en) * | 2021-02-20 | 2021-05-14 | 山东大学 | Biomarker for early detection of heavy hepatitis B virus, detection kit and application thereof |
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| CN106755466A (en) * | 2017-01-12 | 2017-05-31 | 宁夏医科大学 | A kind of its method for building up of specific DNA methylome and application |
| CN112011619A (en) * | 2020-09-17 | 2020-12-01 | 山东大学深圳研究院 | Application of SFRP2 as marker in early diagnosis of hepatitis B virus-related hepatocellular carcinoma and detection kit |
| CN112795643A (en) * | 2021-02-20 | 2021-05-14 | 山东大学 | Biomarker for early detection of heavy hepatitis B virus, detection kit and application thereof |
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