CN101955998A

CN101955998A - Method for detecting necrosis degree of hepatic cells through level of plasma free DNA

Info

Publication number: CN101955998A
Application number: CN 201010233193
Authority: CN
Inventors: 赵英仁; 何英利; 阎志; 李倩; 崔美灵; 王科; 陈天艳; 刘红莉
Original assignee: 赵英仁
Priority date: 2010-07-20
Filing date: 2010-07-20
Publication date: 2011-01-26

Abstract

The invention relates to a method for detecting the necrosis degree of hepatic cells through the level of plasma free DNA, and belongs to the technical field of biology. In the method, through the separation and extraction method and quantification of the plasma free DNA, the necrosis degree of the hepatic cells is reflected by the level of the free DNA. The method comprises the following steps of: preparing a primer beta-globin serving as a standard substance; extracting the plasma DNA in a plasma specimen of hepatopath; performing a real-time fluorescent quantitative polymerase chain reaction on the plasma DNA extracted in the plasma specimen; and measuring and calculating the concentration of the plasma free DNA. The method provides a new diagnostic index for the diagnosis of severe hepatitis.

Description

A kind of plasma free dna level detects the hepatic necrosis degree methods

Technical field

The present invention relates to a kind of plasma free dna level and detect the hepatic necrosis degree methods, belong to biological technical field.

Background technology

Hepatopathy is common clinical disease, only Patients with Viral Hepatitis such as hepatitis B, third liver in the countries in the world number near 500,000,000, account for 1/12 of global population, China is the chronic hepatitis B hotspot, there are every year 350000 people to die from the hepatitis B relative disease approximately, as liver cirrhosis, liver cancer, hepatitis gravis etc., 1% hepatitis B patient generation hepatitis gravis.Hepatitis gravis is that body is under multiple virulence factor effect, one class syndromes of the liver failure of liver a large amount of caused by necrosis in a short time, its clinical manifestation characteristics are that the state of an illness is heavy, development is fast, complication is many, case fatality rate is high, is still a difficult problem that needs to be resolved hurrily in the hepatopathy field so far.

At present, detecting the most direct method of hepatocellular damage clinically is exactly the liver cell puncture, but because it has wound, and may cause certain risk, still can not be widely accepted, and reliable hepatic necrosis sign is not explored clear as yet fully in the body fluid, a lot of blood parameters can be indirect liver damage is estimated arranged, as transaminase, bilirubin, albumin, Pseudocholinesterase, thrombogen mobility or the like, but these lab index all exist some shortcomings separately, as bilirubin and coagulation indexes, all be dead to a great extent liver cell, the residue liver cell just changes in the time of can not be compensatory, and the transformation period is longer, reach 15-19 days as the albumin transformation period, can not in time react the hepar damnification state; Bilirubin is owing to bilirubin direct and the metabolic in vivo characteristics of unconjugated bilirubin, and clinically, obstructive jaundice and hepatocellular jaundice are difficult to differentiate sometimes; Also has only booster action in the detection of hepatic injury in hepatitis gravis such as the variation of transaminase, Pseudocholinesterase etc.Therefore, existing some disputes aspect the hepatitis gravis Case definition always, the chronic type b hepatitis gravis pathological diagnosis standard places 118 routine patients of scholar by Xi'an meeting revision in existing 2000 are arranged, wherein 17.8% do not meet the clinical diagnosis standard, wherein the minority case has presented liver cell bulk, inferior massive necrosis on histology, and PTA is still up to 60%; Total bilirubin has 11 examples not reach Case definition, but pathology has turned out to be hepatitis gravis.So, explore new hepatic necrosis mark, predict that the generation of hepatitis gravis and development seem very important timely and effectively.

At present in the research of circle nucleic acid, the research of circulation nucleosome concentration is generally acknowledged as a kind of vital role of predicting that progression of disease and prediction disease take place, it is as a kind of meta-bolites of body, the secretion of source and apoptosis and necrosis, hemocyte metabolism and lymphocyte and tumour cell, all have related with state, autoimmune disorder, infection, wound, transplanting, tumour, obstetrics' illness of body, though exist arguement on the certain methods at present in quantitative experiment, various quantitative techniques are very ripe.And, before experiment, prepare, use aspects such as serum or blood plasma to reach certain common recognition.And in aspect hepatitis research, existing abroad scholar's research the nucleosome of acute and chronic liver failure change and compared variation before and after the MARS, found that acute hepatic failure patient blood plasma nucleosome significantly raises.The domestic scholar of having is detecting hepatitis gravis patient's blood plasma nucleosome level, think that it is than in acute hepatitis, chronic hepatitis and the liver cirrhosis rising, and itself and ALT carried out dependency relatively, bibliographical information is not seen in otherwise research as yet, and the quantitative assay of nucleosome is feasible in the hepatitis gravis.

Summary of the invention

The purpose of this invention is to provide a kind of plasma free DNA separating and extracting method and quantitative, with dissociative DNA level reflection hepatic necrosis degree.

In order to reach purpose of the present invention, the present invention is achieved in that

The invention provides a kind of plasma free dna level and detect the hepatic necrosis degree methods, comprise primer β-globin standard substance preparation, the extraction of liver problem sufferer's plasma specimen plasma dna, the plasma dna that extracts in the plasma specimen is carried out real-time fluorescence quantitative polymerase chain reaction, the concentration measuring and calculating of plasma free DNA, it is characterized in that described β-globin standard substance are that primer is synthetic, the upstream and downstream primer is respectively:

Upstream primer globin-F:ACACAACTGTGTTCACTAGC

Downstream primer globin-R:CAACTTCATCCACGTTCACC.

Described primer β-preparation method is as follows for the globin standard substance: β-globin primer of reaction cumulative volume 25 μ l carries out pcr amplification, and amplification condition is 95 ℃ of 1min of pre-sex change, 95 ℃ of 30s then, 57 ℃ of 20s, 72 ℃ of 20s, totally 45 circulations; Gel reclaims purified pcr product; The super competent cell of preparation DH5a; Goal gene connects conversion and transforms the back sequence verification; Extract plasmid, and carry out quantitatively; Described β-globin recombinant plasmid length is 2802bp, and concentration is 2.58 * 10 ¹³Copies/ml, being institute behind 10 times of gradient dilutions must standard substance.

