CN106754890A - The extracts kit and extracting method of a kind of viral RNA - Google Patents
The extracts kit and extracting method of a kind of viral RNA Download PDFInfo
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- CN106754890A CN106754890A CN201710052461.7A CN201710052461A CN106754890A CN 106754890 A CN106754890 A CN 106754890A CN 201710052461 A CN201710052461 A CN 201710052461A CN 106754890 A CN106754890 A CN 106754890A
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Abstract
The invention provides a kind of extracts kit of viral RNA, including cracking combines liquid A, cleaning solution B, eluent C and digestion solution D, and cracking contains 20~50mM sucrose with reference to liquid A, 20~50mM ammonium sulfate, 10~50mM Tris HCl, and 1~5%（V/V）Brij 58,0.2~0.5mg/mL magnetic bead；PH value is 4.5~5.5, and cleaning solution B contains 1~5%（V/V）Brij 58,5~25mM Tris HCl, 0.5~1mg/mL Proteinase Ks, pH value are 6~7；The extraction of RNA is carried out using the kit, the method is independent of traditional Chaotropic salt, be cleavable, absorption and washing RNA under less salt ionic environment, while being free of alcohols material in cleaning solution, you can removal related salinity and impurity.Method of the present invention simple possible, it is with low cost, with actual application value.
Description
Technical field
The present invention relates to technical field of molecular biological detection, more particularly, to a kind of extracts kit of viral RNA
And extracting method.
Background technology
Nucleic acid extraction is the first step of the whole techniqueflow of detection of nucleic acids, be also most critical in molecular biology method it
One.It is the basis of downstream nucleic acid detection and scientific research, and the quality and its integrality of the nucleic acid of extracting can directly affect clinic
Research or the result of diagnosis.General nucleic acid extraction process includes two big steps：The cracking and purifying of sample.Sample is instigated in cracking
In nucleic acid be discharged into reaction system, purifying is then to instigate other compositions in nucleic acid and reaction system, such as protein, salt
And other impurities are thoroughly separated.The extracting method of conventional RNA has TRIZOL methods, guanidine isothiocyanate method, CTAB-LiCl methods
And total RNA extraction reagent box of domestic and international each biotech firm etc..The classical way that RNA is extracted is TRIZOL methods, and it is a kind of base
In the method for isothiocyanic acid flesh/phenol/chloroform extraction principles, the method is applied widely, and animals and plants are all suitable for, the RNA bases of extraction
This pollution without genomic DNA, but the method complex operation step, have used the toxic reagents such as phenol, chloroform in extracts reagent,
And the method is not suitable for the extraction rich in polysaccharide polyphenol material RNA.CTAB-LiCl methods are often used to extract plant tissue
RNA, is in itself a reagent similar with SDS functions due to CTAB, although it can form insoluble with RNA and DNA
Compound, but CTAB methods generally will with reference to other operation, such as again through LiCl precipitation step so that the method is cumbersome, and
And use toxic reagent chloroform in extracting.Traditional extracts reagent, it is relatively low due to extracting yield, and step is more numerous
It is miscellaneous, certain influence is brought to work is extracted, therefore, it is difficult to obtain qualified RNA sample.
Paramagnetic particle method is the method for extracting nucleic acid for just growing up in recent years.A diameter of tens nanometers of magnetic bead to a few micrometers,
Magnetic is shown under magnetic field, the profile being made up of inorganic/organic or inorganic/inorganic material is rendered as spherical compound grain
Son.Paramagnetic particle method extracts nucleic acid and does not need the big organic solvent of toxicity such as phenol and chloroform, and the nucleic acid purity of extraction is high, yield is big.
