CN106754881A - A kind of blood disease RNA protective agents and preparation method and application - Google Patents
A kind of blood disease RNA protective agents and preparation method and application Download PDFInfo
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- CN106754881A CN106754881A CN201611214325.5A CN201611214325A CN106754881A CN 106754881 A CN106754881 A CN 106754881A CN 201611214325 A CN201611214325 A CN 201611214325A CN 106754881 A CN106754881 A CN 106754881A
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- 239000003223 protective agent Substances 0.000 title claims abstract description 30
- 208000019838 Blood disease Diseases 0.000 title claims abstract description 26
- 208000014951 hematologic disease Diseases 0.000 title claims abstract description 26
- 208000018706 hematopoietic system disease Diseases 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 5
- 239000008280 blood Substances 0.000 claims abstract description 24
- 210000004369 blood Anatomy 0.000 claims abstract description 24
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 22
- 108020000999 Viral RNA Proteins 0.000 claims abstract description 16
- 239000004471 Glycine Substances 0.000 claims abstract description 11
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims abstract description 11
- MGSKVZWGBWPBTF-UHFFFAOYSA-N aebsf Chemical compound NCCC1=CC=C(S(F)(=O)=O)C=C1 MGSKVZWGBWPBTF-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000002342 ribonucleoside Substances 0.000 claims abstract description 11
- JZBRFIUYUGTUGG-UHFFFAOYSA-J tetrapotassium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].[K+].[K+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O JZBRFIUYUGTUGG-UHFFFAOYSA-J 0.000 claims abstract description 10
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 4
- 230000004224 protection Effects 0.000 claims abstract description 4
- 239000003795 chemical substances by application Substances 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 3
- 230000007774 longterm Effects 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 238000012546 transfer Methods 0.000 claims description 2
- -1 wherein bestain Substances 0.000 claims 1
- 102000006382 Ribonucleases Human genes 0.000 abstract description 3
- 108010083644 Ribonucleases Proteins 0.000 abstract description 3
- 230000023555 blood coagulation Effects 0.000 abstract description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 abstract 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 24
- 108020004707 nucleic acids Proteins 0.000 description 21
- 102000039446 nucleic acids Human genes 0.000 description 21
- 150000007523 nucleic acids Chemical class 0.000 description 21
- 238000000605 extraction Methods 0.000 description 19
- 108090000623 proteins and genes Proteins 0.000 description 17
- 238000012360 testing method Methods 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 9
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 229960002897 heparin Drugs 0.000 description 8
- 229920000669 heparin Polymers 0.000 description 8
- 239000002253 acid Substances 0.000 description 5
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 3
- 241000723873 Tobacco mosaic virus Species 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 241001115402 Ebolavirus Species 0.000 description 1
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- 208000005155 Picornaviridae Infections Diseases 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011814 protection agent Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses a kind of blood disease RNA protective agents and preparation method thereof, the protective agent is that shield agent can not only prevent blood clotting, and can suppress RNase protection viral RNA therein.The protective agent is to be dissolved in DEPC water to be formulated by bestain, glycine, EDTA 3K, IDU, AEBSF, ribonucleoside complex, and wherein concentration of each component in the DEPC water-soluble liquids is respectively：bestain（0.05‑0.2g/L）、glycine（20‑50g/L）、EDTA‑3K（30g‑70g/L）、IDU（100‑400g/L）、AEBSF（0.15‑0.4g/L）, ribonucleoside complex（0.4ml‑1ml/L）.The blood that the present invention will be gathered is with blood disease RNA protective agents with 35:After 2 ratio is slowly fully mixed, blood disease RNA can be preserved 3 days under normal temperature condition, and 20 DEG C to 80 DEG C can preserve for a long time, it is adaptable to the protection and storage of various blood disease RNA.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of blood disease RNA protective agents and preparation method thereof with should
With.
