CN103614371A - Method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs - Google Patents
Method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs Download PDFInfo
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- CN103614371A CN103614371A CN201310594174.0A CN201310594174A CN103614371A CN 103614371 A CN103614371 A CN 103614371A CN 201310594174 A CN201310594174 A CN 201310594174A CN 103614371 A CN103614371 A CN 103614371A
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Abstract
The invention discloses a method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs. The method comprises the following steps of: carrying out lysis on a to-be-extracted substance by using guanidinium isothiocyanate lysate; adsorbing RNA by a silica gel membrane; removing impure protein by washing liquor I; removing impurities by washing liquor II; carrying out DEPC (Diethylpyrocarbonate) water-washing to remove nucleic acid, wherein the guanidinium isothiocyanate lysate comprises 3M-7M guanidinium isothiocyanate, 0.6%-1.0% TriTon-100, 30mM-50mM Tris-Cl, 5mM-15mM DTT (DL-Dithiothreitol), 60 mu g/mL-90 mu g/mL protease K, 10mM-30mM EDTA (Ethylene Diamine Tetraacetic Acid), and PH of the guanidinium isothiocyanate lysate is 4.3-4.6; the washing liquor I comprises 5M-6M guanidine hydrochloride, 53%-59% absolute ethyl alcohol, 70-90 mu g/mL protease K, and PH of the washing liquor I is 6.4-6.6; the washing liquor II comprises 70%-80% alcohol. The method disclosed by the invention has the advantages of being simple in extracting process, short in period, low in cost, and capable of simultaneously extracting RNA virus and DNA virus nucleic acid in an animal blood serum sample and a double-swab sample.
Description
Technical field
The present invention relates to a kind of method of simultaneously extracting animal DNA virus and RNA viruses nucleic acid in serum, two swab, be applicable to biological technical field.
Background technology
Along with molecular biological fast development, gene diagnosis becomes the most accurately and effectively means of detection animal epidemic, carries out in the process of gene diagnosis, and the extraction of viral nucleic acid is the precondition detecting, and the quality of nucleic acid extraction also affects the accuracy of detection.Current, animal virus method for extracting nucleic acid mainly contains classical Trizol extraction method and usings Trizol two kinds of methods of silica gel mould pillar extraction method as lysate.Classical Trizol extraction method process is loaded down with trivial details, the cycle is long, extraction efficiency is low, technical requirements is high, and the Trizol of usining is higher as the silica gel mould pillar extraction method cost of lysate, extract the of low quality of RNA and can only extract RNA viruses.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of simultaneously extracting animal DNA virus and RNA viruses nucleic acid in serum, two swab, and leaching process is simple, the cycle is short, cost is low, can extract RNA viruses and DNA virus nucleic acid in animal serum sample, two swab sample simultaneously.
The present invention is achieved through the following technical solutions.
A method of simultaneously extracting animal DNA virus and RNA viruses nucleic acid in serum, two swab, comprises step: treat extract and use the cracking of guanidinium isothiocyanate lysate; Pellosil absorption RNA; Washing lotion I is except foreigh protein removing; Washing lotion II washes away impurity; DEPC water elution nucleic acid; Above-mentioned guanidinium isothiocyanate lysate comprises 3-7M guanidinium isothiocyanate, 0.6%-1.0%TriTon-100,30-50mM Tris-Cl, 5-15mM DTT, 60-90 μ g/mL Proteinase K, 10-30mM EDTA, and PH is at 4.3-4.6; Above-mentioned washing lotion I comprises 5-6M Guanidinium hydrochloride, 53-59% dehydrated alcohol, and K70-90 μ g/mL proteolytic enzyme, PH is at 6.4-6.6; Above-mentioned washing lotion II comprises 70-80% ethanol.
Further, above-mentioned guanidinium isothiocyanate lysate comprises 5M guanidinium isothiocyanate, 0.8%TriTon-100,40mM Tris-Cl, 10mM DTT, 80 μ g/mL Proteinase Ks, 20mM EDTA, and pH is 4.5.
Further, above-mentioned washing lotion I comprises 5.5M Guanidinium hydrochloride, 56% dehydrated alcohol, and 80 μ g/mL Proteinase Ks, PH is 6.5.