Patent No. 201010281633.6,201510023368.4 etc. discloses the method for extracting viral DNA or RNA, but these sides
Method is all based on Chaotropic salt（Guanidine hydrochloride, guanidinium isothiocyanate, sodium perchlorate etc.）Lower cracking and adsorption of DNA or RNA, then
Washed with a certain proportion of ethanol, nucleic acid is discharged into eluent again finally.Wherein Chaotropic salt is a kind of broken
Bad element's power（Hydrogen bond）A kind of salt of stability, it can make hydrophobic protein be more soluble in water, it is impossible in less salt interference point
Between son by non-covalent bond（Power）The interaction of formation, the ability of magnetic bead absorption nucleic acid can increase in Chaotropic salt
By force, it is essentially all to be derived on this basis to extract RNA in domestic published patent at present.Prior art is few
It is not based on the method for the extraction RNA of Chaotropic salt.
The content of the invention
The technical problems to be solved by the invention are the drawbacks described above for overcoming prior art to exist, there is provided a kind of viral RNA
Extracts kit.
Second object of the present invention is to provide and a kind of carry out nucleic acid to viral RNA using above-mentioned RNA extracts kits and carry
The method for taking.
The purpose of the present invention is achieved by the following technical programs：
A kind of extracts kit of viral RNA, including cracking combines liquid A, cleaning solution B, eluent C and digestion solution D, the cracking
Contain 20~50mM sucrose with reference to liquid A, 20~50mM ammonium sulfate, 10~50mM Tris-HCl, 1~5%（V/V）Brij-58,
0.2~0.5mg/mL magnetic beads；PH value is 4.5~5.5, and the cleaning solution B contains 1~5%（V/V）Brij-58,5~25mM
Tris-HCl, 0.5~1mg/mL Proteinase K, pH value are 6~7.
The operation principle of kit of the present invention：In the presence of digestion solution D, the shell of virus is deteriorated, viral nucleic acid
RNA is released, while in the presence of cracking combines liquid A, RNA is can be incorporated on magnetic bead, then again by washing
Liquid B is cleaned to the RNA on magnetic bead, removes unnecessary salinity and protein molecular, is made on magnetic bead finally by eluent C
RNA is eluted.
Here the pH value cracked with reference to liquid A ensures 4.5~5.5, it is ensured that RNA is not dissolved, while the ring of low pH value
Nucleic acid is adsorbed in border beneficial to magnetic bead, and nucleic acid can be made farthest to combine on magnetic bead.
Preferably, the eluent C contains 10~20 mM Tris-HCl, 0.5mM EDTA, and pH value is 8.5.
Preferably, the digestion solution D contains 20mg/mL Proteinase Ks.
Preferably, the particle diameter of the magnetic bead is 0.5~1 μm.
The present invention also provides a kind of method that nucleic acid extraction is carried out using the kit, comprises the following steps：
S1. sampling originally, adds digestion solution D and cracking to combine liquid A, and after mixing centrifugation, supernatant is abandoned in suction；
S2. cleaning solution B is added, after mixing 10min centrifugations, is inhaled abandon supernatant again；
S3. cleaning solution B is added, after mixing 1min centrifugations, is inhaled abandon supernatant again；
S4. eluent C is added, warm bath is shaken mix for a period of time, during warm bath, after centrifugation, Aspirate supernatant to new centrifugation
Pipe obtains final product nucleic acid.
Preferably, the mixed volume ratio that sample described in S1 and cracking combine liquid A is 1：3.
Preferably, the condition of warm bath described in S4 is 56~75 DEG C, and the addition volume of eluent C is 50~100 μ L.
Preferably, the addition volume of cleaning solution B described in S2 and S3 is 300~600 μ L.
Used as a kind of specific embodiment, the method for carrying out nucleic acid extraction of the present invention is comprised the following steps：
S1. 200 μ L fresh plasmas or serum sample are added in 1.5ml centrifuge tubes, to adding the digestion solutions of 20 μ 1 in centrifuge tube
D and 600 μ 1 cracking combines liquid A, and room temperature is mixed 15 minutes, then centrifuge tube is placed on magnetic frame, magnetic separation 1min, and suction is abandoned
Clearly；
S2. to the cleaning solution B of 500 μ 1 are added in centrifuge tube, room temperature is mixed 10 minutes, then centrifuge tube is placed on magnetic frame, magnetic force
1min is separated, supernatant is abandoned in suction；
S3. to the cleaning solution B of 500 μ 1 are added in centrifuge tube, room temperature is mixed 1 minute, then centrifuge tube is placed on magnetic frame, magnetic force
1min is separated, supernatant is abandoned in suction；
S4. to adding the eluent C of 100 μ 1, warm bath 6 minutes in centrifuge tube（Period need to not stop concussion and mix）, after brief centrifugation again
Centrifuge tube is placed on magnetic frame, magnetic separation 1min, in absorption supernatant to new centrifuge tube.