Background technology
Viral (Virus) is made up of or only by protein a kind of nucleic acid molecules (DNA or RNA) and protein (Protein)
Constitute (such as prion).Viral volume is very small, structure and its simple, but the parasitics with height, is completely dependent on host
The energy and metabolic system of cell.When it touches host cell, just deproteinising overcoat, its nucleic acid (gene) is invaded
In host cell, by the dubbing system of the latter, new virus is replicated according to the instruction of viral gene, eventually cause host cell
It is dead.DNA virus are essentially all RNA virus seldom in nature, such as AIDS virus, tobacco mosaic virus (TMV), SARS diseases
Poison, MERS viruses, Ebola virus（EBV）, spanish influenza virus, H1N1virus, avian influenza virus etc..
After human body is by picornavirus infection, with the amount reproduction of virus, viral RNA is can detect in human blood, therefore can
Just can be confirmed whether to have infected virus by carrying out corresponding correlated virus detection to blood.So, the detection of blood disease RNA
And its aspects such as gene diagnosis application for RNA virus etc. diagnosis and monitoring tool be of great significance.
But viral RNA content in blood is few, and RNase content is more in blood, viral RNA is in the state without protection
Under be easy to degraded and disappear, so a kind of protective agent of effective and feasible extension viral RNA pot-life of research and development is necessary
's.
The content of the invention
It is an object of the invention to propose a kind of blood disease RNA protective agents, the protective agent can not only prevent blood clotting,
And viral RNA molecules can be protected, it is to avoid it is degraded by RNase, and the change of gene expression variation in vivo is minimized.
Protectant composition is：Bestain, glycine, EDTA-3K, IDU, AEBSF, ribonucleoside complex, DEPC water
Lysate, blood and blood disease RNA protective agents with 35:After 2 ratio is slowly fully mixed, room temperature places about half an hour
, room temperature can preserve 3 days, and -20 DEG C to -80 DEG C long-term preserve.
The purpose of the present invention can be achieved through the following technical solutions：A kind of blood disease RNA protective agents, the protection
Agent is to be dissolved in DEPC water by bestain, glycine, EDTA-3K, IDU, AEBSF, ribonucleoside complex to be formulated, its
Concentration of the middle each component in the DEPC water-soluble liquids is respectively：bestain（0.05-0.2g/L）、glycine（20-50g/
L）、EDTA-3K（30g-70g/L）、IDU（100-400g/L）、AEBSF（0.15-0.4g/L）, ribonucleoside complex
（0.4ml-1ml/L）.
Preferably, blood disease RNA protective agents of the present invention be by bestain, glycine, EDTA-3K,
IDU, AEBSF, ribonucleoside complex are dissolved in DEPC water and are formulated according to certain ratio, wherein bestain,
The concentration of glycine, EDTA-3K, IDU, AEBSF, ribonucleoside complex in the solution be respectively 0.1g/L, 30g/L,
50g/L、250g/L、0.235g/L、0.7ml/L。
It is of the present invention to prepare the protectant method of viral RNA, comprise the following steps that：
1）Claim by formula（Inhale）Take during each group assigns to container；
2）To 1）The sterilized DEPC water of middle addition 800ml, after after all components dissolving, 7.35-7.45 is adjusted to by solution ph,
Transfer the solution into again in 1L volumetric flasks, be settled to 1L, obtain viral RNA protective agent.
The application of blood disease RNA protective agents described in claim any one of 1-2 in blood disease RNA preservations, its
It is characterised by, in the application, the blood that will be gathered is with viral RNA protective agent with 35:After 2 ratio is slowly fully mixed,
Room temperature places about half an hour.
After half an hour according to claim 4, it is characterised in that room temperature can be preserved 3 days, and -20 DEG C to -80 DEG C can grow
Phase preserves.
Advantages of the present invention：
1. the viral RNA in blood can be protected in the very first time；
2. the integrality of viral RNA is better ensured that.
