CN108676794A - A kind of rapid extracting method for animal tissue's pathological material of disease nucleic acid - Google Patents
A kind of rapid extracting method for animal tissue's pathological material of disease nucleic acid Download PDFInfo
- Publication number
- CN108676794A CN108676794A CN201810755759.9A CN201810755759A CN108676794A CN 108676794 A CN108676794 A CN 108676794A CN 201810755759 A CN201810755759 A CN 201810755759A CN 108676794 A CN108676794 A CN 108676794A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- lysate
- animal tissue
- pathological material
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- 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
Abstract
The invention discloses a kind of rapid extracting methods for animal tissue's pathological material of disease nucleic acid, are related to nucleic acid extraction technical field.Lysate is prepared including (1)；(2) Tissue Lysis；(3) initial gross separation；(4) nucleic acid precipitates；(5) nucleic acid purification.The present invention is by improving the lysate used during nucleic acid extraction, the method for enormously simplifying nucleic acid extraction, it is easy to operate and safe, it is of low cost, it solves existing method for extracting nucleic acid and is not suitable with outdoor environment, detection time is longer and extracts reagent used contains poisonous and harmful substance and there are problems that security risk；The extraction to DNA and RNA respectively can be realized by adjusting the pH value of lysate, solve the problems, such as that the methods of available reagent box can only extract single nucleic acid.
Description
Technical field
The invention belongs to nucleic acid extraction technical fields, more particularly to a kind of quickly carrying for animal tissue's pathological material of disease nucleic acid
Take method.
Background technology
Nucleic acid (including DNA and RNA) extraction purification is a basic operation in molecular biology, Protocols in Molecular Biology
With it quickly, it is sensitive, special the advantages that and in the detection of animal epidemic, make a definite diagnosis and more and more applied in work, at present
The extraction of nucleic acid includes that phenol extraction method, salt wash method, TRIZOL methods, paramagnetic particle method and RNA isolation kit etc., but traditional detection method
It is higher to instrument and environmental requirement, outdoor work is cannot be satisfied, to further increase the level of port animal quarantine, invention is a kind of
The new method that can meet port Fast Detection Technique but also meet the accurate detection technique requirement in laboratory is particularly important；Patent
Application 201010621890 discloses a kind of cell cracking agent for biological sample amplifying nucleic acid extraction purification, the reagent solution
In alkalinity and contain guanidine salt, ammonium ion and surfactant component, biological sample can be more fully cracked under alkaline liquid phase environment
Cell in product, and the combination that can promote to be discharged into extracellular nucleic acid (DNA/RNA) and solid phase material is adsorbed, finally by washing
De- step separates nucleic acid from solid phase material.Although disclosing the alkaline bleach liquor cleavage condition of pH8~13 in the patent application,
But it is only capable of making the pH of lysate to reach 9 or so by adding ammonium ion.Although and higher alkaline item can be reached by being passed through ammonia
Part, but ammonia is volatile, is not easy to preserve steadily in the long term, be unfavorable in outdoor work.
Invention content
The purpose of the present invention is to provide a kind of rapid extracting methods for animal tissue's pathological material of disease nucleic acid, by improving core
The lysate used in sour extraction process, the method for enormously simplifying nucleic acid extraction is easy to operate and safe, of low cost, solves
Existing method for extracting nucleic acid is not suitable with outdoor environment, and detection time is longer and extracts reagent used contains poisonous and harmful substance
There are problems that security risk, can realize the extraction to DNA and RNA respectively by adjusting the pH value of lysate, solve existing
The methods of kit can only extract the problem of single nucleic acid.
In order to solve the above technical problems, the present invention is achieved by the following technical solutions：
The present invention is a kind of rapid extracting method for animal tissue's pathological material of disease nucleic acid, is included the following steps：
(1) lysate is prepared；The lysate includes guanidine salt, buffer, surfactant, PH conditioning agents；
(2) Tissue Lysis：Lysate is added in tissue samples, is also homogenized to no visible tissue block with Syrup-homogenizing instrument；
(3) initial gross separation：It places 15 minutes, 13000g is centrifuged 5 minutes, detaches supernatant；
(4) nucleic acid precipitates：If the isopropanol of dry volume is added into supernatant, 10000g is centrifuged 10 minutes, reject supernatant
Liquid obtains nucleic acid precipitation；
(5) nucleic acid purification：It using a concentration of 75% ethyl alcohol washing precipitate, is purified, obtains the nucleic acid to be extracted.
Further, the guanidine salt is guanidinium isothiocyanate；The buffer is sodium citrate；The surfactant is the moon
Osmanthus base sodium sulphate；The PH conditioning agents are hydrochloric acid.