The extracting method of described liver problem sufferer's plasma sample plasma dna is as follows: get 300 μ l blood plasma, add lysate 900 μ l (0.1M Tri-HCL 90 μ l, 0.05M EDTA90 μ l, 1%SDS 450 μ l, ddH by 1: 3 volume ₂O 270 μ l), adding 6 μ l Proteinase Ks (Proteinase K 20mg/ml) is 100 μ g/ml to final concentration, mixing 10s on vibrator, and of short duration centrifugal back is in 56 ℃ of water-bath 2h; The water-bath mixing liquid is in 95 ℃ of water-bath 10min, sex change Proteinase K; Of short duration centrifugal, add the saturated phenol of equal-volume Tris: chloroform: primary isoamyl alcohol (volume ratio 25: 24: 1) mixed solution 1200 μ l, abundant mixing on the vibrator, about 2-3min; 12, the centrifugal 15min of 000rpm carefully draws supernatant, places new EP pipe (import EP pipe is used in suggestion); In supernatant liquor, add the dehydrated alcohol (about 3000 μ l) of its 2.5 times of volumes, put upside down mixing, in-20 ℃ of precipitations (more than the 12h) of spending the night; Normal temperature 12, the centrifugal 20min of 000rpm carefully removes supernatant; 70% ethanol, the 800 μ l that add 2 times of volumes, normal temperature 12, the centrifugal 10min of 000rpm; Carefully remove supernatant, in 56 ℃ of baking ovens, place 20min, be placed on the super clean bench 1h alcohol that thoroughly volatilizees; Add 40 μ l ddH ₂O ₂Dissolving DNA, slight concussion, of short duration centrifugal, place 4 ℃ of abundant dissolving DNAs of 1h; The DNA sample is in-20 ℃ of preservations.

The plasma dna that extracts in the described plasma specimen carries out real-time fluorescence quantitative polymerase chain reaction and may further comprise the steps: β-globin primer of reaction cumulative volume 20 μ l carries out pcr amplification, amplification condition is 95 ℃ of 1min of pre-sex change, 95 ℃ of 30s then, 57 ℃ of 20s, 72 ℃ of 32s, totally 45 circulations.

The concentration measuring and calculating of described plasma free DNA comprises that with concentration known be 2.58 * 10 ¹³The β of copies/ml-globin recombinant plasmid makes its final concentration be respectively 2.58 * 10 as 10 times of proportional diluted of standard substance series ⁵Copies/ μ l, 2.58 * 10 ⁴Copies/ μ l, 2.58 * 10 ³Copies/ μ l, 2.58 * 10 ²Copies/ μ l sets up typical curve and establishes linear relationship between the logarithm of Ct value and standard substance starting template; The amount of circulation nucleosome is calculated as follows in the blood plasma: C=Q * (V _DNA/ V _PCR) * (1/V _Ext), wherein C is a testing sample plasma circulation nucleosome concentration (GE/ml), Q is the DNA amount (copies) that instrumental analysis software is calculated, V _DNABe the DNA total amount (40 μ l) that extracting goes out, V _PCRFor joining DNA amount (9.5 μ l) in the reaction system, V _ExtBe the total plasma volume (300 μ l) that is used for extracting and purify DNA.

The present invention carries out real-time fluorescence quantitative polymerase chain reaction by primer β-globin standard substance in the plasma specimen plasma dna that the liver problem sufferer is extracted, the concentration of measuring and calculating plasma free DNA is for the diagnosis of hepatitis gravis provides new diagnosis index.

Embodiment

Fig. 1 is that 2% agarose gel electrophoresis is identified PCR figure as a result, and 1,2,3,4,5 is the pcr amplification product of complete genome DNA among the figure, and Maker is DL500 marker.

Fig. 2 is that 1% agarose gel electrophoresis is identified plasmid figure as a result, and Marker is DL2000 marker among the figure, and 1,2 is β-globin recombinant plasmid.

Fig. 3 is the sample amplification curve diagram.

Fig. 4 is the sample canonical plotting, and slope is among the figure :-3.664065, and intercept is: 44.995626, R ²Be 0.996965.

Fig. 5 is plasma free DNA distribution plan between each hepatopathy group, and a among the figure: dissociative DNA mainly is distributed in 15-20 (log in the hepatitis patient blood plasma ₂); B:log ₂(cell free DNA)＜15 mainly are distributed in the DLC group; C:log ₂(cell free DNA)＞20 mainly are distributed in LF, AH and HCC group and DLC group.

Fig. 6 is plasma free DNA mean value distribution plan in each group.

Fig. 7 is dissociative DNA level and each test item correlation figure, plasma free DNA log among the figure ₂＞20:a. dissociative DNA and MELD scoring: R ²=0.72, P value: 0.002; B. there are dependency in dissociative DNA and serum cholesterol level: R ²=0.575, P value: 0.011; C. there are dependency in dissociative DNA and serum direct bilirubin concentration: R ²=0.591, P value: 0.009; D. there are dependency in dissociative DNA and serum N a ion concentration: R ²=0.707, P value: 0.002; E. there are dependency in dissociative DNA and leucocyte level: R ²=0.588, P value: 0.010; F. there are dependency in dissociative DNA and monocyte level: R ²=0.934, P value: 0.000.

When Fig. 8 was ALT＞500U/L, there were dependency: R in a. plasma free DNA and MELD scoring ²=0.615, the P value: there are dependency in 0.000 b. plasma free DNA and blood plasma cholesterol level: R ²=0.353, P value: 0.001.

Fig. 9 is a creatinine during greater than 3mg/dl, and there are dependency in plasma free DNA and serum creatinine, R ²=0.756, p value: 0.005.