Compared with prior art, the invention has the advantages that：
The invention provides a kind of extracts kit of viral RNA, including crack with reference to liquid A, cleaning solution B, eluent C and clear up
Liquid D, the cracking combination liquid A contain 20~50mM sucrose, 20~50mM ammonium sulfate, 10~50mM Tris-HCl, and 1~5%（V/
V）Brij-58,0.2~0.5mg/mL magnetic bead；PH value is 4.5~5.5, and the cleaning solution B contains 1~5%（V/V）Brij-
58,5~25mM Tris-HCl, 0.5~1mg/mL Proteinase Ks, pH value is 6~7；The extraction of RNA is carried out using the kit,
The method is independent of traditional Chaotropic salt, is cleavable, absorption and washing RNA under less salt ionic environment, while washing
Wash in liquid without alcohols material（Ethanol）Can remove related salinity and impurity.Method of the present invention simple possible, low cost
It is honest and clean and without it is any pollution environment composition.
Specific embodiment
The present invention is expanded on further with reference to specific embodiment.These embodiments be merely to illustrate the present invention rather than
Limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in lower example embodiment, generally according to this area normal condition
Or the condition advised according to manufacturer.Unless otherwise defined, all specialties used in text and scientific words and this area
Meaning is identical familiar to technical staff.
Embodiment 1
A kind of extracts kit of viral RNA, including cracking combines liquid A, cleaning solution B, eluent C, digestion solution D（Herein refer to
20mg/mL Proteinase Ks）.
Cracking is constituted with reference to liquid A：20mM sucrose, 50 mM ammonium sulfate, 10mM Tris-HCl（pH 4.0）, 1%（V/
V）Brij-58,0.5mg/ml magnetic bead, solvent are autoclaving water, and the whole pH that cracking combines liquid A is 4.5.
The composition of cleaning solution B is：1% （V/V）Brij-58,5 mM Tris-HCl（pH 6.6）, 1mg/ml Proteinase Ks,
Solvent is autoclaving water, and the whole pH of cleaning solution B is 6.6.
The composition of eluent C is：10mM Tris-HCl, the 0. 5 mM EDTA aqueous solution, the whole pH of eluent C is 8.5.
HIV-1 blood plasma or serum sample RNA are extracted using mentioned reagent box, is comprised the following steps：
（1）The fresh HIV-1 blood plasma of 200 μ L or serum sample are added in 1.5ml centrifuge tubes, are disappeared to 20 μ L are added in centrifuge tube
Solution liquid D and 600 μ L cracking combines liquid A, and room temperature is mixed 15 minutes, then centrifuge tube is placed on magnetic frame, magnetic separation 1min, inhales
Abandon supernatant；
（2）To 500 μ L cleaning solution B are added in centrifuge tube, room temperature is mixed 10 minutes, then centrifuge tube is placed on magnetic frame, magnetic force
1min is separated, supernatant is abandoned in suction；
（3）To 500 μ L cleaning solution B are added in centrifuge tube, room temperature is mixed 1 minute, then centrifuge tube is placed on magnetic frame, magnetic force point
From 1min, supernatant is abandoned in suction；
（4）To adding the eluent C of 100 μ 1, warm bath 6 minutes in centrifuge tube（Period need to not stop concussion and mix）, after brief centrifugation again
Centrifuge tube is placed on magnetic frame, magnetic separation 1min, in absorption supernatant to new centrifuge tube.
