CN101586162B - Method of extracting target nucleic acid and performing PCR amplification - Google Patents
Method of extracting target nucleic acid and performing PCR amplification Download PDFInfo
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- CN101586162B CN101586162B CN2009100436522A CN200910043652A CN101586162B CN 101586162 B CN101586162 B CN 101586162B CN 2009100436522 A CN2009100436522 A CN 2009100436522A CN 200910043652 A CN200910043652 A CN 200910043652A CN 101586162 B CN101586162 B CN 101586162B
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Abstract
A method of extracting target nucleic acid and performing PCR augmentation comprises the following steps: adding a cracking liquid, a sample containing target nucleic acid and a reaction liquid which performs PCR augmentation into a PCR reaction tube and evenly mixing; placing in the PCR apparatus for performing PCR augmentation. The invention only needs simple instrument to rapidly and high efficiently extract target nucleic acid from the sample and to synchronously perform real-time fluorescence PCR augmentation detection; the invention has wide detection linear range, accurate quantification for samples of different concentrations, good specificity and good repetitiveness; the invention is extremely simple in operation and suitable for the PCR apparatus of different types; it can be widely applied to the fields of detection of pathogenic microorganism, medicolegal expertise, tissue and blood typing, genetic mutation detection.
Description
Technical field
The present invention relates to a kind of go forward side by side method of performing PCR augmentation detection of target nucleic acid of extracting.
Background technology
Since round pcr in 1985 came out, this technology had been applied to each message area of life science, because of its susceptibility height, high specificity, easy and simple to handle, advantage such as required time is short, had been widely used in the detection of pathogenic micro-organism at present.Owing to contain materials such as protein, lipid in the clinical samples, can disturb the PCR reaction, so before doing the PCR reaction, must carry out nucleic acid extraction.The first step of PCR detection technique is exactly the preparation of template DNA, i.e. the extraction of DNA in the sample, and this directly affects the result of PCR reaction.For a long time, the extraction of DNA and purifying are consuming time, loaded down with trivial details processes always in the sample, and detection speed has seriously slowed down.
At present, the extracting method of DNA comprises two steps in the sample: lysing cell and extraction nucleic acid.Lysing cell, the common method that DNA is discharged comprise physics method (boil, freeze thawing, microwave, ultrasonic, grinding etc.), chemical process (high salt, surfactant SDS, hot phenol etc.) and enzymolysis process (lyase, N,O-Diacetylmuramidase, Proteinase K etc.).
Phenol extraction process, isopropanol precipitating method and methane amide cracking process are to extract DNA classic methods the most, the improvement of at present a lot of methods all is to carry out on the basis of these methods, and these three kinds of methods all are to utilize Proteinase K and sodium lauryl sulphate (SDS) digestion smudge cells.In preceding two kinds of methods, lysate is removed protein with phenol/chloroform earlier, more respectively with ethanol or isopropanol precipitating DNA.The methane amide method is to utilize the methane amide depolymerization protein of high density and combining of DNA, utilizes dialysis to handle the DNA sample then.The DNA purity that these classical ways obtain is very high, can satisfy the requirement of various tests, but complex operation is used duration, and agents useful for same has certain toxicity.
The method for extracting nucleic acid that is applied to the real-time fluorescence quantitative PCR detection at present mainly contains three kinds, i.e. alkaline lysis, boiling lysis and paramagnetic particle method.
Alkaline lysis is under high pH (12.0～12.6) condition that NaOH provides, and destroys cell walls with strong cation washing agent SDS, and lysing cell makes the protein generation sex change of cell jointly with NaOH, discharges DNA.After the cracking, the protein of cell wall fragments and sex change and other impurity form big mixture, and these mixtures can effectively precipitate, and DNA remain in the supernatant liquor under high sylvite condition, again by step purify DNAs such as dehydrated alcohol precipitation, washing with alcohol.Though alkaline lysis is fast and convenient, the Deproteinization effect is not good enough, has obviously influenced the PCR effect.
The boiling lysis principle, be when boiling, the DNA in the sample to be discharged by the effect that contains lysis buffers such as Proteinase K, be dissolved in the damping fluid, in the time of the boiling water bath lysing cell, the base pairing of DNA chain be can destroy, and the protein and the chromosomal DNA sex change of cell made.Remove protein and other impurity of sex change by centrifugation, the DNA that reclaims then in the supernatant liquor is used for pcr amplification.Yet through high-temperature boiling, protein coagulation is wrapped part nucleic acid and loses with centrifugation, directly causes template nucleic acid amount minimizing in the supernatant liquor; In addition, though, still contain composition such as albumen, heparin and the micro-oxyphorase etc. of a small amount of suppression of amplification in the supernatant liquor, suppressed amplification efficiency through boiling and precipitating at a high speed.