Below in conjunction with accompanying drawing the present invention is described in further detail, but not as a limitation of the invention.

Embodiment one, the preparation of primer β-globin standard substance.

1, experiment main agents and equipment

1) chemical reagent commonly used such as sodium hydroxide, potassium hydroxide, sodium-chlor, Repone K, dehydrated alcohol, glacial acetic acid, acetate ammonia, sodium acetate, primary isoamyl alcohol are the production of Xi'an chemical reagent factory, and purity grade is an analytical pure;

2) EB (the product packing of Sigma company is purchased in Huamei Bio-Engrg Co.);

3) loading buffer (bio-engineering corporation in Xi'an Zhou Dingguo is purchased in the packing of precious biological TaKaRa product);

4) agar powder (GEGE TECH Shanghai company limited purchases the bio-engineering corporation in Xi'an Zhou Dingguo);

5) agarose (Amresco company original product is purchased the bio-engineering corporation in Xi'an Zhou Dingguo);

6) nucleic acid molecular weight object of reference 500bp DNA maker, 2000bp DNA maker (the gloomy bio-engineering corporation in Wal, Xi'an);

7) the UNIQ-10 pillar plasmid of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd a small amount of extraction agent box;

8) measure plasmid extraction kit among the Promega;

9) Shanghai is given birth to worker's biotechnology UNIQ-5 of company limited pillar DNA glue and is reclaimed test kit;

10) β-globin primer is synthetic: upstream primer globin-F:ACACAACTGTGTTCACTAGC, and downstream primer globin-R:CAACTTCATCCACGTTCACC gives birth to the synthetic and mark of worker Bioisystech Co., Ltd by Shanghai;

11) PCR reaction reagent: contain the Taq enzyme, reaction buffer, magnesium ion, dNTP (precious biological TaKaRa product is purchased in precious Tyke bio-engineering corporation);

Mentioned reagent stores according to explanation.

Main apparatus model or specification production company

The adjustable micropipet 1000 μ l of single track 200 μ l 10 μ l Germany Eppendorf company

Disposable centrifuge tube 0.5ml 1.5ml U.S. company BD

Constant incubator PXY-DHS 400-BS Shanghai Bo Tai company limited

Constant temperature oscillator CHA-S state China business corporation

Automatic clinical chemistry analyzer AU5400 Japan OLYMPUS company

Pressure steam sterilizer LDZX-50KB Shenan Medical Appliances Factory, Shanghai

Program control pcr amplification instrument 2104142 types of thermograde Germany Biometra company

Ultraviolet gel imaging instrument system T2A type Italy Bio-RAD company

Refrigerated centrifuge Sigma, 3-18K Germany SIGMA company

2, pcr amplification

With β-globin primer people's complete genome DNA is increased, reaction cumulative volume 25 μ l, undertaken by following system:

Reaction solution component dosage (μ l) final concentration

PCR reagent mixture 2x 12.5 1x

Upstream primer F10umol 0.5 0.125umol

Downstream primer R10umol 0.5 0.125umol

Testing sample (or standard substance) 2.0

Aseptic deionized water 9.5

Amplification condition: pre-95 ℃ of 1min of sex change, 95 ℃ of 30s then, 57 ℃ of 20s, 72 ℃ of 20s, totally 45 circulations.

2% agarose gel electrophoresis is identified PCR result, occurs clear band in about 110bp, and promptly the purpose band as Fig. 1, is established β-globin gene amplification condition.

3, gel reclaims

It is as follows that glue purification method (giving birth to worker's biotechnology UNIQ-5 of company limited pillar DNA glue by Shanghai reclaims the test kit explanation and carry out) step is cut in employing:

1) getting fresh, a sterilization 1.5ml centrifuge tube weighs.

2) in the fresh tin foil of ultraviolet transilluminator middle berth lastblock, gel (seeing Fig. 3-1) is placed on (to improve the purity that DNA reclaims as far as possible) on the tin foil, opens long-wave ultra violet lamp and fast purpose fragment (about 110bp) is scaled off (removing unnecessary agarose gel as far as possible) from sepharose with clean scalpel.The blob of viscose that scales off is put into the centrifuge tube of 1.5ml, with micro-scales/electronic balance weighing and perform record.

3) amount that adds 400 μ l Binding buffer by every 100mg sepharose is calculated, and adds the Binding buffer of respective volume, and mixing is placed on 10min in the 50-60 ℃ of water-bath, makes glue thoroughly melt (when adding hot melt adhesive, every 2min mixing once).

4) sol solution that melts is transferred to cover and be put in the UNIQ-10 post of 2ml collection tube, room temperature is placed 2min, with desk centrifuge high speed (6000rpm) centrifugal 1min.

5) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put back in the collection tube, add 500 μ l Wash solution, centrifugal 8000rpm 1min.

6) repeating step 5 once.

7) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put back in the collection tube, 12,000rpm 1min.

8) the UNIQ-10 post is put into the 1.5ml centrifuge tube of a fresh cleaning, added 30 μ l Elution Buffer in the UNIQ-10 post in post film central authorities, room temperature is placed 2min.(improving eluting temperature) to 55-80 ℃ of elution efficiency that helps improving DNA

9) 12, the centrifugal 1min of 000rpm room temperature, the liquid in the centrifuge tube is the dna fragmentation of recovery, be stored in-20 ℃ standby.

10) get 3 μ l PCR purified products and add an amount of sample loading buffer mixing, 1.5% agarose electrophoresis is checked the rate of recovery, single band lighter about 110bp occurs, and DNA reclaims the purifying success.

4, prepare super competent cell

1) prepare before the preparation:

(1) preparation SOB substratum:

Prepare every 1L substratum, in the 950ml deionized water, add: tryptone 20g, yeast extract 5g, NaCl 0.5g.Magnetic stirring apparatus dissolves solute fully.Add 10ml 250 mmol/L KCl solution.With 5mol/L NaOH adjust pH to 7.0.Be settled to 1L with deionized water.4 ℃ of preservations of steam sterilizing 20min under the high pressure.Note this solution before use, add the 2mol/L MgCl of 5ml sterilization ₂

(2) preparation SOC substratum:

The glucose solution of preparation 1mol/L: the glucose of 18g is dissolved in the deionized water of 90ml, is settled to 100ml, with 0.22 μ m membrane filtration degerming.The 1mol/L glucose solution 2ml that in 100ml SOB substratum, adds degerming, uniform mixing.4 ℃ of preservations.

