CN103184214A - Hepatitis B virus nucleic acid rapid extraction reagent - Google Patents
Hepatitis B virus nucleic acid rapid extraction reagent Download PDFInfo
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- CN103184214A CN103184214A CN2011104485243A CN201110448524A CN103184214A CN 103184214 A CN103184214 A CN 103184214A CN 2011104485243 A CN2011104485243 A CN 2011104485243A CN 201110448524 A CN201110448524 A CN 201110448524A CN 103184214 A CN103184214 A CN 103184214A
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- rapid extraction
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- 241000700721 Hepatitis B virus Species 0.000 title claims abstract description 59
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 37
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 37
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 37
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 35
- 238000000605 extraction Methods 0.000 title claims abstract description 32
- 210000002966 serum Anatomy 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 7
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 claims abstract description 7
- 229940107698 malachite green Drugs 0.000 claims abstract description 7
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims abstract description 7
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 7
- 239000013504 Triton X-100 Substances 0.000 claims abstract description 5
- 229920004890 Triton X-100 Polymers 0.000 claims abstract description 5
- 210000002381 plasma Anatomy 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- 238000013016 damping Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 238000010009 beating Methods 0.000 abstract 1
- 238000007664 blowing Methods 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 37
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000000284 extract Substances 0.000 description 9
- 238000007400 DNA extraction Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 230000003321 amplification Effects 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 238000003753 real-time PCR Methods 0.000 description 6
- 239000012472 biological sample Substances 0.000 description 5
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a reagent for rapid extraction of hepatitis B virus (HBV) nucleic acid in serum and plasma samples. The extraction reagent contains 1-5% KCl (w/v), 0.01-0.5mg/ml surface active peptide, 0.1-2% Triton X-100 or Tween 20 (v/v), and 0.001-0.005% malachite green (w/v). During application, HBV positive serum or plasma with an equal volume to the nucleic acid extraction reagent is added into it, a pipettor is employed to conduct blowing and beating for 5-10 times under room temperature to mix them uniformly. At the moment, HBV DNA is released into the mixed liquid, and a PCR buffer solution can be added into the liquid to carry out a fluorescent PCR detection. The HBV nucleic acid rapid extraction reagent provided in the invention has the advantages of simple operation, safe use, and low cost.
Description
Technical field
The invention belongs to technological field of biochemistry, be specifically related to hbv nucleic acid extraction in the biological sample.
Background technology
(Hepatitis B Virus, during HBV) nucleic acid quantification detected, the HBV DNA extraction was the first step operation of at first carrying out clinical hepatitis B virus.At present, the HBV method for extracting nucleic acid of clinical application both at home and abroad mainly contains: (1) alkaline lysis boiling method, or carry out saccharan concentrating and precipitating HBV virus earlier simultaneously, in precipitation, add lysate and boil extraction HBV DNA; (2) pellosil adsorption column method adopts the absorption of pellosil chromatography column, cleaning, wash-out to obtain HBV DNA; (3) magnetic bead absorption method adopts magnetic bead absorption, cleaning, wash-out to obtain HBV DNA.These three kinds of methods all need through multistep suddenly centrifugal (or negative pressure-pumping, bar magnet separate), replace tubes or pipetting could obtain nucleic acid, have the deficiency that operation steps is various, the process amplifying nucleic acid is lost easily or polluted.
Summary of the invention
The purpose of this invention is to provide a kind of HBV nucleic acid rapid extraction reagent, it adopts the single stage method operation to finish the quick release extraction of HBV DNA in biological sample serum, the blood plasma, the nucleic acid that obtains is applicable to detected downstream such as fluorescent PCR, has easy and simple to handle, use safety, advantage with low cost is adapted at applying in the clinical gene test.
Technical scheme
The invention provides a kind of HBV nucleic acid rapid extraction reagent, it contains 1-5%KCl (mass/volume ratio, w/v), 0.01-0.5mg/ml surfactant peptides, 0.1-2%Triton X-100 or Tween 20 (quality/plasmid ratio, v/v) and 0.001-0.005% malachite green (w/v), described quality or volume percent serve as to calculate benchmark to extract reagent volume.