Further, above-mentioned washing lotion II comprises 75% ethanol.
Further, aforesaid operations step comprises:
(1) get a clean centrifuge tube, 100 μ L samples add in the guanidinium isothiocyanate lysate of 200 μ L, and vibration mixes, and places 5 minutes;
(2) add and the isopyknic dehydrated alcohol of guanidinium isothiocyanate lysate, 200 μ L, put upside down and repeatedly mix 15 seconds;
(3) Filter column is enclosed within on 2mL collection tube, all mixed solution moves on on Filter column, centrifugal 1 minute of 12,000rpm;
(4) abandon the liquid in collection tube, toward Filter column, add the washing lotion 1 of 600 μ L, centrifugal 1 minute of 12000rpm;
(5) abandon the liquid in collection tube, toward Filter column, add the washing lotion 2 of 600 μ L, centrifugal 1 minute of 12000rpm;
(6) repeating step (5) once;
(7) abandon the liquid in collection tube, Filter column is enclosed within on collection tube, centrifugal 1 minute of 12000rpm;
(8) Filter column on the 1.5mL centrifuge trunnion renewing, adds 20 μ LDEPC to Filter column film, and room temperature is placed 1 minute, and centrifugal 1 minute of 12,000rpm collects filtrate, preserves.
Beneficial effect of the present invention:
Extract RNA viruses and DNA virus nucleic acid in animal serum sample, two swab sample simultaneously, have the advantages that method is easy, quick, cost is low.The method is for setting up animal RNA viruses and DNA virus multiple PCR method is established nucleic acid extraction basis.
Embodiment
According to embodiment, the present invention is described in further detail below.
Case study on implementation 1:
Liquor is used in preparation:
Guanidinium isothiocyanate lysate: composition is 5M guanidinium isothiocyanate, 0.8%TriTon-100,40mM Tris-Cl, 10mM DTT, 80 μ g/mL Proteinase Ks, 20mM EDTA, and pH is 4.5.
Washing lotion I: composition is 5.5M Guanidinium hydrochloride, 56% dehydrated alcohol, 80 μ g/mL Proteinase Ks, PH is 6.5.
Washing lotion II: composition is 75% ethanol.
Case study on implementation 2:
The method of simultaneously extracting animal DNA virus and RNA viruses nucleic acid in serum, two swab, comprises step:
(1) get a clean centrifuge tube, 100 μ L samples add in the guanidinium isothiocyanate lysate of 200 μ L, and vibration mixes, and places 5 minutes;
(2) add and the isopyknic dehydrated alcohol of guanidinium isothiocyanate lysate, 200 μ L, put upside down and repeatedly mix 15 seconds;
(3) Filter column is enclosed within on 2mL collection tube, all mixed solution moves on on Filter column, centrifugal 1 minute of 12,000rpm;
(4) abandon the liquid in collection tube, toward Filter column, add the washing lotion 1 of 600 μ L, centrifugal 1 minute of 12000rpm;
(5) abandon the liquid in collection tube, toward Filter column, add the washing lotion 2 of 600 μ L, centrifugal 1 minute of 12000rpm;
(6) repeating step (5) once;
(7) abandon the liquid in collection tube, Filter column is enclosed within on collection tube, centrifugal 1 minute of 12000rpm;
(8) Filter column on the 1.5mL centrifuge trunnion renewing, adds 20 μ LDEPC to Filter column film, and room temperature is placed 1 minute, and centrifugal 1 minute of 12,000rpm collects filtrate, preserves.
Above-described embodiment is only explanation technical conceive of the present invention and feature, and its object is to allow the personage who is familiar with this art can understand content of the present invention and be implemented, and can not limit the scope of the invention with this.All equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (5)
1. a method of simultaneously extracting animal DNA virus and RNA viruses nucleic acid in serum, two swab, is characterized in that, comprises step: treat extract and use the cracking of guanidinium isothiocyanate lysate; Pellosil absorption RNA; Washing lotion I is except foreigh protein removing; Washing lotion II washes away impurity; DEPC water elution nucleic acid; Described guanidinium isothiocyanate lysate comprises 3-7M guanidinium isothiocyanate, 0.6%-1.0%TriTon-100,30-50mM Tris-Cl, 5-15mM DTT, 60-90 μ g/mL Proteinase K, 10-30mM EDTA, and PH is at 4.3-4.6; Described washing lotion I comprises 5-6M Guanidinium hydrochloride, 53-59% dehydrated alcohol, and K70-90 μ g/mL proteolytic enzyme, PH is at 6.4-6.6; Described washing lotion II comprises 70-80% ethanol.