Brief description of the drawings
Fig. 1 carries out core for the whole blood of infected by HIV is placed in after blood disease as herein described is preserved 0 day in RNA protective agents
Acid is extracted, and the nucleic acid that extraction is obtained carries out the testing result of HIV gene real-time fluorescence PCRs
Fig. 2 carries out nucleic acid and carries for the whole blood of infected by HIV is placed in after blood disease as herein described is preserved 1 day in RNA protective agents
Take, the nucleic acid that extraction is obtained carries out the testing result of HIV gene real-time fluorescence PCRs
Fig. 3 carries out nucleic acid and carries for the whole blood of infected by HIV is placed in after blood disease as herein described is preserved 2 days in RNA protective agents
Take, the nucleic acid that extraction is obtained carries out the testing result of HIV gene real-time fluorescence PCRs
Fig. 4 carries out nucleic acid and carries for the whole blood of infected by HIV is placed in after blood disease as herein described is preserved 3 days in RNA protective agents
Take, the nucleic acid that extraction is obtained carries out the testing result of HIV gene real-time fluorescence PCRs
Fig. 5 carries out nucleic acid extraction, the core that extraction is obtained after being preserved 0 day in EDTA-Na2 heparin tubes for the whole blood of infected by HIV is placed in
Acid carries out the testing result of HIV gene real-time fluorescence PCRs
Fig. 6 carries out nucleic acid extraction, the core that extraction is obtained after being preserved 1 day in EDTA-Na2 heparin tubes for the whole blood of infected by HIV is placed in
Acid carries out the testing result of HIV gene real-time fluorescence PCRs
Fig. 7 carries out nucleic acid extraction, the core that extraction is obtained after being preserved 2 days in EDTA-Na2 heparin tubes for the whole blood of infected by HIV is placed in
Acid carries out the testing result of HIV gene real-time fluorescence PCRs
Fig. 8 carries out nucleic acid extraction, the core that extraction is obtained after being preserved 3 days in EDTA-Na2 heparin tubes for the whole blood of infected by HIV is placed in
Acid carries out the testing result of HIV gene real-time fluorescence PCRs
Specific implementation method
Following case study on implementation is only used for explaining the present invention, and not limits the present invention.
Embodiment 1：
5 parts of people's whole blood samples of infected by HIV are randomly selected, is divided into 8 person-portions by 5ml/ person-portions respectively, wherein 4 person-portions are 1 group, point
Into 2 groups.Wherein 1 group is placed in conventional EDTA-Na2In heparin tube, another 1 group is placed in blood disease RNA protective agents of the present invention
In, respectively room temperature 0 day, 1 day, 2 days, after 3 days carry out nucleic acid extraction（RNeasy Mini Kit (50) of Qiagen companies
The kit of-article No. 74104）, enter the real-time PCR detection of pedestrian's HIV genes as sample with the nucleic acid for extracting.
Testing result：
It is placed in conventional EDTA-Na2The whole blood for having infected HIV in heparin tube and blood disease RNA protective agents of the present invention
Sample, after room temperature different time, carries out nucleic acid extraction respectively, and the nucleic acid of extraction carries out the real-time fluorescence PCR of HIV genes
Detection, the Ct values such as table 1 of its testing result.As shown in Table 1, blood disease RNA protective agents can effectively protect blood in 3 days
Viral RNA in liquid, only extremely slight degraded；And routine EDTA-Na2Stability maintenance time of the heparin tube to viral RNA
Less than 1 day and degraded situation than more serious.
Table 1
Fig. 1 ~ Fig. 8 is the real-time PCR detection result figure of the corresponding HIV genes of table 1, and wherein Fig. 1 ~ Fig. 4 is placed in this for whole blood
Nucleic acid extraction is carried out after different time is preserved in free virus RNA protective agents described in text, the nucleic acid that extraction is obtained carries out HIV bases
Because of the testing result figure of real-time fluorescence PCR, as seen from the figure, in the detection time of 0 ~ 3 day, all detection samples are all correctly examined
Go out, and have good stability, virus in blood RNA is effectively protected；Fig. 5 ~ Fig. 8 conventional EDTA-Na for whole blood is placed in2Adopt
Nucleic acid extraction is carried out after different time is preserved in blood vessel, the nucleic acid that extraction is obtained carries out the detection knot of HIV gene real-time fluorescence PCRs
Fruit is schemed, and testing result shows, conventional EDTA-Na2The viral RNA stability of 1 day in heparin tube can not all be maintained, feelings of degrading
Condition is extremely serious.