Further, the cracking liquid making method is：Weigh the lemon of a certain amount of guanidinium isothiocyanate, 0.75mol/L
Sour sodium and 10% NaLS, be added deionized water heating stirring dissolving, using hydrochloric acid conditioning solution pH value be 7-9 it
Between, it is packed into brown bottle, room temperature preservation.
Further, the lysate includes the lemon of the guanidinium isothiocyanate of final concentration of 4-6mol/L, 20-25mol/L
The NaLS of sour sodium and 0.2-0.5%.
Further, the nucleic acid is DNA and RNA.
The invention has the advantages that
1, the lysate that the present invention is used in the process by improving nucleic acid extraction, the method for enormously simplifying nucleic acid extraction,
Detection time is short, completes only to need 30 minutes from be homogenized to extracting.
2, easy to operate, outdoor environment is adapted to, the rapid quarantine operation of port animal is convenient for.
3, the extraction to DNA and RNA respectively can be realized by adjusting the pH value of lysate, solving existing method can only carry
The problem of taking single nucleic acid.
4, safe operation, used reagent is without organic solvents such as poisonous and hazardous phenol.
5, economical cheap, it is low to experimental facilities requirement, effectively save cost.
Certainly, it implements any of the products of the present invention and does not necessarily require achieving all the advantages described above at the same time.
Specific implementation mode
It is right below to extract the method for sending qin worm DNA and foot and mouth disease virus and PPR virus RNA in clam tissue
Technical solution in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only the present invention one
Section Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making
Go out all other embodiment obtained under the premise of creative work, shall fall within the protection scope of the present invention.
Implementation column 1
(1) prepare lysate, accurately weigh guanidinium isothiocyanate 35.45g, 0.75mol/L sodium citrate 1.67ml and
10% NaLS 2.5ml is added the dissolving of 10ml deionized water heating stirrings, is using the pH value of hydrochloric acid conditioning solution
8.5, be configured to the guanidinium isothiocyanate of final concentration of 6mol/L, the sodium citrate of 25mol/L and 0.2% NaLS it is molten
Liquid is settled to 50ml, is put into brown bottle, room temperature preservation；
(2) it takes 4 clam sons to weigh cheek tissue about 50mg respectively, respectively with Syrup-homogenizing instrument homogenate is held, is separately added into 1ml preparations
Lysate, homogenate is to no visible tissue block；
(3) 15 minutes are placed at room temperature for, 13000g is centrifuged 5 minutes, and supernatant is moved to new pipe；
(4) isopropanol of 0.5ml is added into supernatant, 10000g is centrifuged 10 minutes, reject supernatant, and sediment is
DNA；
(5) use 1.5ml a concentration of 75% ethyl alcohol washing precipitate, DNA is purified, obtain to be extracted send qin
Worm DNA；55 degrees Celsius of 50uL sterile waters can be added to place 2 minutes, dissolving DNA precipitation, -20 degrees Celsius save backup.
Embodiment 2
(1) prepare lysate, accurately weigh guanidinium isothiocyanate 29.54g, 0.75mol/L sodium citrate 1.67ml and
The dissolving of 10ml deionized water heating stirrings is added in 10% NaLS 2.5ml, and the pH value using hydrochloric acid conditioning solution is 7,
Be configured to the guanidinium isothiocyanate of final concentration of 5mol/L, the sodium citrate of 25mol/L and 0.2% sodium lauryl sulfate solution,
It is settled to 50ml, is put into brown bottle, room temperature preservation；
(2) 4 parts of foot and mouth disease viruses and each 200uL of PPR virus culture solution are taken, splitting for 1ml preparations is separately added into
Solve liquid；
(3) 10 minutes are placed at room temperature for, 10000g is centrifuged 5 minutes, and supernatant is moved to new pipe；
(4) isopropanol of 0.5ml is added into supernatant, 10000g is centrifuged 8 minutes, reject supernatant, and sediment is
RNA；
(5) the ethyl alcohol washing precipitate for using 1.5ml a concentration of 75%, qin is sent to what RNA carried out purifying to be extracted
Worm DNA；55 degrees Celsius of 50uL sterile waters can be added to place 2 minutes, dissolving DNA precipitation, -80 degrees Celsius save backup.
Comparative example 1
TRIZOL methods：The cheek tissue for taking 1 identical 4 clam son of same embodiment carries out DNA extractions using TRIZOL methods.
Comparative example 2
RNA isolation kit：The cheek tissue for taking 1 identical 4 clam son of same embodiment carries out DNA extractions using RNA isolation kit.