Paramagnetic particle method is the method for extracting nucleic acid that just grew up in recent years, it is by the cell pyrolysis liquid lysing cell that paramagnetic particle method extracts nucleic acid, the nucleic acid molecule that dissociates out from cell is by the special magnetic-particle surface that is adsorbed onto, and impurity such as protein are not adsorbed, and stays in the solution.After the reaction certain hour, under the action of a magnetic field, magnetic-particle and liquid are separated again, reclaim particle (being magnetic bead-DNA mixture), use the elutriant wash-out again, obtain purified DNA.Advantages such as because its reagents series do not contain the big organic solvents of toxicity such as chloroform, phenol, extraction step is simpler, and high and purity is good to the rate of recovery of sample of nucleic acid and extremely the investigator favor.But product is essentially external and monopolizes, and price is very expensive, thereby has limited its widespread use in China.
No matter be classical way or various improved method, all must pay close attention to following three main points: the first, the yield of goal gene; The second, the simplified and traditional degree of schedule of operation; Three, the consumption of material and time.Above the whole bag of tricks all must just can carry out PCR and detect after extracting purification of nucleic acid, it is painstaking to compare unavoidably when handling a large amount of clinical sample.
Summary of the invention
The object of the present invention is to provide a kind of used equipment simple, easy and simple to handle, the go forward side by side method of performing PCR augmentation detection of the extraction target nucleic acid that cost is low.
The go forward side by side method of performing PCR amplification of the present invention's extraction nucleic acid comprises the steps: a kind of lysate, a kind of sample and a kind of reaction solution that carries out pcr amplification that contains target nucleic acid are added in the PCR reaction tubes, mixes, and places the enterprising performing PCR amplification of PCR instrument.
Behind lysate, the sample that contains target nucleic acid and the pcr amplification reaction liquid mixing, should leave standstill 3-8 minute, and then place the enterprising performing PCR amplification of PCR instrument.
Described lysate is dissolved in the KCl (mass volume ratio) of 50～200mM by the surfactant peptides of 0.01～0.5mM (mass volume ratio), the SDS of 0.01%～2% (volume), LLS, Chelex-100 (mass volume ratio), and the ethanol of 0.05%～1% (volume) is formed.
The testing sample that contains target nucleic acid can be serum, blood plasma, juice or sputum.
Described nucleic acid can be DNA or RNA.
Pcr amplification reaction liquid (hereinafter to be referred as " PCR reaction solution ") comprises the pcr amplification damping fluid, is selected from the mixture of 4 kinds of dNTP among dATP, dCTP, dGTP, dTTP or the dUTP, archaeal dna polymerase, and magnesium ion is at the upstream and downstream primer of target gene design; According to the various objectives of real-time fluorescence quantitative PCR, also comprise fluorescence dye or according to the custom-designed primer of target sequence and with the probe of target sequence specific hybridization.The proportioning of these materials generally is the reaction system adjustment according to different experiments, as dNTPs, the 1 * PCR buffer of Mg2+, the 0.2-0.4mM of the archaeal dna polymerase of 1-5U, 3-5.5mM; The upstream primer of 50-900nM, the downstream primer of 50-900nM are determined to determine to add amount of substance (referring to aftermentioned embodiment) again after the concentration
The mixture ratio of described 4 kinds of dNTP, the ratio of dATP: dCTP: dGTP: dTTP was generally 1: 1: 1: 1; The ratio of dATP: dCTP: dGTP: dUTP is 1: 1: 1: 2.
Proportioning is unrestricted between described lysate, the testing sample that contains target nucleic acid, these three kinds of materials of PCR reaction solution, promptly can be any ratio, but more preferably proportioning is 1: 1-3: 10-25; The order that is mixed does not have strict restriction yet, but suitable last interpolation of PCR reaction solution.
Except that lysate, the testing sample that contains target nucleic acid, PCR reaction solution, also can add interior mark as required.
Be designated as the section of DNA fragment similar of known low levels in described to template amplification efficient, its is added in PCR reaction solution, increase jointly, can react every pipe situation that increases really with template, if interior mark and sample all do not amplify the result, represent that then this pipe amplification is invalid.
Described pcr amplification comprises simple PCR, linear amplification and real-time fluorescence quantitative PCR.
Advantage of the present invention is: only need simple equipment, as liquid-transfering gun, PCR reaction tubes; Two kinds of simple reagent (lysate, PCR reaction solution) can rapidly and efficiently extract target nucleic acid and carry out the real-time fluorescence quantitative PCR augmentation detection synchronously from sample.