2. the method for simultaneously extracting animal DNA virus and RNA viruses nucleic acid in serum, two swab according to claim 1, it is characterized in that, described guanidinium isothiocyanate lysate comprises 5M guanidinium isothiocyanate, 0.8%TriTon-100,40mM Tris-Cl, 10mM DTT, 80 μ g/mL Proteinase Ks, 20mM EDTA, and pH is 4.5.
3. the method for simultaneously extracting animal DNA virus and RNA viruses nucleic acid in serum, two swab according to claim 1, is characterized in that, described washing lotion I comprises 5.5M Guanidinium hydrochloride, 56% dehydrated alcohol, and 80 μ g/mL Proteinase Ks, PH is 6.5.
4. the method for simultaneously extracting animal DNA virus and RNA viruses nucleic acid in serum, two swab according to claim 1, is characterized in that, described washing lotion II comprises 75% ethanol.
5. according to time described in claim 1-4, extract the method for animal DNA virus and RNA viruses nucleic acid in serum, two swab, it is characterized in that, described operation steps comprises:
(1) get a clean centrifuge tube, 100 μ L samples add in the guanidinium isothiocyanate lysate of 200 μ L, and vibration mixes, and places 5 minutes;
(2) add and the isopyknic dehydrated alcohol of guanidinium isothiocyanate lysate, 200 μ L, put upside down and repeatedly mix 15 seconds;
(3) Filter column is enclosed within on 2mL collection tube, all mixed solution moves on on Filter column, centrifugal 1 minute of 12,000rpm;
(4) abandon the liquid in collection tube, toward Filter column, add the washing lotion 1 of 600 μ L, centrifugal 1 minute of 12000rpm;
(5) abandon the liquid in collection tube, toward Filter column, add the washing lotion 2 of 600 μ L, centrifugal 1 minute of 12000rpm;
(6) repeating step (5) once;
(7) abandon the liquid in collection tube, Filter column is enclosed within on collection tube, centrifugal 1 minute of 12000rpm;
(8) Filter column on the 1.5mL centrifuge trunnion renewing, adds 20 μ LDEPC to Filter column film, and room temperature is placed 1 minute, and centrifugal 1 minute of 12,000rpm collects filtrate, preserves.
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| CN201310594174.0A CN103614371A (en) | 2013-11-21 | 2013-11-21 | Method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs |
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| CN201310594174.0A CN103614371A (en) | 2013-11-21 | 2013-11-21 | Method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs |
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Cited By (15)
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| CN104611324A (en) * | 2014-06-09 | 2015-05-13 | 南京美宁康诚生物科技有限公司 | Whole blood sample fast pyrolysis kit and detection method thereof |
| CN104805073A (en) * | 2015-04-21 | 2015-07-29 | 益百尚(北京)生物技术有限责任公司 | Kit for extracting viral genome nucleic acid and use method thereof |
| CN105316317A (en) * | 2015-12-08 | 2016-02-10 | 山东莱博生物科技有限公司 | Viral nucleic acid lysate and application thereof in extraction of viral nucleic acid by utilizing silica membrane adsorption column |
| CN107916262A (en) * | 2016-10-09 | 2018-04-17 | 上海怡泽生物科技有限公司 | A kind of method and reagent for being used to extract nucleic acid from cell |
| CN108070584A (en) * | 2016-11-16 | 2018-05-25 | 江苏然科生物技术有限公司 | The silica gel adsorption column extracts kit and method of a kind of blood plasma or free serum DNA and RNA |
| CN109863393A (en) * | 2016-08-15 | 2019-06-07 | 格雷斯坎私人有限公司 | Elution and detection |