Claims (5)
1. a kind of blood disease RNA protective agents, it is characterised in that the protective agent be by bestain, glycine, EDTA-3K,
IDU, AEBSF, ribonucleoside complex are formulated in being dissolved in DEPC water, and wherein each component is in the DEPC water-soluble liquids
Concentration be respectively：bestain（0.05-0.2g/L）、glycine（20-50g/L）、EDTA-3K（30g-70g/L）、IDU
（100-400g/L）、AEBSF（0.15-0.4g/L）, ribonucleoside complex（0.4ml-1ml/L）.
2. blood disease RNA protective agents according to claim 1, it is characterised in that the free RNA protections of described blood
Agent is dissolved according to certain ratio by bestain, glycine, EDTA-3K, IDU, AEBSF, ribonucleoside complex
It is formulated in DEPC water, wherein bestain, glycine, EDTA-3K, IDU, AEBSF, ribonucleoside complex are described
Concentration in solution is respectively 0.1g/L, 30g/L, 50g/L, 250g/L, 0.235g/L, 0.7ml/L.
3. one kind prepares the protectant methods of blood disease RNA described in claim any one of 1-2, it is characterised in that described
Preparation method is comprised the following steps that：
1）Claim by formula（Inhale）Take during each group assigns to container；
2）To 1）The sterilized DEPC water of middle addition 800ml, after after all components dissolving, 7.35-7.45 is adjusted to by solution ph,
Transfer the solution into again in 1L volumetric flasks, be settled to 1L, obtain viral RNA protective agent.
4. 1 in claim 3）-2）Blood disease RNA protective agents answering in blood disease RNA preservations described in any one
With, it is characterised in that in the application, the blood that will be gathered is with viral RNA protective agent with 35:2 ratio is slowly fully mixed
After even, room temperature places about half an hour.
5. after half an hour according to claim 4, it is characterised in that room temperature can be preserved 3 days, and -20 DEG C to -80 DEG C can be long-term
Preserve.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611214325.5A CN106754881A (en) | 2016-12-26 | 2016-12-26 | A kind of blood disease RNA protective agents and preparation method and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611214325.5A CN106754881A (en) | 2016-12-26 | 2016-12-26 | A kind of blood disease RNA protective agents and preparation method and application |
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| Publication Number | Publication Date |
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| CN106754881A true CN106754881A (en) | 2017-05-31 |
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ID=58920697
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679946A (en) * | 2019-01-04 | 2019-04-26 | 宁波艾捷康宁生物科技有限公司 | A kind of blood disease RAN protective agent and heparin tube |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010134246A1 (en) * | 2009-05-20 | 2010-11-25 | オリンパス株式会社 | Method for preparation of nucleic acid-containing sample |
| CN103789202A (en) * | 2014-01-26 | 2014-05-14 | 付士明 | Container for collecting nucleic acid preserved at normal temperature |
| CN105431536A (en) * | 2013-08-16 | 2016-03-23 | 通用电气公司 | Methods and compositions for extracting and storing nucleic acids |
-
2016
- 2016-12-26 CN CN201611214325.5A patent/CN106754881A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010134246A1 (en) * | 2009-05-20 | 2010-11-25 | オリンパス株式会社 | Method for preparation of nucleic acid-containing sample |
| CN105431536A (en) * | 2013-08-16 | 2016-03-23 | 通用电气公司 | Methods and compositions for extracting and storing nucleic acids |
| CN103789202A (en) * | 2014-01-26 | 2014-05-14 | 付士明 | Container for collecting nucleic acid preserved at normal temperature |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679946A (en) * | 2019-01-04 | 2019-04-26 | 宁波艾捷康宁生物科技有限公司 | A kind of blood disease RAN protective agent and heparin tube |
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Application publication date: 20170531 |