(3) LB substratum preparation:

Prepare every 1L substratum, should in the 950ml deionized water, add: tryptone 10g, yeast extract 5g, NaCl 10g.Magnetic stirring apparatus dissolves solute fully.Transfer pH to 7.0 with 5mol/LNaOH.Be settled to 1L with deionized water, steam sterilizing 20min under the high pressure.

(a). preparation LB-AMP resistance substratum:

Preparation LB liquid nutrient medium 100ml, step is the same.

Add 1.5% agar powder in the LB liquid nutrient medium, autoclaving sterilization 20min.

Take out substratum from pressure kettle, turn makes dissolved agar powder uniform distribution gently.

The LB solid medium that contains penbritin Amp:, add penbritin storage liquid (original content is 50mg/ml) 120 μ l with about the LB liquid nutrient medium autoclaving postcooling to 60 for preparing ℃.

(b) .LB/AMP/X-Gal/IPTG culture dish preparation:

The same preparation LB-AMP resistance substratum is got the 100mm sterile petri dish, pours about 35ml in each into, solidifies.Drip the X-gal 50 μ l of 20mg/ml and the IPTG 100 μ l of 24mg/ml, evenly coat on the plate culture medium.

(4) preparation of Inoue conversion fluid:

Preparation 1L Inoue conversion fluid:, take by weighing the MnCl of 10.88g respectively ₂2H ₂The CaCl of O, 1.665g ₂, 18.65g KCl add in the deionized water of 800ml, the dissolving back adds the PIPES of 20ml fully, is settled to 1L with deionized water then, 0.45 μ m membrane filtration degerming ,-20 ℃ of preservations are standby after the packing.

2) use the Inoue method to prepare super competent cell

Step is as follows:

(1) (diameter 2～3mm) is seeded in the 25ml LB nutrient solution in the 250ml Erlenmeyer flask, cultivates 8h in 37 ℃ of shaking table 250rpm at the single bacterium colony of the DH5 of 37 ℃ of incubated overnight α intestinal bacteria for one of picking;

(2) behind the 8h, above-mentioned initial incubation thing is inoculated in three 1L Erlenmeyer flasks that fill 250ml LB nutrient solution, first adds 10ml, and second adds 4ml, and the 3rd adds 2ml, in the shaking table incubated overnight of 18 ℃ of 200rpm rotating speeds;

(3) morning next day, measure the OD of three flask culture things ₆₀₀Value, every 45min measures once, as one bottle culture OD ₆₀₀=0.55 o'clock, culturing bottle is placed ice-water bath 10min, the wave and culture bottle is fully lowered the temperature culture, discards two flask culture things in addition;

(4) collect thalline in 4 ℃ with the centrifugal 10min of 2500g;

(5) remove nutrient solution, remaining liq in the centrifuge tube is dried as far as possible, blot with thieving paper;

(6) Inoue of adding 40ml precooling transforms damping fluid re-suspended cell precipitation, rotates mixing gently, and not with vibrator or pressure-vaccum mixing, 4 ℃ of centrifugal 10min of 2500g collect thalline, supernatant discarded;

(7) remaining liq in the centrifuge tube is dried as far as possible, the Inoue that adds the 10ml precooling transforms the resuspended gently precipitation of damping fluid;

(8) add 750 μ l DMSO.Mixing is placed 10min in the ice-water bath gently;

(9) rapidly suspension is installed in the aseptic 1.5ml centrifuge tube of refrigerative with every pipe 100 μ l branch, seal the tight mouth of pipe, quick freezing competent cell (more than half an hour) in the liquid nitrogen that submerges.Be stored in-80 ℃ standby.

5, the connection of goal gene and conversion

Connect: set up 10 μ l ligation systems (according to TaKaRa pMD18T carrier specification sheets)

The PMD18T carrier: 1 μ l,

β-globin DNA (front is through purifying): 1 μ l,

ddH2O：3μl

The solution I that adds 5 μ l reflects 30min behind the mixing gently in 16 ℃ of water baths; Negative control and positive control are set simultaneously.

Transform: 1) get 100 μ l (1 pipe) DH5 α competent cell and be held in the palm of the hand and melt postposition 10min on ice rapidly from-80 ℃;

2) the rifle head of the ligation thing (10 μ l) behind the reaction 30min with precooling moved in the competent cell suspension, place 30min gently behind the mixing on ice;

3) 42 ℃ of water-bath heat shock 90s;

4) forward ice bath 2min fast to; Add 900 μ l SOC substratum then;

5) 37 ℃, 225rpm shaking culture 60min;

6) coating: the conversion fluid of getting 50 μ l drops on the dull and stereotyped LB/AMP/X-Gal/IPTG culture dish of 37 ℃ of preheatings, with aseptic spreader it evenly is coated with out, and places 37 ℃ of incubators to cultivate, and after liquid to be transformed absorbs fully, is inverted culture dish, continues to cultivate 12-16h;

7) culture plate is taken out, place 5h for 4 ℃, make blue fully colour developing.The purpose bacterium colony occurs;

8) shake bacterium: get 2 50ml centrifuge tubes, each adds 5ml LB/AMP substratum.With 2 white clones of aseptic toothpick difference picking bacterium colonies, be inoculated in the 50ml centrifuge tube, be labeled as 1,2;

9) 37 ℃, 225rpm shaking culture.Behind the 8h, survey the OD value: OD1:0.885, OD2:1.320 (get 2ml bacterium liquid and be used to extract recombinant plasmid, use 30% glycerine to preserve bacterium liquid in addition, use) in order to follow-up confirmatory experiment.