Simultaneously, the inventive method detects linear wide ranges, and for samples of different concentrations, quantitatively accurately, specificity is good, good reproducibility.
Compare with present existing nucleic acid extraction and PCR detection method, the present invention's operation is especially easy, operation near automatization has guaranteed that experimental result is stable, difference and the mistake of avoiding manual operation to cause, be applicable to dissimilar PCR instrument, can be widely used in the detection of pathogenic micro-organism, medical jurisprudence is identified, tissue and blood grouping, fields such as the detection of transgenation.
Description of drawings
Fig. 1 (a) detects the positive reference material of HBV DNA national standard product for the inventive method;
Fig. 1 (b) detects the negative reference material of HBV DNA national standard product for the inventive method;
Fig. 2 detects HBV DNA positive serum for the inventive method, and concentration is respectively 1.0 * 10 from left to right
7IU/ml, 1.0 * 10
6IU/ml, 1.0 * 10
5IU/ml, 1.0 * 10
4IU/ml
Fig. 3 (a) detects HBV DNA positive serum for the inventive method, and concentration is 500IU/ml
Fig. 3 (b) detects HBV DNA positive serum for the inventive method, and concentration is 300IU/ml
Fig. 3 (c) detects HBV DNA positive serum for the inventive method, and concentration is 200IU/ml
Fig. 4 detects HBV DNA and corresponding interior mark for the inventive method.
Embodiment
Below in conjunction with embodiment the present invention is made and to further specify:
The fluorescence quantitative PCR detection embodiment of embodiment 1:HBV DNA
Draw 2 microlitre lysates (surfactant peptides of 0.1mM is dissolved in 80mM KCl, 0.1%SDS, 0.5% ethanol) with band filter core suction nozzle, 3 microlitre samples to be tested (known HBV DNA male serum or blood plasma), add in the PCR reaction tubes, the liquid-transfering gun head is inhaled and is beaten mixing, after leaving standstill 5min, draw the PCR reaction solution that 45ul has prepared with band filter core suction nozzle, on Stratagene Mx3000P or ABI7300 PCR instrument, carry out the real-time fluorescence quantitative PCR amplification.
Each constituent concentration of PCR reaction solution and ratio are as follows:
| Composition | Single part of consumption |
| (upstream) primer HBV-F1 (50pmol/ μ l) | 0.2μl |
| (downstream) primer HBV-F1 (50pmol/ μ l) | 0.2μl |
| HBV probe HBV-P1 (50pmol/ μ l) | 0.1ul |
| ROX solution (10 μ mol/L) | 1ul |
| dNTPs | 0.4 |
| 10×PCR?buffer | 5μl |
| The sterilization purified water | 38.1μl |
| Total amount | 45μl |
The PCR response procedures is:
1) with the yin and yang attribute reference material in the HBV national standard product as sample to be checked, adopt the inventive method on the StratageneMx3000P instrument, to detect, detect the yin and yang attribute coincidence rate.Be 9 positive reference materials in the national standard product as Fig. 1 (a), utilization the present invention detects positive entirely, and Fig. 1 (b) is 8 negative reference materials, and is negative entirely after testing.Utilization the inventive method is carried out HBV DNA fluorescence quantitative PCR detection and is had good specificity.
2) the HBV DNA positive serum of 4 kinds of concentration of demarcating with Chinese biological goods calibratings institute (middle inspection institute) hepatitis B virus (HBV) nucleic acid quantification standard substance is a sample to be tested, and concentration is respectively 1.0 * 10
7IU/ml, 1.0 * 10
6IU/ml, 1.0 * 10
5IU/ml, 1.0 * 10
4IU/ml, each concentration repeated experiments 3 times, the result is as shown in Figure 2, the variation coefficient of CT value is respectively: 0.25%, 0.44%, 0.48%, 0.36%, the variation coefficient all in 1%, this shows that utilization the inventive method detects the repeated fine of serum sample.
3) the HBV positive serum of demarcating with middle inspection institute hepatitis B virus (HBV) nucleic acid quantification standard substance is initial sample, be diluted to 500IU/ml, 300IU/ml, 200IU/ml, each concentration repeats 5 times, as Fig. 3,500IU/ml, the amplification of the sample standard deviation 100% of 300IU/ml, and only 1 the sample of 200IU/ml does not have amplification, can reach 300IU/ml so utilization the inventive method is carried out fluorescence quantitative PCR detection sensitivity.
