CN104673783A - Kit for extracting DNA/RNA of virus through magnetic bead method and using method - Google Patents
Kit for extracting DNA/RNA of virus through magnetic bead method and using method Download PDFInfo
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- CN104673783A CN104673783A CN201510023368.4A CN201510023368A CN104673783A CN 104673783 A CN104673783 A CN 104673783A CN 201510023368 A CN201510023368 A CN 201510023368A CN 104673783 A CN104673783 A CN 104673783A
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Abstract
The invention discloses a kit for extracting DNA/RNA of a virus by utilizing magnetic beads and a using method. The kit comprises the following eight components: an extracting solution I (cracking), an extracting solution II (binding), an extracting solution III (scrubbing), an extracting solution IV (scrubbing), an extracting solution V (eluting), magnetic bead suspension, a protease K working solution and a nucleic acid settling agent. The kit extraction method comprises the following steps: cracking, binding, scrubbing and eluting. The kit and the extraction method can be used for extracting the DNA/RNA of the viruses of different types of samples, the impurities such as proteins and lipids in the samples are effectively removed, the extracted nucleic acid is high in purity and complete in fragments, and the extraction process is safe and non-toxic.
Description
Technical field
The invention belongs to technical field of molecular biology, particularly relate to reagent and using method thereof that a kind of paramagnetic particle method extracts viral DNA/RNA.
Background technology
Background technology describes paragraph Protocols in Molecular Biology and can be applicable to the research of heredopathia and the detection of pathogenic agent, and gene molecule level has been brought up in the research of the aspects such as the nosetiology of tumour, pathogenesis, Diagnosis and Treat, the isolation and purification technology of nucleic acid is a basic fundamental of Biochemistry and Molecular Biology.Nucleic acid is the carrier of genetic information, is the basic substance of genetic expression.No matter carry out nucleic acid construct or functional study, first need to be separated nucleic acid.
Although current separate nucleic acid technology development rapidly, but all there are some shortcomings: such as: although phenol chloroform extraction method nucleic acid purity is high, but it is harmful, and easily cause environmental pollution, leaching process needs extracting repeatedly simultaneously, repeatedly replace tubes, complex steps, can cause loss and the pollution of sample.Alkaline lysis: the nucleic acid that this method is extracted is not easy to preserve, and can not be used for extracting RNA.Pellosil adsorption column method extract nucleic acid DNA/RNA concentration purity low, realize automatization difficulty, unit time flux is low.
Magnetic bead is by the magnetic particle such as Z 250 or ferric oxide and the various spherical particle be composited containing the earth silicon material of activity functional groups, magnetic bead surfaces can be endowed various active functional group, as-COOH ,-OH etc., also can the biologically active substance such as covalent attachment enzyme, cell, antibody.Magnetic bead can occur to assemble or dispersion under magnetic field condition, when particle diameter is less than certain value, magnetic bead is just provided with good instantaneous magnetic responsiveness, namely superparamagnetism, the magnetic that in occurring, particle is such can be avoided to reunite, because have superparamagnetism, magnetic bead can be decided to be, lead and be separated under the effect of externally-applied magnetic field.The specific groups of surface bonding and active substance can Specific adsorption nucleic acid, and reach the effect of isolating nucleic acid under the influence of a magnetic field.
Summary of the invention
The present invention is devoted to a kind of quick, automatable extensive extraction nuclei aoid methods of exploitation, and provides a kind of test kit adopting magnetic bead to extract also purification of nucleic acid from sample.This test kit is easy and simple to handle, and extraction nucleic acid fragment is complete, purity is high, is applicable to the downstream experiments such as detection, clone, sequential analysis, pcr amplification, molecular hybridization and order-checking.
This reagent constituents is as follows: extracting solution I (cracking), extracting solution II (combination), extracting solution III (washing), extracting solution IV (washing), extracting solution V (wash-out), bead suspension, Proteinase K working fluid, nucleic acid settling agent.
Described extracting solution I (cracking) is the solution containing tensio-active agent, preferably 2M-5M GuSCN, 60-95mM Tris-HCl, 0.5%-2% Triton X-100,20 ~ 100mM EDTA, the pH value=6-7 of described extracting solution I.
Described extracting solution II (combination) preferably 0.6-3 M NaCl, 0.6%-3% beta-mercaptoethanol, 50-200mM HEPES.
Described extracting solution III (washing) preferably 50%-65% ethanol, 20-200mM NaCl and 20-150mM Tris-HCl.