6, extract recombinant plasmid in a small amount

1) with the centrifugal 1min of cultured 2ml bacterium liquid 12000rpm, removes supernatant;

2) add 250 μ l Solution I, with rifle head or the vibrator bacterium that fully suspends;

3) add 250 μ l Solution II, turn upside down immediately 5～10 times, make the bacterium cracking, room temperature is placed 2min;

4) add 350 μ l Solution III, turn upside down immediately 5～10 times, make it abundant neutralization, room temperature is placed 2min;

5) 12,000rpm is centrifugal, 5min;

6) supernatant in the step 5 is transferred to overlaps in the UNIQ-10 post that is put in the 2.0ml collection tube, 12, the centrifugal 1min of 000rpm room temperature;

7) abandon waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, draw 500 μ l Wash Solution to the UNIQ-10 post, 12, the centrifugal 1min of 000rpm room temperature;

8) repeating step 7);

9) abandon waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, 12, the centrifugal 2min of 000rpm room temperature is with thorough removal Wash Solution;

10) the UNIQ-10 post is put into new clean 1.5ml centrifuge tube, added 50 μ l DW (or Elution Buffer), after room temperature is placed 2min, 10, the centrifugal 1min of 000rpm room temperature.Solution in the centrifuge tube is extractive plasmid DNA.

1% agarose gel electrophoresis identifies whether propose plasmid, the homogeneous band occurs, as Fig. 2.

7, identify institute's upgrading grain (PCR method preliminary identification)

Use β-globin primer to increase: system 25 μ l, specifically composed as follows:

Reaction solution component dosage (μ l) final concentration

PCR reagent mixture (2x) 12.5 1x

Upstream primer 10umol 0.5 0.2umol

Downstream primer 10umol 0.5 0.2umol

Plasmid DNA 2

Aseptic deionized water 9.5

The PCR instrument is provided with condition: pre-sex change: 95 ℃ of 1min; 95 ℃ of 30s of sex change, the 57 ℃ of 20s that anneal extend 72 ℃ of 20s, totally 45 circulations.

1% agarose gel electrophoresis is identified PCR result: size occurring is 110bp purpose band.

(prove that tentatively goal gene transforms the DH5a success, for further checking, send company's order-checking after the bacterium liquid of preserving increased bacterium again) sequencing result shows through comparison: β-globin has been connected on the T carrier and has transformed successfully.

8, middle amount is extracted recombinant plasmid

Operating process is as follows:

(1) get the LB/AMP substratum that 50 μ L recombinant plasmid transformed bacterium liquid are inoculated into 5mL, 6-8h is cultivated in 37 ℃ of concussions.Get 2ml bacterium liquid then and add incubated overnight in the 100ml SOB/AMP substratum;

(2) next day, survey OD ₆₀₀=1.983.The centrifugal 10min of 5000g under the room temperature collects thalline, and inhales as much as possible and remove supernatant;

(3) add the resuspended liquid of 3mL cell (cell resuspension), the eddy current concussion guarantees that bacterial precipitation suspends again;

(4) add 3mL cell pyrolysis liquid (cell lysis solution), put upside down 5-10 time lightly, leave standstill 3min then and clarify to the solution thickness to mix;

(5) add 5mL neutralizer (Neutralization), softly put upside down immediately 5 times, leave standstill 2min;

(6) the centrifugal post of blueness is put into the 50ml centrifuge tube, the mixed solution of the 4th step gained is moved in the pillar, at room temperature centrifugal 5 minutes of 1500g (if still have liquid, but recentrifuge);

(7) white adsorption column is put into clean 50ml centrifuge tube, the liquid that the 5th step was collected is transferred in the white adsorption column the centrifugal 30min of 3000rmp;

(8) add 5mL endotoxin removal liquid in white adsorption column, the centrifugal 3min of 3000rpm outwells the waste liquid in the collection tube under the room temperature, pillar is relay get back in the collection tube;

(9) add the 20ml purifying in white adsorption column and wash the centrifugal 5min of post liquid 3000rpm, abandon waste liquid, pillar is relay the recovery collector, the centrifugal 5min of 3000rpm is to remove the ethanol in the washings;

(10) white adsorption column is moved on to a new 50ml centrifuge tube, under the room temperature 5, the 000g centrifugal 10min that uncaps is with the residual ethanol of thorough removal;

(11) white adsorption column is gone in the new 50ml centrifuge tube, add the ddH of 0.6ml to the center of DNA post film ₂O (pH is between 7.0-8.5), room temperature is placed 1min, and the centrifugal 5min of 2000g is with the wash-out plasmid DNA;

(12) improve yield, the liquid of available the 10th step gained wash-out is more once collected in the centrifuge tube after centrifugal, i.e. the plasmid DNA of being extracted.

9, quantitative

Survey the OD value, adopt ultraviolet absorption method, measure OD ₂₆₀Absorption value.The concentration that computer is directly measured β-globin recombinant plasmid is 79.4ng/ μ l; Ng/ μ l is scaled copies/ml:copies/ml=(6.02 * 10 ²³) * (ng/ μ l * 10 ^-6)/(DNA length * 660);

β-globin recombinant plasmid length is that β-globin and T carrier base are counted sum, is 2802bp, and finally conversing concentration is 2.58 * 10 ¹³Copies/ml, being institute behind 10 times of gradient dilutions must standard substance.

Embodiment two, the extraction of blood plasma nucleosome and quantitative

1, experiment main raw

Fluorescent PCR reaction mixture SYBR

Premix Ex Taq ^TM, contain SYBR

Green I, reaction buffer, dNTP, TaKaRa Ex Taq TM HS, magnesium ion (precious biological TaKaRa product is purchased in precious Tyke bio-engineering corporation), reagent stores according to explanation.