4) be sample to be tested with the linear sensitivity reference material in the HBV national standard product, (concentration is respectively 1.0 * 10 with the positive criteria product of gradient dilution
7IU/ml, 1.0 * 10
6IU/ml, 1.0 * 10
5IU/ml, 1.0 * 10
4IU/ml) adopt fast method of the present invention to detect the accordance of analyzing and testing value and theoretical value:
| Linear sensitivity reference material | Quality standard (IU/ml) | The theoretical concentration logarithmic value | Detected value | The detectable level logarithmic value |
| L0 | 7.762×10 7～6.165×10 8 | 8.342 | 2.55×10 8 | 8.407 |
| L1 | 1.479×10 7～1.175×10 8 | 7.619 | 3.54×10 7 | 7.549 |
| L2 | 1.585×10 6～1.259×10 7 | 6.649 | 4.43×10 6 | 6.646 |
| L3 | 1.659×10 5～1.318×10 6 | 5.672 | 3.41×10 5 | 5.382 |
| L4 | 1.820×10 4～1.479×10 5 | 4.715 | 1.38×10 4 | 4.140 |
| L5 | 1.514×10 3～1.230×10 4 | 3.636 | 1.71×10 3 | 3.233 |
| L6 | 3.890×10 1～3.090×10 2 | 2.043 | 5.64×10 1 | 1.751 |
The double logarithmic curve linearly dependent coefficient (r) of theoretical concentration and detectable level is 0.9993, and it is quantitatively very accurate that utilization this method is carried out the real-time fluorescence quantitative PCR detection.
Embodiment 2:HBV DNA and corresponding interior target rapid fluorescence quantitative PCR detection embodiment
Method as embodiment 1, draw 2 microlitre lysates (surfactant peptides of 0.1mM is dissolved in 80mM KCl, 0.1%SDS, 0.5% ethanol) with band filter core suction nozzle, 3 microlitre samples to be tested (known HBV DNA male serum or blood plasma), add in the PCR reaction tubes, the liquid-transfering gun head is inhaled and is beaten mixing, leaves standstill about 5min.Add mark (10 in the 0.3ul according to stoichiometric number according to every reaction
4Copies/ul) ratio adds interior mark in the PCR reaction solution.Present embodiment has selected for use lower concentration HBV dna sample (concentration is 500IU/ml, 200IU/ml) and a negative sample as sample to be tested.As Fig. 4, higher one group of fluorescent signal is interior target amplification curve, and one group that fluorescent signal is lower is the amplification curve of sample to be tested, and interior mark amplification is normal and detect sample for lower concentration and do not have influence.In be designated as the section of DNA fragment similar of known low levels to template amplification efficient, it is added in the PCR reaction solution, increase jointly with template, can react every pipe situation that increases really, if interior mark and sample all do not amplify the result, represent that then this pipe amplification is invalid, mark monitoring in this fast method can add and have good effect.
Experiment showed, that by hepatitis B virus thymus nucleic acid (HBV DNA) utilization Taqman real-time fluorescence quantitative PCR is carried out absolute quantitation the inventive method detects linear wide ranges, for samples of different concentrations, quantitatively accurately, specificity is good, good reproducibility.
Reference examples 1: boiling method extracts HBV DNA and fluorescence quantitative PCR detection
Boiling method the inventive method
At present domestic have a lot of employing boiling methods extraction nucleic acid and carry out the HBV detection by quantitative, but sensitivity only can reach 500IU/ml or 10
3More than the IU/ml, and the inventive method sensitivity is than the boiling method height, and operation is easier.
Reference examples 2: paramagnetic particle method extracts HBV DNA and fluorescence quantitative PCR detection
Paramagnetic particle method: add cell pyrolysis liquid in the sample to be tested, after treating lysis, in lysate, add magnetic bead, when the pH value of solution less than 6.5 the time, magnetic bead optionally combines with the DNA that optimizes, the magnetic bead that will be adsorbed with DNA this moment places magnetic field, remove the impurity (albumen etc.) that is not adsorbed by damping fluid, then magnetic bead is placed the damping fluid of pH value 8.5, the DNA of purifying can enter damping fluid, and the machine of going up in the DNA adding PCR reaction solution with wash-out carries out real-time fluorescence quantitative PCR and detects.
In the present known data, it is low slightly than paramagnetic particle method that the sensitivity of utilization paramagnetic particle method extraction nucleic acid and amplification can reach the sensitivity of 10IU/ml the inventive method, but easy relatively a lot of in operation.