Contrast agents are 200 μ l from the sample size that QIAamp Viral RNA Mini Kit are extracted, and elution volume is
100µl。
After the completion of extraction, the above-mentioned μ l of nucleic acid RNA extract solutions 20 are taken as template, using the human immune deficiency up to peace gene
The type kit for detecting nucleic acid of syndrome virus 1（PCR fluorescence probe methods）Detected, testing result see the table below 1.
The different sample CT values of table 1 compare
| Sample number | Testing result of the present invention（CT values） | Qiagen reagent testing results（CT values） |
| 1 | 24.21 | 24.35 |
| 2 | 35.98 | 35.32 |
| 3 | 30.47 | 31.05 |
| 4 | 27.12 | 27.31 |
| 5 | 32.34 | 32.67 |
| 6 | 36.21. | 36.01 |
Result shows, using the efficiency and existing nucleic acid extracting reagent of kit extraction nucleic acid described in the embodiment of the present invention 1
Extraction efficiency is in same level, therefore, the present invention has a clear superiority in HIV-1 RNA nucleic acid extraction application aspects.
Embodiment 2
A kind of extracts kit of viral RNA, including cracking combines liquid A, cleaning solution B, eluent C, digestion solution D（Herein refer to
20mg/mL Proteinase Ks）.
Cracking is constituted with reference to liquid A：30mM sucrose, 30 mM ammonium sulfate, 30mM Tris-HCl（pH 4.0）, 3%（V/
V）Brij-58,0.4mg/ml magnetic bead, solvent are autoclaving water, and the whole pH that cracking combines liquid A is 5.0.
The composition of cleaning solution B is：3% （V/V）Brij-58,18 mM Tris-HCl（pH 6.6）, 0.8mg/ml protease
K, solvent is autoclaving water, and the whole pH of cleaning solution B is 6.0.
The composition of eluent C is：15mM Tris-HCl, the 0. 5 mM EDTA aqueous solution, the whole pH of eluent C is 8.5.
Embodiment 3
A kind of extracts kit of viral RNA, including cracking combines liquid A, cleaning solution B, eluent C, digestion solution D（Herein refer to
20mg/mL Proteinase Ks）.
Cracking is constituted with reference to liquid A：50mM sucrose, 20 mM ammonium sulfate, 50mM Tris-HCl（pH 4.0）, 5%（V/
V）Brij-58,0.5mg/ml magnetic bead, solvent are autoclaving water, and the whole pH that cracking combines liquid A is 5.5.
The composition of cleaning solution B is：5%（V/V）Brij-58,25 mM Tris-HCl（pH 6.6）, 0.5mg/ml protease
K, solvent is autoclaving water, and the whole pH of cleaning solution B is 7.0.
The composition of eluent C is：20mM Tris-HCl, the 0. 5 mM EDTA aqueous solution, the whole pH of eluent C is 8.5.
Comparative example 1
RNA kits used by the comparative example with RNA kits described in embodiment one, it is unique unlike, cracking combines liquid A's
PH value is 7.0, is detected that its result shows as using the methods described of embodiment 1：The result of comparative example 1 is significantly worse than implementation
Example 1, low concentration sample is without testing result.Testing result such as table 2.
The different sample CT values of table 2 compare
| Sample number | The result of embodiment 1（CT values） | The testing result of comparative example 1（CT values） |
| 1 | 24.58 | 28.98 |
| 2 | 35.12 | - |
| 3 | 30.06 | 38.05 |
| 4 | 27.32 | 32.14 |
| 5 | 32.54 | 38.37 |
| 6 | 36.83 | - |
Comparative example 2
RNA kits used by the comparative example with RNA kits described in embodiment one, it is unique unlike, Brij- in lysate
58 concentration is（V/V）10%, detected that its result shows as using the methods described of embodiment 1：The result of comparative example 2 is obvious
It is worse than embodiment 1, it was demonstrated that extraction efficiency is not reaching to optimal.Testing result such as table 3.