HBV nucleic acid rapid extraction reagent compound method of the present invention is: adding KCl final concentration reaches 1-5% (w/v) in the sterilization deionized water, adding surfactant peptides final concentration reaches 0.01-0.5mg/ml, adding TritonX-100 or Tween 20 final concentrations reach 0.1-2% (v/v), adding malachite green final concentration reaches 0.001-0.005% (w/v).The reagent of preparation can be in room temperature preservation.
HBV nucleic acid rapid extraction reagent using method of the present invention is: get the nucleic acid rapid extraction reagent of above-mentioned preparation, add in the pending biological sample of equal-volume, with 5-10 mixing of pipettor piping and druming, HBV DNA namely extracts from virion and discharges.Described biological sample is serum, blood plasma.Extract after reagent and the sample mix liquid and transferred to colourlessly by blue-greenish colour, can be used as template and directly add the PCR reaction solution and carry out augmentation detection.
Nucleic acid rapid extraction reagent of the present invention adopts protein denaturant and sanitas, and energy rapid damage HBV virion coat protein structure discharges HBV DNA, and extraction reagent contains the component that suppresses nuclease and follow-up PCR experiment do not exerted an influence; Extract add sample in the reagent after color change, whether avoid causing obscuring of application of sample.Adopt HBV nucleic acid rapid extraction reagent of the present invention and at present method commonly used relatively need not heating, centrifugally operated, only need a pipettor at ambient temperature single stage method finish the extraction of HBV DNA in the biological sample.The nucleic acid that obtains can be widely used in fields such as follow-up fluorescent PCR detection by quantitative, the detection of PCR-gene chip hybridization.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can do various technical changes or modification to the present invention by technology general knowledge after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Main agents related among the present invention illustrates:
KCl is analytical pure, and tensio-active agents such as Triton X-100 and Tween 20 are biological level purity, and malachite green is chemical pure, and mentioned reagent is available from Chemical Reagent Co., Ltd., Sinopharm Group, and surfactant peptides is available from Sigma.Highpure Total Nucleic Acid Kit (silica gel embrane method) used in the embodiment is available from Luo Shi diagnostic products (Shanghai) Co., Ltd.; Platform is moulded DNA/RNA extraction agent (paramagnetic particle method) available from Taisushengyi Science Technology Co., Ltd.; Hepatitis B virus (HBV) nucleic acid fluorescent quantitative PCR detection kit is Shanghai clone Biology high technology Ltd's product.
Embodiment 1: and the alkaline lysis boiling method relatively is used for the detection of serum HBV DNA extraction fluorescent PCR
(1) HBV nucleic acid rapid extraction reagent preparation
According to summary of the invention, at first prepare HBV nucleic acid rapid extraction reagent: adding KCl final concentration reaches 2% (w/v) in the sterilization deionized water, adding surfactivity peptide concentration reaches 0.5mg/ml, adding Triton X-100 or Tween 20 concentration reach 0.5% (v/v), adding indicator malachite green concentration reaches 0.001% (w/v).
(2) HBV positive serum nucleic acid extraction
To 5 routine HBV positive serums, adopt the extracting method in the summary of the invention to handle: get 3.5 μ l and extract reagent adding 0.2ml pcr amplification pipe, add 3.5 μ l serum respectively, blow and beat 5-10 time repeatedly with pipettor, the operation required time is 5min.
Simultaneously, boiling extraction HBV DNA method with traditional virus precipitation-alkaline lysis in hepatitis B virus (HBV) the nucleic acid fluorescent quantitative PCR detection kit is contrast: respectively get 50 μ l serum, add 50 μ l nucleic acid extraction liquid A respectively, 10 seconds kinds of vibration mixing, 13, centrifugal 10 minutes of 000rpm abandons supernatant; Add 50 μ l nucleic acid extraction liquid B respectively to precipitation, vibration 10 seconds of mixing, 100 ℃ of boiling water baths 10 minutes, centrifugal 2 minutes of 13,000rpm, the operation required time is 30min.