The aqueous solution of described extracting solution IV (washing) preferably 20-200mM Tris-HCl.
Described extracting solution V (wash-out) containing 10 mM Tris-HCl, 1mM EDTA, pH value=8.0.
Described bead suspension preferably magnetic bead content is 30 ~ 60mg/ml, and magnetic bead particles diameter is 0.2-1.5 μm.
Described Proteinase K working fluid preferably final concentration is 1-20mg/ml.
Described nucleic acid settling agent is molecular biology grade 5-30mg/mlAcryl polymer solution.
Above-mentioned paramagnetic particle method extracts the test kit extracting method of viral nucleic acid, and it comprises the following steps:
1) in biological specimen, add extracting solution I, magnetic bead, Proteinase K, extracting solution II, make sample cracking discharge DNA/RNA, DNA/RNA is combined with nanometer magnetic bead, and under the action of a magnetic field, magnetic bead is assembled, magnetic bead-nucleic acid complexes and liquid separation;
2) add extracting solution III to magnetic bead-nucleic acid complexes, wash away the impurity such as composite surface protein, lipid and salt ion, under the action of a magnetic field, obtain the magnetic bead-nucleic acid complexes after washing;
3) add extracting solution IV to magnetic bead-nucleic acid complexes, wash away the impurity such as composite surface protein, lipid and salt ion, under the action of a magnetic field, obtain the magnetic bead-nucleic acid complexes after washing;
4) add extracting solution V to the magnetic bead-nucleic acid complexes after washing, change magnetic bead and nucleic acid are in conjunction with situation, and eluted on magnetic bead by nucleic acid, under the action of a magnetic field, magnetic bead and nucleic acid DNA/RNA solution separating, obtain the nucleic acid DNA/RNA of purifying.
This test kit is tried out sample and is comprised serum, blood plasma, tissue extract, swab washing lotion, secretory product, ight soil, virus-culturing fluid and other acellular body fluid samples.
The nucleic acid that the present invention extracts directly can carry out the research of PCR and RT-PCR equimolecular biological experiment and clinical detection.
This test kit can with coordinating self-reacting device, and high-throughput extracts viral nucleic acid.
Accompanying drawing explanation
The middle inspection institute National reference L0-L7 amplification that Fig. 1 extracts for employing test kit of the present invention.
Fig. 2 is the different extent of dilution amplification of HCV adopting test kit of the present invention to extract.
Embodiment
Reference once embodiment is also described in further detail the present invention by reference to the accompanying drawings; But following examples are only illustrations, and the present invention is not limited to these embodiments.
The Making and banking of serum or plasma lipid sample:
1) serum preparation: extract person under inspection's venous blood 2ml with disposable sterilized injector, inject aseptic dry glass tube, room temperature places 30-60 minute, but be no more than 4 hours, blood specimen can separate out serum by self-solidifying, or the centrifugal 5-10 minute of 1500rpm, draw upper serum and be transferred in sterile centrifugation tube;
2) blood plasma preparation: extract person under inspection's venous blood 2ml with disposable sterilized injector, inject the Glass tubing containing disodium ethylene diamine tetraacetate (EDTA) or sodium citrate anticoagulant, putting upside down Glass tubing immediately gently makes venous blood and antithrombotics fully mix, room temperature places 5-10 min, the centrifugal 5-10 minute of 1500rpm, draws upper plasma and is transferred in sterile centrifugation tube.
The serum prepared or blood plasma room temperature are placed and are no more than 2 hours, and 4 DEG C of preservations are no more than 48 hours, and-20 DEG C of preservations are no more than half a year.Need add ice bag sealing with 0 DEG C of curling stone or bubble chamber during transport, the time limit in transit was no more than 48 hours.
Embodiment 1: with middle inspection institute hepatitis B (HBV) national standard linear sensitivity L0-L7 for sample to be tested, L7 is that L6 10 times dilutes gained, concentration is 3-30 IU/ml, carries out extraction and fluorescence quantitative PCR detection by test kit of the present invention and using method to above-mentioned sample.