Claims (9)
1. one kind is extracted the go forward side by side method of performing PCR augmentation detection of target nucleic acid, it is characterized in that, comprises the steps: lysate, the reaction solution that contains the sample of target nucleic acid and carry out pcr amplification are added in the PCR reaction tubes, mixes, and places the enterprising performing PCR amplification of PCR instrument;
Described lysate is dissolved in the KCl (mass volume ratio) of 50～200mM by the surfactant peptides of 0.01～0.5mM (mass volume ratio), the SDS of 0.01%～2% (volume), LLS, Chelex-100 (mass volume ratio), and the ethanol of 0.05%～1% (volume) is formed.
2. the go forward side by side method of performing PCR augmentation detection of extraction target nucleic acid according to claim 1 is characterized in that, behind lysate, the sample that contains target nucleic acid and pcr amplification reaction liquid mixing, leaves standstill 3-8 minute earlier, and then places the enterprising performing PCR amplification of PCR instrument.
3. the go forward side by side method of performing PCR augmentation detection of extraction target nucleic acid according to claim 1 and 2 is characterized in that described nucleic acid is DNA or RNA.
4. the go forward side by side method of performing PCR augmentation detection of extraction target nucleic acid according to claim 1 and 2, it is characterized in that, the reaction solution that carries out pcr amplification comprises the pcr amplification damping fluid, be selected from the mixture of 4 kinds of dNTP among dATP, dCTP, dGTP, dTTP and the dUTP, archaeal dna polymerase, magnesium ion is at the upstream and downstream primer of target gene design.
5. the go forward side by side method of performing PCR augmentation detection of extraction target nucleic acid according to claim 4 is characterized in that the mixture ratio of described 4 kinds of dNTP, the ratio of dATP: dCTP: dGTP: dTTP are 1: 1: 1: 1; The ratio of dATP: dCTP: dGTP: dUTP is 1: 1: 1: 2.
6. the go forward side by side method of performing PCR augmentation detection of extraction target nucleic acid according to claim 4 is characterized in that, also comprise fluorescence dye or according to the custom-designed primer of target sequence and with the probe of target sequence specific hybridization.
7. the go forward side by side method of performing PCR augmentation detection of extraction target nucleic acid according to claim 1 and 2 is characterized in that the testing sample that contains target nucleic acid is serum, blood plasma, juice or sputum.
8. the go forward side by side method of performing PCR augmentation detection of extraction target nucleic acid according to claim 1 and 2 is characterized in that pcr amplification is simple PCR, linear amplification or real-time fluorescence quantitative PCR.
9. the go forward side by side method of performing PCR augmentation detection of extraction target nucleic acid according to claim 4 is characterized in that the proportioning between lysate, the testing sample that contains target nucleic acid, the PCR reaction solution is 1: 1-3: 10-25.
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| CN101701267B (en) * | 2009-11-26 | 2012-09-19 | 戴立忠 | Fluorescence quantitative PCR detection kit of hepatitis B virus and application thereof |
| KR20160088958A (en) * | 2010-02-23 | 2016-07-26 | 루미넥스 코포레이션 | Apparatus and methods for integrated sample preparation, reaction and detection |
| CN102807975B (en) * | 2011-05-30 | 2014-10-29 | 熊慧 | Method for rapidly extracting nucleic acid from biological sample |
| CN103184214B (en) * | 2011-12-27 | 2016-02-10 | 上海星耀医学科技发展有限公司 | A kind of hbv nucleic acid rapid extraction reagent |
| CN104450674A (en) * | 2013-09-24 | 2015-03-25 | 上海艾迪康医学检验所有限公司 | Nucleic acid extraction and preservation solution |
| CN108531563A (en) * | 2018-02-05 | 2018-09-14 | 深圳市尚维高科有限公司 | The purposes and lysate of porous microsphere and the application method of lysate |
| CN108977436A (en) * | 2018-08-10 | 2018-12-11 | 邵忠民 | Biotinylated nucleic acid DNA quick release extracts reagent and preparation method and application |
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| CN1712547A (en) * | 2005-02-01 | 2005-12-28 | 合肥中科大生物技术有限公司 | Fluorescent quantitative RT-PCR detecting kit of 2-f(o)etoprotein (AFP)mRNA |
| CN1873023A (en) * | 2006-04-14 | 2006-12-06 | 武汉大学 | Fluorescence quantitative kit PCR for quick testing chlamydia trachomatis |
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| CN1712547A (en) * | 2005-02-01 | 2005-12-28 | 合肥中科大生物技术有限公司 | Fluorescent quantitative RT-PCR detecting kit of 2-f(o)etoprotein (AFP)mRNA |
| CN1873023A (en) * | 2006-04-14 | 2006-12-06 | 武汉大学 | Fluorescence quantitative kit PCR for quick testing chlamydia trachomatis |
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