(3) HBV DNA fluorescence quantitative PCR detection
Test kit HBV PCR damping fluid n * 30 μ l, MgCl got in two kinds of HBV DNA HBV nucleic acid fluorescent quantitative PCR test kit detections that method is extracted
2N * 5 μ l, HBV fluorescent probe n * 5 μ l, Taq enzyme n * 3 μ l mix in a centrifuge tube (n is sample number to be amplified), and the vibration mixing is 10 seconds on the vortex vibrator, and the low-speed centrifugal several seconds is by every pipe 43 μ l packing PCR reaction solutions.Nucleic acid rapid extraction reagent of the present invention is extracted the HBV DNA that obtains, as long as the PCR reaction solution is added above-mentioned containing in the amplification pipe that extracts reagent and serum mixed solution; To contrast method, then draw in the PCR reaction solution amplification pipe that 7 μ l templates add 43 μ l packing.
Carry out augmentation detection at ABI7500 amplified fluorescence instrument, program is: behind 50 ℃ of 2min, the 94 ℃ of 5min according to 93 ℃ of 30 seconds → 60 ℃ 90 seconds cyclic amplifications 40 times.Reaction finishes the back and uses the instrument software analytical results, and two kinds of methods are to 5 routine serum HBV DNA quantitative result such as tables 1, and two kinds of method quantitative results are close as can be known, and the method operation of reagent is easier, the time is shorter, has advantage but employing the present invention extracts.
Table 1 and alkaline lysis boiling method relatively are used for serum HBV DNA extraction fluorescent PCR detected result
Embodiment 2: and post method, paramagnetic particle method relatively are used for the detection of blood plasma HBV DNA extraction fluorescent PCR
(1) HBV nucleic acid rapid extraction reagent preparation
According to summary of the invention, at first prepare HBV nucleic acid rapid extraction reagent: adding KCl final concentration reaches 5% (w/v) in the sterilization deionized water, adding surfactivity peptide concentration reaches 0.01mg/ml, adding TritonX-100 or Tween 20 concentration reach 2% (v/v), adding indicator malachite green concentration reaches 0.004% (w/v).
(2) the positive blood plasma nucleic acid extraction of HBV
To the positive blood plasma of 5 routine HBV, adopt the extracting method in the summary of the invention to handle: get 3.5 μ l and extract reagent adding 0.2ml pcr amplification pipe, add 3.5 μ l serum respectively, blow and beat 5-10 time repeatedly with pipettor, the operation required time is 5min.
Simultaneously, moulding living doctor's DNA/RNA extraction agent (paramagnetic particle method) processing HBV positive blood paste-making method with Luo Shi Highpure Total Nucleic Acid Kit (pellosil post method), platform is contrast, operate according to specification sheets separately respectively, through the HBV nucleic acid that cracking, cleaning, elution step acquisition are extracted, each about 30min of operation required time.
(3) HBV DNA fluorescence quantitative PCR detection
The HBV DNA that above-mentioned three kinds of methods are extracted detects with HBV nucleic acid fluorescent quantitative PCR test kit, gets test kit HBV PCR damping fluid n * 30 μ l, MgCl
2N * 5 μ l, HBV fluorescent probe n * 5 μ l, Taq enzyme n * 3 μ l mix in a centrifuge tube (n is sample number to be amplified), and the vibration mixing is 10 seconds on the vortex vibrator, and the low-speed centrifugal several seconds is by every pipe 43 μ l packing PCR reaction solutions.Nucleic acid rapid extraction reagent of the present invention is extracted the HBV DNA that obtains, as long as the PCR reaction solution is added above-mentioned containing in the amplification pipe that extracts reagent and blood plasma mixed solution; To pellosil post method, paramagnetic particle method contrast method, the template of then drawing 7 μ l wash-outs adds in the PCR reaction solution amplification pipe of 43 μ l packing.
Carry out augmentation detection at ABI7500 amplified fluorescence instrument, program is: behind 50 ℃ of 2min, the 94 ℃ of 5min according to 93 ℃ of 30 seconds → 60 ℃ 90 seconds cyclic amplifications 40 times.Reaction finishes the back and uses the instrument software analytical results, and three kinds of methods are to 5 routine blood plasma HBV DNA quantitative result such as tables 2, and three kinds of method quantitative results are close as can be known, and the method operation of reagent is easier, the time is shorter, has advantage but employing the present invention extracts.