1) in 1.5ml nuclease free centrifuge tube, extracting solution I 300ul is added, Proteinase K 60ul, nucleic acid settling agent 4ul, magnetic bead 20ul, country is with reference to dish each sensitivity reference material 200ul, extracting solution II 100ul, room temperature places 15min, and period ceaselessly puts upside down mixing, and magnetic bead is fully contacted with nucleic acid, 1500rpm brief centrifugation, centrifuge tube is placed in magnetic frame 2min, magnetic bead in pipe is adsorbed, siphons away liquid in pipe with pipettor.2) add extracting solution III 550ul, vibration 5s, makes magnetic bead suspend, brief centrifugation, centrifuge tube is placed in 2min on magnetic frame, magnetic bead in pipe is adsorbed, siphons away liquid in pipe with pipettor, then repeat aforesaid operations 1 time.3) keep centrifuge tube on magnetic frame, leave standstill 1min, siphon away raffinate in pipe, add extracting solution IV 600ul, magnetic frame is blown and beaten 5-6 time repeatedly with pipettor, magnetic frame absorption 1min, makes magnetic bead in pipe be adsorbed, leaves standstill 1min, siphon away raffinate in pipe with pipettor.4) add 50ul extracting solution V wash-out, pressure-vaccum mixes, then 55 DEG C of temperature bath 10min, and period puts upside down mixing 4 times, centrifuge tube is placed in 2min on magnetic frame, magnetic bead in pipe is adsorbed, is moved on in new centrifuge tube by liquid rotating.
Embodiment 2: with hepatitis C (HCV) strong positive serum sample for sample to be tested, 10 times of gradient dilutions, carry out extraction and fluorescence quantitative PCR detection by this test kit and inventive method to above-mentioned sample.
1) in 1.5ml nuclease free centrifuge tube, extracting solution I 300ul is added, Proteinase K 60ul, nucleic acid settling agent 4ul, magnetic bead 20ul, containing serum sample 200ul, extracting solution II 100ul of hepatitis C virus, room temperature places 15min, and period ceaselessly puts upside down mixing, and magnetic bead is fully contacted with nucleic acid, 1500rpm brief centrifugation, centrifuge tube is placed in magnetic frame 2min, magnetic bead in pipe is adsorbed, siphons away liquid in pipe with pipettor.2) add extracting solution III 550ul, vibration 5s, makes magnetic bead suspend, brief centrifugation, centrifuge tube is placed in 2min on magnetic frame, magnetic bead in pipe is adsorbed, siphons away liquid in pipe with pipettor, then repeat aforesaid operations 1 time.3) keep centrifuge tube on magnetic frame, leave standstill 1min, siphon away raffinate in pipe, add extracting solution IV 600ul, magnetic frame is blown and beaten 5-6 time repeatedly with pipettor, magnetic frame absorption 1min, makes magnetic bead in pipe be adsorbed, leaves standstill 1min, siphon away raffinate in pipe with pipettor.4) add 50ul extracting solution V wash-out, pressure-vaccum mixes, then 55 DEG C of temperature bath 10min, and period puts upside down mixing 4 times, centrifuge tube is placed in 2min on magnetic frame, magnetic bead in pipe is adsorbed, is moved on in new centrifuge tube by liquid rotating.
Claims (5)
1. paramagnetic particle method extracts a method of viral DNA/RNA, it is characterized in that, comprises the following steps:
In biological specimen, add extracting solution I, magnetic bead, Proteinase K, extracting solution II, make sample cracking discharge DNA/RNA, DNA/RNA is combined with nanometer magnetic bead, and under the action of a magnetic field, magnetic bead is assembled, magnetic bead-nucleic acid complexes and liquid separation;
Add extracting solution III to magnetic bead-nucleic acid complexes, wash away the impurity such as composite surface protein, lipid and salt ion, under the action of a magnetic field, obtain the magnetic bead-nucleic acid complexes after washing;
Add extracting solution IV to magnetic bead-nucleic acid complexes, wash away the impurity such as composite surface protein, lipid and salt ion, under the action of a magnetic field, obtain the magnetic bead-nucleic acid complexes after washing;
Add extracting solution V to the magnetic bead-nucleic acid complexes after washing, change magnetic bead and nucleic acid are in conjunction with situation, and eluted on magnetic bead by nucleic acid, under the action of a magnetic field, magnetic bead and nucleic acid DNA/RNA solution separating, obtain the nucleic acid DNA/RNA of purifying.