2, research object

The acute and chronic hepatic injury patient that hepatitis B, hepatitis C and the drug induced hepatitis that No.1 Hospital Attached to Medical College, Xi'an Communication Univ Infectious Disease hospitalized between year June in March, 2009 to 2010 causes, liver cancer patient be totally 204 examples.Clinical diagnosis meets the viral hepatitis of Xi'an meeting revision in 2000 and prevents and treats scheme.Age 5-79 year, average 45.14 ± 14.547 years old; The male sex's 146 examples wherein, age 5-79 year, women's 58 examples, age 16-73 year.Systemic autoimmune diseases such as equal removal system lupus erythematosus of all cases and rheumatoid arthritis.

3, the collection of sample

Do not gather patient and volunteer's sample before the feed after rising morning, 3ml places the EDTA anticoagulant tube in the ulnar vein venous blood samples, and sample is for further processing in 2h.

4, sample disposal and preservation

1) the sample trim is placed on the low temperature ultracentrifuge in 4 ℃ of centrifugal 10min of following 1600g;

2) take out test tube lightly, draw upper plasma, place the micro-centrifuge tube after the handling of 1.5ml through autoclave sterilization with 1000 μ l adjustable pipettes (using disposable autoclave sterilization rifle head);

3) behind the trim micro-centrifuge tube, place the low temperature ultracentrifuge to descend 1, the centrifugal 10min of 6000g in 4 ℃;

4) take out centrifuge tube lightly, draw supernatant liquor, be loaded in the centrifuge tube after the handling of 1.5ml, and indicate numbering according to the sample registration through autoclave sterilization with 1000 μ l adjustable pipettes (using disposable autoclave sterilization rifle head);

5) it is standby to preserve above-mentioned plasma specimens in-80 ℃, more than in the standby sample preservation process, strictly note preservation condition, prevent multigelation.

5, detect clinical indices

Collection of specimens is taken a blood sample simultaneously and carried out following project detection: routine blood test mensuration, ten mensuration of liver function, four mensuration of blood coagulation, renal function are measured, and all patient serum sample detect and finish in my clinical laboratory of institute.

6, plasma dna extracts

The plasma free DNA extraction adopts classical phenol, chloroform, primary isoamyl alcohol (volume ratio 25: 24: 1) method for extracting.

1) gets 300 μ l blood plasma, add lysate 900 μ l (0.1MTri-HCL 90 μ l, 0.05M EDTA 90 μ l, 1% SDS, 450 μ l, ddH by 1: 3 volume ₂O 270 μ l), adding 6 μ l Proteinase Ks (Proteinase K 20mg/ml) is 100 μ g/ml to final concentration, mixing 10s on vibrator, and of short duration centrifugal back is in 56 ℃ of water-bath 2h;

2) the first step mixing liquid is in 95 ℃ of water-bath 10min, sex change Proteinase K; Of short duration centrifugal, add the saturated phenol of equal-volume Tris: chloroform: primary isoamyl alcohol (volume ratio 25: 24: 1) mixed solution 1200 μ l, abundant mixing on the vibrator, about 2-3min;

3) 12, the centrifugal 15min of 000rpm carefully draws supernatant, places new EP pipe (import EP pipe is used in suggestion);

4) dehydrated alcohol (about 3000 μ l) of its 2.5 times of volumes of adding in supernatant liquor is put upside down mixing, in-20 ℃ of precipitations (more than the 12h) of spending the night;

5) normal temperature 12, and the centrifugal 20min of 000rpm carefully removes supernatant;

6) 70% ethanol, the 800 μ l of 2 times of volumes of adding, normal temperature 12, the centrifugal 10min of 000rpm;

7) carefully remove supernatant, in 56 ℃ of baking ovens, place 20min, be placed on the super clean bench 1h alcohol that thoroughly volatilizees;

8) add 60 μ l ddH ₂The O dissolving DNA, slight concussion, of short duration centrifugal, place 4 ℃ of abundant dissolving DNAs of 1h;

9) the DNA sample is in-20 ℃ of preservations.

7, the plasma dna to all extractions carries out real-time fluorescence quantitative polymerase chain reaction

1) primer, i.e. β-globin primer.Upstream primer globin-F:ACACAACTGTGTTCACTAGC, downstream primer globin-R:CAACTTCATCCACGTTCACC.Give birth to the synthetic and mark of worker Bioisystech Co., Ltd by Shanghai.

2) reaction system: cumulative volume 20 μ l, quantitative criterion product, the positive, negative control and sample to be tested are all undertaken by following system.

3) amplification condition: pre-sex change, 95 ℃ of 1min, 95 ℃ of 30s then, 57 ℃ of 20s, 72 ℃ of 32s, totally 45 circulations (since this experiment use be ABI 7500, the instrument requirement PCR extension time is that the fluorescence acquisition time must not be lower than 32s).

4) typical curve: with concentration known is 2.58 * 10 ¹³The β of copies/ml-globin recombinant plasmid makes its final concentration be respectively 2.58 * 10 as 10 times of proportional diluted of standard substance series ⁵Copies/ μ l, 2.58 * 10 ⁴Copies/ μ l, 2.58 * 10 ³Copies/ μ l, 2.58 * 10 ²Copies/ μ l sets up typical curve.

5) data processing: the each analysis comprises a typical curve and two no template negative controls.After reaction finishes, analyze automatically and calculation result by computer.Institute responds and adopts ABI7500 fluorescent quantitative PCR instrument and SDS 14.0 analysis software, the drawing standard curve, linear relationship between the logarithm of establishment Ct value and standard substance starting template according to the amplification situation of sample to be tested, is calculated the initial amount of sample to be tested in the reaction system.

The amount of circulation nucleosome is calculated as follows in the blood plasma:

C=Q * (V _DNA/ V _PCR) * (1/V _Ext), wherein C is a testing sample plasma circulation nucleosome concentration (GE/ml), Q is the DNA amount (copies) that instrumental analysis software is calculated, V _DNABe the DNA total amount (40 μ l) that extracting goes out, V _PCRFor joining DNA amount (9.5 μ l) in the reaction system, V _ExtBe the total plasma volume (300 μ l) that is used for extracting and purify DNA.