| CN110760567A (en) * | 2019-11-12 | 2020-02-07 | 杭州昱鼎生物科技有限公司 | Urine sample RNA stabilizing solution and preparation method thereof |
| CN111041024A (en) * | 2019-12-31 | 2020-04-21 | 阿吉安(福州)基因医学检验实验室有限公司 | Nucleic acid extraction kit and method for extracting nucleic acid |
| CN111534510A (en) * | 2020-05-21 | 2020-08-14 | 上海领骏生物科技有限公司 | Kit and method for extracting pathogen nucleic acid from throat swab sample |
| 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 |
| CN112501158A (en) * | 2020-12-04 | 2021-03-16 | 麦凯(上海)生物科技有限公司 | Human liquid sample whole nucleic acid extraction kit and application thereof |
| CN112931487A (en) * | 2021-02-08 | 2021-06-11 | 中国科学院合肥物质科学研究院 | Virus preserving fluid 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 |
| CN114351261A (en) * | 2022-02-28 | 2022-04-15 | 江苏先声医学诊断有限公司 | Method for detecting respiratory tract sample difficultly-detected pathogenic microorganisms based on nanopore sequencing platform |
| CN114517196A (en) * | 2022-02-25 | 2022-05-20 | 杭州诺辉健康科技有限公司 | Extraction method and application of plasma free miRNA |
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| CN104611324A (en) * | 2014-06-09 | 2015-05-13 | 南京美宁康诚生物科技有限公司 | Whole blood sample fast pyrolysis kit and detection method thereof |
| CN104805073A (en) * | 2015-04-21 | 2015-07-29 | 益百尚(北京)生物技术有限责任公司 | Kit for extracting viral genome nucleic acid and use method thereof |
| CN105316317A (en) * | 2015-12-08 | 2016-02-10 | 山东莱博生物科技有限公司 | Viral nucleic acid lysate and application thereof in extraction of viral nucleic acid by utilizing silica membrane adsorption column |
| CN109863393A (en) * | 2016-08-15 | 2019-06-07 | 格雷斯坎私人有限公司 | Elution and detection |
| CN107916262A (en) * | 2016-10-09 | 2018-04-17 | 上海怡泽生物科技有限公司 | A kind of method and reagent for being used to extract nucleic acid from cell |
| CN108070584A (en) * | 2016-11-16 | 2018-05-25 | 江苏然科生物技术有限公司 | The silica gel adsorption column extracts kit and method of a kind of blood plasma or free serum DNA and RNA |
| CN110760567A (en) * | 2019-11-12 | 2020-02-07 | 杭州昱鼎生物科技有限公司 | Urine sample RNA stabilizing solution and preparation method thereof |
| CN111041024A (en) * | 2019-12-31 | 2020-04-21 | 阿吉安(福州)基因医学检验实验室有限公司 | Nucleic acid extraction kit and method for extracting nucleic acid |
| CN111534510A (en) * | 2020-05-21 | 2020-08-14 | 上海领骏生物科技有限公司 | Kit and method for extracting pathogen nucleic acid from throat swab sample |
| 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 |
| CN112501158A (en) * | 2020-12-04 | 2021-03-16 | 麦凯(上海)生物科技有限公司 | Human liquid sample whole nucleic acid extraction kit and application thereof |
| CN112931487A (en) * | 2021-02-08 | 2021-06-11 | 中国科学院合肥物质科学研究院 | Virus preserving fluid and application thereof |
| CN112931487B (en) * | 2021-02-08 | 2023-12-22 | 中国科学院合肥物质科学研究院 | Virus preservation solution 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 |
| CN114517196A (en) * | 2022-02-25 | 2022-05-20 | 杭州诺辉健康科技有限公司 | Extraction method and application of plasma free miRNA |
| CN114351261A (en) * | 2022-02-28 | 2022-04-15 | 江苏先声医学诊断有限公司 | Method for detecting respiratory tract sample difficultly-detected pathogenic microorganisms based on nanopore sequencing platform |
| CN114351261B (en) * | 2022-02-28 | 2023-12-15 | 江苏先声医学诊断有限公司 | Detection method for difficult-to-detect pathogenic microorganisms in respiratory tract sample based on nanopore sequencing platform |
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