2. the method for extracting nucleic acid of using nanometer magnetic beads according to claim 1, it is characterized in that, reagent constituents comprises extracting solution I (cracking), extracting solution II (combination), extracting solution III (washing), extracting solution IV (washing), extracting solution V (wash-out), bead suspension, Proteinase K working fluid, nucleic acid settling agent;
Described extracting solution I (cracking) is containing 2M-5M GuSCN, 60-95mM Tris-HCl, 0.5%-2% Triton X-100,20 ~ 100mM EDTA;
Described extracting solution II (combination) is 0.6-3 M NaCl, 0.6%-3% beta-mercaptoethanol, 50-200mM HEPES;
Described extracting solution III (washing) is containing 50%-65% ethanol, 20-200mM NaCl and 20-150mM Tris-HCl;
The aqueous solution that described extracting solution IV (washing) is 20-200mM Tris-HCl;
Described extracting solution V (wash-out) containing 10mM Tris-HCl, 1mM EDTA, pH value=8.0;
Described bead suspension is magnetic bead content is 30-60mg/ml, and magnetic bead particles diameter is 0.2-1.5 μm;
Described Proteinase K working fluid final concentration is 1-20mg/ml;
Described nucleic acid settling agent is molecular biology grade 5-30mg/mlAcryl polymer solution.
3. according to claims 2 reagent constituents, it is characterized in that, extracting solution I (cracking) pH value=6-7.
4. the using nanometer magnetic beads according to claims 1,2 extracts the test kit of viral nucleic acid, it is characterized in that, biological sample can be extracted and comprise serum, blood plasma, tissue extract, swab washing lotion, secretory product, ight soil, virus-culturing fluid and other acellular body fluid samples.
5. the bead suspension according to claims 2, is characterized in that, nanometer magnetic bead surface has nucleocapsid structure, i.e. superparamagnetism monox nanometer magnetic micro-beads, and magnetic bead particle diameter is 0.2-1.5 μm.
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925568A (en) * | 2016-06-20 | 2016-09-07 | 杭州市第人民医院 | Method for co-extracting DNA/RNA (Deoxyribonucleic Acid/Ribonucleic Acid) virus nucleic acid |
| CN105925569A (en) * | 2016-06-27 | 2016-09-07 | 北京卓诚惠生生物科技股份有限公司 | Kit and method for rapidly extracting bacterial genomic DNA from clinical sample |
| CN106754890A (en) * | 2017-01-24 | 2017-05-31 | 南方医科大学 | The extracts kit and extracting method of a kind of viral RNA |
| CN107058286A (en) * | 2016-12-29 | 2017-08-18 | 苏州英芮诚生化科技有限公司 | The kit and its application method of oral cavity sample genomic dna are extracted based on paramagnetic particle method |
| CN107460190A (en) * | 2017-09-06 | 2017-12-12 | 成都晟博源生物工程有限公司 | A kind of extracting method of bacteria RNA |
| CN108642044A (en) * | 2018-04-28 | 2018-10-12 | 广州奕昕生物科技有限公司 | Kit and method for extracting viral nucleic acid |
| CN108676791A (en) * | 2018-04-25 | 2018-10-19 | 浙江迪恩生物科技股份有限公司 | A kind of kit and extracting method of paramagnetic particle method extraction DNA |
| CN108753767A (en) * | 2018-08-02 | 2018-11-06 | 广州奕昕生物科技有限公司 | A kind of Viral nucleic acid extraction reagent box and extracting method |
| CN109439655A (en) * | 2018-12-25 | 2019-03-08 | 北京优迅医学检验实验室有限公司 | The kit and method extracted suitable for ultramicron cellular nucleic acid |
| CN109722431A (en) * | 2019-01-21 | 2019-05-07 | 上海科华生物工程股份有限公司 | It is a kind of based on paramagnetic particle method without alcohol Viral nucleic acid extraction reagent box |
| CN109750030A (en) * | 2019-01-21 | 2019-05-14 | 上海科华生物工程股份有限公司 | A kind of quick, nucleic acid extraction kit for not heating based on paramagnetic particle method |
| CN109762874A (en) * | 2018-12-30 | 2019-05-17 | 北京优迅医疗器械有限公司 | Nucleic acid settling agent, pregnant woman blood plasma dissociative DNA extracts kit and method |
| CN110819626A (en) * | 2019-11-29 | 2020-02-21 | 上海之江生物科技股份有限公司 | Lysis solution for extracting HCMV nucleic acid by paramagnetic particle method and application thereof |