Embodiment three, clinical experiment

1, research object

The hepatitis B virus that No.1 Hospital Attached to Medical College, Xi'an Communication Univ hospital contagious department was hospitalized between year June in March, 2009 to 2010, hepatitis C virus and drug-induced acute, chronic hepatic injury patient and liver cancer patient be totally 204 examples, gets rid of various immunological diseases.Clinical diagnosis chronic hepatitis and compensatory cirrhosis do not have absolute boundary on pathology, part clinical diagnosis chronic hepatitis may present early stage liver cirrhosis, and the two is all based on inflammatory reaction; Hepatitis gravis, acute hepatitis and liver cancer all present liver cell or cancer cellular necrosis or apoptosis, based on necrocytosis.The patient is divided into three groups: first group is CH and LC group, comprises the compensatory phase patient of chronic hepatitis and liver cirrhosis; Second group is the DLC group, comprises the cirrhosis patients in decompensation patient; The 3rd group is LF, and AH and HCC group comprises heavy type (acute, subacute and chronic heavy type) hepatitis, primary hepatocarcinoma and acute icterohepatitis.(CH, Chronic Hepatitis chronic hepatitis; LC, the compensatory phase of Liver cirrhosis liver cirrhosis; DLC, Decompensated liver cirrhosis cirrhosis patients in decompensation; AH, Acute Hepatitis acute hepatitis; HCC, hepatocellular carcinoma primary hepatocarcinoma; LF, Liver Failure hepatitis gravis).

2, real-time quantitative PCR result:

Amplification curve is seen Fig. 3.

Typical curve is seen Fig. 4.

3, patient's physical data and laboratory examination result

Patient's physical data and laboratory examination result are as shown in the table:

There is notable difference in plasma free DNA concentration, P=0.002 between each group; LF, there is notable difference in plasma free DNA concentration between AH and HCC group and CH and LC group and DLC group, P=0.000,0.049, statistical significance is all arranged, as shown in the table.

4. plasma free DNA distributes between each hepatopathy group

Plasma free DNA distributes between each hepatopathy group and sees Fig. 5, and plasma free DNA mean value in each group distributes and sees Fig. 6.

5. dissociative DNA level and each test item dependency

Its each dependency is seen Fig. 7, Fig. 8 and Fig. 9.

The present invention uses real-time fluorescence quantitative PCR and detects 204 routine liver problem sufferer's plasma free dna levels, comprising five kinds of acute hepatitis, chronic hepatitis, compensatory phase and decompensated liver cirrhosis, hepatitis gravis, liver cancer in various degree with the type hepatopathy, 204 routine patients were analyzed by sex and age, find that sex and age are little to the influence of blood plasma nucleosome; The liver problem sufferer is found by different clinical fractional analysis, LF, AH and HCC group dna level is in the highest, be followed successively by CH and LC group and DLC group (Fig. 6), LF, there is significant difference in AH and HCC group with CH and LC group and DLC group dissociative DNA level, and the P value is respectively 0.049,0.000 (chart 2).A large amount of hepatic necrosis under the acute liver damage state, the inflammatory reaction that occurs together, a large amount of DNA are released into blood; In liver cancer patient, have and studies confirm that malignant tumor patient plasma free DNA level is higher than the normal people; A large amount of fibers and pseudolobuli form in the decompensated cirrhosis, liver, present the symptom that shows with the forfeiture of liver compensation, and liver inner cell degree of necrosis is lighter relatively; All still show as the master with inflammation in chronic hepatitis patient liver and the compensatory cirrhosis liver, hepatic necrosis or apoptosis are released into blood with respect to the compensatory phase height of mistake, but the still too late LF of hepatocellular injury degree, AH and HCC group.

Hepatitis gravis is a large amount of liver cell necrosis on pathology, discharge a large amount of DNA and go into blood, the plasma free dna level is higher than other chronic inflammatory diseases damages in theory, but because part patient hepatic necrosis degree is heavier, liver volume is seriously dwindled, the most of cell of liver is downright bad, and part patient has passed through regular treatment, and the state of an illness is steady; And the plasma free DNA transformation period is 16.3 minutes, makes that collect sample becomes an important factor in order opportunity, and therefore the sample that detects does not present the hepatitis gravis dna level and all is higher than other hepatitis samples.The plasma free dna level is boundary (Fig. 5) with 20, be higher than 20 10 routine patients only, comprise hepatitis gravis, liver cancer and decompensated liver cirrhosis, and hepatitis gravis and liver cancer account for 50%, plasma free DNA has extensive studies at tumor area, prove that all plasma free DNA is higher than normal population, and a large amount of downright bad released dnas of hepatitis gravis liver cell is gone into blood; High level DNA appears at decompensated liver cirrhosis patient blood plasma, and may point out the state of an illness to increase the weight of or transform to hepatitis gravis may.

The MELD scoring can accurately be reacted the liver function state, MELD is a digital scope that changes from 6 (minor ailments) to 40 (serious diseases), its calculation formula is: R=3.8 * ln[bilirubin (mg/dl)]+11.2 * ln (INR)+9.6 * ln[creatinine (mg/dl)]+6.4 * (cause of disease: bile or alcohol 0, other are 1 years old).All there are not dependency in plasma free dna level and every inspection item, but log ₂(cell free DNA)＞20 o'clock, there are strong correlation in dna level and MELD scoring, and prompting is when DNA high level state, and plasma free DNA can accurately react liver function state, is an index comparatively accurately; During the liver cell major injury, cholesterol is synthetic the minimizing in liver, and the low more prompting prognosis of cholesterol is poor more, and there are negative correlation in dna level and cholesterol; One of the renal function of most of patient with liver cirrhosis is functional unusual, is the reaction to the disorder of systemic blood flow power, and retention of sodium and water is the not normal modal result of patient with liver cirrhosis renal function, and na concn is negative correlation in the indirect reaction liver function, dna level and body; There is dependency in white corpuscle also and in the body simultaneously, studies confirm that the interior inflammatory reaction of dissociative DNA level and body is proportionate.