Table 2 and post method, paramagnetic particle method relatively are used for blood plasma HBV DNA extraction fluorescent PCR detected result
Claims (2)
1. the present invention is a kind of reagent for serum, plasma sample hepatitis B virus (HBV) nucleic acid rapid extraction, it is characterized in that, this extraction reagent contains 1-5%KCl (w/v), 0.01-0.5mg/ml surfactant peptides, 0.1-2%Triton X-100 or Tween 20 (v/v) and 0.001-0.005% malachite green (w/v), and described quality or volume percent serve as to calculate benchmark to extract reagent volume.
2. according to claim 1, it is characterized in that, get this HBV nucleic acid rapid extraction reagent during use, add equal-volume HBV positive serum or blood plasma, blow and beat 5-10 mixing with pipettor under the room temperature condition, HBV DNA will be discharged in the mixing liquid this moment, can add the PCR damping fluid and carry out the fluorescent PCR detection in this liquid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201110448524.3A CN103184214B (en) | 2011-12-27 | 2011-12-27 | A kind of hbv nucleic acid rapid extraction reagent |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201110448524.3A CN103184214B (en) | 2011-12-27 | 2011-12-27 | A kind of hbv nucleic acid rapid extraction reagent |
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| CN103184214A true CN103184214A (en) | 2013-07-03 |
| CN103184214B CN103184214B (en) | 2016-02-10 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105002175A (en) * | 2015-08-20 | 2015-10-28 | 北京鑫诺美迪基因检测技术有限公司 | Primer combined fluorescent probe for detecting HBV (hepatitis B virus) amplification as well as kit |
| CN105063235A (en) * | 2015-07-28 | 2015-11-18 | 宝瑞源生物技术(北京)有限公司 | Kit for quickly and quantitatively detecting hepatitis b virus nucleic acid DNA (deoxyribonucleic acid) and using method of kit |
| CN106350610A (en) * | 2016-11-02 | 2017-01-25 | 中国科学院动物研究所 | Quantitative detection method of Plutella xylostella granulosis virus |
| CN107299097A (en) * | 2017-05-25 | 2017-10-27 | 北京立科技发展有限公司 | A kind of micro-nucleic acid releasing agent, preparation method and applications |
| CN112391445A (en) * | 2020-11-30 | 2021-02-23 | 江苏默乐生物科技股份有限公司 | Sample releasing agent, kit and pretreatment method for virus nucleic acid detection |
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| CN101586162A (en) * | 2009-06-10 | 2009-11-25 | 戴立忠 | Method of extracting target nucleic acid and performing PCR augmentation |
| CN101712954A (en) * | 2009-11-19 | 2010-05-26 | 戴立忠 | Reagent and method for quick release of nucleic acid |
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2011
- 2011-12-27 CN CN201110448524.3A patent/CN103184214B/en active Active
Patent Citations (3)
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| CN101586162A (en) * | 2009-06-10 | 2009-11-25 | 戴立忠 | Method of extracting target nucleic acid and performing PCR augmentation |
| CN101712954A (en) * | 2009-11-19 | 2010-05-26 | 戴立忠 | Reagent and method for quick release of nucleic acid |
| CN101713002A (en) * | 2009-11-19 | 2010-05-26 | 戴立忠 | Fluorescent quantitative PCR detection method of pathogen nucleic acid by one-step method |
Non-Patent Citations (1)
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| 岳振峰: "《食品中兽药残留检测指南》", 30 September 2010 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105063235A (en) * | 2015-07-28 | 2015-11-18 | 宝瑞源生物技术(北京)有限公司 | Kit for quickly and quantitatively detecting hepatitis b virus nucleic acid DNA (deoxyribonucleic acid) and using method of kit |
| CN105002175A (en) * | 2015-08-20 | 2015-10-28 | 北京鑫诺美迪基因检测技术有限公司 | Primer combined fluorescent probe for detecting HBV (hepatitis B virus) amplification as well as kit |
| CN106350610A (en) * | 2016-11-02 | 2017-01-25 | 中国科学院动物研究所 | Quantitative detection method of Plutella xylostella granulosis virus |
| CN107299097A (en) * | 2017-05-25 | 2017-10-27 | 北京立科技发展有限公司 | A kind of micro-nucleic acid releasing agent, preparation method and applications |
| CN112391445A (en) * | 2020-11-30 | 2021-02-23 | 江苏默乐生物科技股份有限公司 | Sample releasing agent, kit and pretreatment method for virus nucleic acid detection |
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| CN103184214B (en) | 2016-02-10 |
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