| CN111500677A (en) * | 2020-05-18 | 2020-08-07 | 浙江树人学院（浙江树人大学） | Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA |
| CN111826374A (en) * | 2020-08-18 | 2020-10-27 | 上海派森诺生物科技股份有限公司 | RNA virus nucleic acid extracting solution and extracting method |
| CN112226432A (en) * | 2020-12-11 | 2021-01-15 | 北京健为医学检验实验室有限公司 | Rapid nucleic acid extraction kit by magnetic bead method and application thereof |
| CN113493783A (en) * | 2021-07-23 | 2021-10-12 | 杭州圣庭医疗科技有限公司 | Method for co-extracting DNA and RNA of different samples |
| CN114457069A (en) * | 2022-03-07 | 2022-05-10 | 江苏迅睿生物技术有限公司 | Magnetotherapeutic bead method pathogen nucleic acid extraction kit and use method thereof |
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| CN105925568A (en) * | 2016-06-20 | 2016-09-07 | 杭州市第人民医院 | Method for co-extracting DNA/RNA (Deoxyribonucleic Acid/Ribonucleic Acid) virus nucleic acid |
| CN105925569A (en) * | 2016-06-27 | 2016-09-07 | 北京卓诚惠生生物科技股份有限公司 | Kit and method for rapidly extracting bacterial genomic DNA from clinical sample |
| CN107058286A (en) * | 2016-12-29 | 2017-08-18 | 苏州英芮诚生化科技有限公司 | The kit and its application method of oral cavity sample genomic dna are extracted based on paramagnetic particle method |
| CN106754890B (en) * | 2017-01-24 | 2020-05-08 | 南方医科大学 | Extraction kit and extraction method of virus RNA |
| CN106754890A (en) * | 2017-01-24 | 2017-05-31 | 南方医科大学 | The extracts kit and extracting method of a kind of viral RNA |
| CN107460190A (en) * | 2017-09-06 | 2017-12-12 | 成都晟博源生物工程有限公司 | A kind of extracting method of bacteria RNA |
| CN108676791A (en) * | 2018-04-25 | 2018-10-19 | 浙江迪恩生物科技股份有限公司 | A kind of kit and extracting method of paramagnetic particle method extraction DNA |
| CN108676791B (en) * | 2018-04-25 | 2022-03-25 | 浙江迪恩生物科技股份有限公司 | Kit for extracting DNA by paramagnetic particle method and extraction method |
| CN108642044A (en) * | 2018-04-28 | 2018-10-12 | 广州奕昕生物科技有限公司 | Kit and method for extracting viral nucleic acid |
| CN108753767A (en) * | 2018-08-02 | 2018-11-06 | 广州奕昕生物科技有限公司 | A kind of Viral nucleic acid extraction reagent box and extracting method |
| CN109439655A (en) * | 2018-12-25 | 2019-03-08 | 北京优迅医学检验实验室有限公司 | The kit and method extracted suitable for ultramicron cellular nucleic acid |
| CN109762874A (en) * | 2018-12-30 | 2019-05-17 | 北京优迅医疗器械有限公司 | Nucleic acid settling agent, pregnant woman blood plasma dissociative DNA extracts kit and method |
| CN109750030A (en) * | 2019-01-21 | 2019-05-14 | 上海科华生物工程股份有限公司 | A kind of quick, nucleic acid extraction kit for not heating based on paramagnetic particle method |
| CN109722431A (en) * | 2019-01-21 | 2019-05-07 | 上海科华生物工程股份有限公司 | It is a kind of based on paramagnetic particle method without alcohol Viral nucleic acid extraction reagent box |
| CN109722431B (en) * | 2019-01-21 | 2024-01-23 | 上海科华生物工程股份有限公司 | Non-alcohol virus nucleic acid extraction kit based on magnetic bead method |
| CN110819626A (en) * | 2019-11-29 | 2020-02-21 | 上海之江生物科技股份有限公司 | Lysis solution for extracting HCMV nucleic acid by paramagnetic particle method and application thereof |
| CN110819626B (en) * | 2019-11-29 | 2021-11-26 | 上海之江生物科技股份有限公司 | Lysis solution for extracting HCMV nucleic acid by paramagnetic particle method and application thereof |
| CN111500677A (en) * | 2020-05-18 | 2020-08-07 | 浙江树人学院（浙江树人大学） | Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA |
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| CN112226432A (en) * | 2020-12-11 | 2021-01-15 | 北京健为医学检验实验室有限公司 | Rapid nucleic acid extraction kit by magnetic bead method and application thereof |
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| CN113493783A (en) * | 2021-07-23 | 2021-10-12 | 杭州圣庭医疗科技有限公司 | Method for co-extracting DNA and RNA of different samples |
| CN114457069A (en) * | 2022-03-07 | 2022-05-10 | 江苏迅睿生物技术有限公司 | Magnetotherapeutic bead method pathogen nucleic acid extraction kit and use method thereof |
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Application publication date: 20150603 |