Select the higher patient of transaminase, with 500U/L is boundary (Fig. 8), greater than 500U/L person, free dna level is marked with MELD and there is dependency on the statistics in cholesterol in the blood plasma, ALT mainly is present in the liver cell, and behind the hepatocellular injury, transaminase is released into blood, be the common counter that reacts hepatocyte function at present clinically, the high low energy of transaminase level is reacted the hepar damnification degree to a certain extent.When hepar damnification was serious, plasma free dna level and the MELD strong positive correlation of marking can accurately be estimated liver function state and hepatitis prognosis in conjunction with liver function test.

Hepatorenal syndrome occurs in the patient of the liver failure and the portal hypertension in chronic hepatopathy and later stage, shows as renal tubal dysfunction, the unusual and endogenous vasoactive system activity of tangible arterial circulation, and significantly vasoconstriction causes low glomerular filtration rate(GFR.Serum creatinine is as an index of evaluating liver function and severity extent, when greater than 3mg/dL (Fig. 9), there is strong correlation in plasma free DNA with it, when the renal function major injury, the dissociative DNA energy may be relevant through renal excretion with the part dissociative DNA as an index of reaction kidney merit.

Claims

1. a plasma free dna level detects the hepatic necrosis degree methods, comprise primer β-globin standard substance preparation, the extraction of liver problem sufferer's plasma specimen plasma dna, the plasma dna that extracts in the plasma specimen is carried out real-time fluorescence quantitative polymerase chain reaction, the concentration measuring and calculating of plasma free DNA, it is characterized in that described β-globin standard substance are that primer is synthetic, the upstream and downstream primer is respectively:

Upstream primer globin-F:ACACAACTGTGTTCACTAGC

Downstream primer globin-R:CAACTTCATCCACGTTCACC.

2. a kind of plasma free dna level according to claim 1 detects the hepatic necrosis degree methods, it is characterized in that, described primer β-preparation method is as follows for the globin standard substance: β-globin primer of reaction cumulative volume 25 μ l carries out pcr amplification, amplification condition is 95 ℃ of 1min of pre-sex change, 95 ℃ of 30s then, 57 ℃ of 20s, 72 ℃ of 20s, totally 45 circulations; Gel reclaims purified pcr product; The super competent cell of preparation DH5a; Goal gene connects conversion and transforms the back sequence verification; Extract plasmid, and carry out quantitatively; Described β-globin recombinant plasmid length is 2802bp, and concentration is 2.58 * 10 ¹³Copies/ml, being institute behind 10 times of gradient dilutions must standard substance.

3. a kind of plasma free dna level according to claim 1 detects the hepatic necrosis degree methods, it is characterized in that, the extracting method of described liver problem sufferer's plasma sample plasma dna is as follows: get 300 μ l blood plasma, add lysate 900 μ l (0.1MTri-HCL 90 μ l by 1: 3 volume, 0.05M EDTA 90 μ l, 1%SDS 450 μ l, ddH ₂O 270 μ l), adding 6 μ l Proteinase Ks (Proteinase K 20mg/ml) is 100 μ g/ml to final concentration, mixing 10s on vibrator, and of short duration centrifugal back is in 56 ℃ of water-bath 2h; The water-bath mixing liquid is in 95 ℃ of water-bath 10min, sex change Proteinase K; Of short duration centrifugal, add the saturated phenol of equal-volume Tris: chloroform: primary isoamyl alcohol (volume ratio 25: 24: 1) mixed solution 1200 μ l, abundant mixing on the vibrator, about 2-3min; 12, the centrifugal 15min of 000rpm carefully draws supernatant, places new EP pipe (import EP pipe is used in suggestion); In supernatant liquor, add the dehydrated alcohol (about 3000 μ l) of its 2.5 times of volumes, put upside down mixing, in-20 ℃ of precipitations (more than the 12h) of spending the night; Normal temperature 12, the centrifugal 20min of 000rpm carefully removes supernatant; 70% ethanol, the 800 μ l that add 2 times of volumes, normal temperature 12, the centrifugal 10min of 000rpm; Carefully remove supernatant, in 56 ℃ of baking ovens, place 20min, be placed on the super clean bench 1h alcohol that thoroughly volatilizees; Add 40 μ l ddH ₂O ₂Dissolving DNA, slight concussion, of short duration centrifugal, place 4 ℃ of abundant dissolving DNAs of 1h; The DNA sample is in-20 ℃ of preservations.

4. a kind of plasma free dna level according to claim 1 detects the hepatic necrosis degree methods, it is characterized in that, the plasma dna that extracts in the described plasma specimen carries out real-time fluorescence quantitative polymerase chain reaction and may further comprise the steps: β-globin primer of reaction cumulative volume 20 μ l carries out pcr amplification, amplification condition is 95 ℃ of 1min of pre-sex change, 95 ℃ of 30s then, 57 ℃ of 20s, 72 ℃ of 32s, totally 45 circulations.

5. a kind of plasma free dna level according to claim 1 detects the hepatic necrosis degree methods, it is characterized in that, the concentration measuring and calculating of described plasma free DNA comprises that with concentration known be 2.58 * 10 ¹³The β of copies/ml-globin recombinant plasmid makes its final concentration be respectively 2.58 * 10 as 10 times of proportional diluted of standard substance series ⁵Copies/ μ l, 2.58 * 10 ⁴Copies/ μ l, 2.58 * 10 ³Copies/ μ l, 2.58 * 10 ²Copies/ μ l sets up typical curve and establishes linear relationship between the logarithm of Ct value and standard substance starting template; The amount of circulation nucleosome is calculated as follows in the blood plasma: C=Q * (V _DNA/ V _PCR) * (1/V _Ext), wherein C is a testing sample plasma circulation nucleosome concentration (GE/ml), Q is the DNA amount (copies) that instrumental analysis software is calculated, V _DNABe the DNA total amount (40 μ l) that extracting goes out, V _PCRFor joining DNA amount (9.5 μ l) in the reaction system, V _ExtBe the total plasma volume (300 μ l) that is used for extracting and purify DNA.