The different sample CT values of table 3 compare
| Sample number | The result of embodiment 1（CT values） | The testing result of comparative example 2（CT values） |
| 1 | 24.58 | 27.17 |
| 2 | 35.12 | 38.72 |
| 3 | 30.06 | 32.15 |
| 4 | 27.32 | 29.52 |
| 5 | 32.54 | 34.37 |
| 6 | 36.83 | 38.85 |
Claims (8)
1. a kind of extracts kit of viral RNA, it is characterised in that including cracking with reference to liquid A, cleaning solution B, eluent C and disappearing
Solution liquid D, the cracking combination liquid A contain 20~50mM sucrose, 20~50mM ammonium sulfate, 10~50mM Tris-HCl, and 1~5%
（V/V）Brij-58,0.2~0.5mg/mL magnetic bead；PH value is 4.5~5.5, and the cleaning solution B contains 1~5%（V/V）
Brij-58,5~25mM Tris-HCl, 0.5~1mg/mL Proteinase K, pH value are 6~7.
2. the extracts kit of viral RNA according to claim 1, it is characterised in that the eluent C contains 10~
20 mM Tris-HCl, 0.5mM EDTA, pH value is 8.5.
3. the extracts kit of viral RNA according to claim 1, it is characterised in that the digestion solution D contains 20mg/
ML Proteinase Ks.
4. the extracts kit of viral RNA according to claim 1, it is characterised in that the particle diameter of the magnetic bead is 0.5~
1μm。
5. a kind of method for carrying out nucleic acid extraction using kit described in any one of claim 1 to 4, it is characterised in that including
Following steps：
S1. sampling originally, adds digestion solution D and cracking to combine liquid A, and after mixing centrifugation, supernatant is abandoned in suction；
S2. cleaning solution B is added, after mixing 10min centrifugations, is inhaled abandon supernatant again；
S3. cleaning solution B is added, after mixing 1min centrifugations, is inhaled abandon supernatant again；
S4. eluent C is added, warm bath is shaken mix for a period of time, during warm bath, after centrifugation, Aspirate supernatant to new centrifugation
Pipe obtains final product nucleic acid.
6. method according to claim 5, it is characterised in that sample described in S1 and cracking combine the mixed volume ratio of liquid A
It is 1：3.
7. method according to claim 5, it is characterised in that the condition of warm bath described in S4 is 56~75 DEG C, eluent C's
Addition volume is 50~100 μ L.
8. method according to claim 5, it is characterised in that the addition volume of cleaning solution B described in S2 and S3 is 300~
600μL。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN112899266A (en) * | 2021-02-04 | 2021-06-04 | 北京中科医学检验实验室有限公司 | Cracking binding solution for nucleic acid extraction, kit and application thereof |
| CN112941066A (en) * | 2021-02-04 | 2021-06-11 | 天津诺道医学检验中心有限公司 | Lysis binding solution for bacterial nucleic acid extraction and kit and application thereof |
| CN112941067A (en) * | 2021-02-04 | 2021-06-11 | 天津诺道医学检验中心有限公司 | Lysis binding solution for whole blood nucleic acid extraction and kit and application thereof |
| EP3978623A1 (en) * | 2020-10-02 | 2022-04-06 | Luminultra Technologies Ltd. | Virus detection in wastewater |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| EP3978623A1 (en) * | 2020-10-02 | 2022-04-06 | Luminultra Technologies Ltd. | Virus detection in wastewater |
| CN112899266A (en) * | 2021-02-04 | 2021-06-04 | 北京中科医学检验实验室有限公司 | Cracking binding solution for nucleic acid extraction, kit and application thereof |
| CN112941066A (en) * | 2021-02-04 | 2021-06-11 | 天津诺道医学检验中心有限公司 | Lysis binding solution for bacterial nucleic acid extraction and kit and application thereof |
| CN112941067A (en) * | 2021-02-04 | 2021-06-11 | 天津诺道医学检验中心有限公司 | Lysis binding solution for whole blood nucleic acid extraction and kit and application thereof |
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