Using common PCR detection method respectively to method for extracting nucleic acid provided by the invention, TRIZOL methods and kit
What method was extracted sends qin worm DNA to be detected, and real-time fluorescence quantitative PCR accurate quantitative analysis original template copy number is used in combination, according to Ct
It is worth (critical cycle number) and CV values (coefficient of variation) compares the sensitivity of three kinds of methods, the results are shown in Table 1；
Table 1.Ct values and CV Data-Statistics
By table one it can be seen that the qin worm DNA coefficient of variation of sending of TRIZOL methods extraction is 7.63%, RNA isolation kit extraction
The qin worm DNA coefficient of variation of sending be 2.68%, the qin worm DNA coefficient of variation of sending of method for extracting nucleic acid of the present invention extraction is
1.78%, it is not difficult to find out that the method for extracting nucleic acid coefficient of variation provided by the present invention is small, has absolutely proved provided by the present invention
Method for extracting nucleic acid is with good stability compared to traditional method and repeated.
In the description of this specification, the description of reference term " one embodiment ", " example ", " specific example " etc. means
Particular features, structures, materials, or characteristics described in conjunction with this embodiment or example are contained at least one implementation of the present invention
In example or example.In the present specification, schematic expression of the above terms may not refer to the same embodiment or example.
Moreover, particular features, structures, materials, or characteristics described can be in any one or more of the embodiments or examples to close
Suitable mode combines.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.There is no detailed for preferred embodiment
All details are described, are not limited the invention to the specific embodiments described.Obviously, according to the content of this specification,
It can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is in order to preferably explain the present invention
Principle and practical application, to enable skilled artisan to be best understood by and utilize the present invention.The present invention is only
It is limited by claims and its full scope and equivalent.
Claims (5)
1. a kind of rapid extracting method for animal tissue's pathological material of disease nucleic acid, which is characterized in that include the following steps：
(1) lysate is prepared；The lysate includes guanidine salt, buffer, surfactant and PH conditioning agents；
(2) Tissue Lysis：Lysate is added in tissue samples, is also homogenized to no visible tissue block with Syrup-homogenizing instrument；
(3) initial gross separation：It places 15 minutes, 13000g is centrifuged 5 minutes, detaches supernatant；
(4) nucleic acid precipitates：If the isopropanol of dry volume is added into supernatant, 10000g is centrifuged 10 minutes, and reject supernatant obtains
Nucleic acid precipitates；
(5) nucleic acid purification：It using a concentration of 75% ethyl alcohol washing precipitate, is purified, obtains the nucleic acid to be extracted.
2. the rapid extracting method according to claim 1 for animal tissue's pathological material of disease nucleic acid, which is characterized in that the guanidine
Salt is guanidinium isothiocyanate；The buffer is sodium citrate；The surfactant is NaLS；The PH conditioning agents
For hydrochloric acid.
3. the rapid extracting method according to claim 1 for animal tissue's pathological material of disease nucleic acid, which is characterized in that described to split
Solving liquid making method is：A certain amount of guanidinium isothiocyanate, the sodium citrate of 0.75mol/L and 10% NaLS are weighed,
The dissolving of deionized water heating stirring is added, using the pH value of hydrochloric acid conditioning solution between 7-9, is packed into brown bottle, room
Temperature preserves.
4. the rapid extracting method according to claim 2 or 3 for animal tissue's pathological material of disease nucleic acid, which is characterized in that institute
State the moon that lysate includes the guanidinium isothiocyanate of final concentration of 4-6mol/L, the sodium citrate of 20-25mol/L and 0.2-0.5%
Osmanthus base sodium sulphate.
5. the rapid extracting method according to claim 1 for animal tissue's pathological material of disease nucleic acid, which is characterized in that the core
Acid is DNA and RNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810755759.9A CN108676794A (en) | 2018-07-11 | 2018-07-11 | A kind of rapid extracting method for animal tissue's pathological material of disease nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810755759.9A CN108676794A (en) | 2018-07-11 | 2018-07-11 | A kind of rapid extracting method for animal tissue's pathological material of disease nucleic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108676794A true CN108676794A (en) | 2018-10-19 |
Family
ID=63813897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810755759.9A Withdrawn CN108676794A (en) | 2018-07-11 | 2018-07-11 | A kind of rapid extracting method for animal tissue's pathological material of disease nucleic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108676794A (en) |
-
2018
- 2018-07-11 CN CN201810755759.9A patent/CN108676794A/en not_active Withdrawn
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7727718B2 (en) | Reagents for storage and preparation of samples for DNA analysis | |
| US10308929B2 (en) | Method of preparing biological material | |
| EP3636769A1 (en) | Sample nucleic acid measurement test kit, reagent, and application thereof | |
| CN105176969B (en) | A kind of method for extracting nucleic acid and reagent for biological sample | |
| CN101200716B (en) | Nucleic acid isolation using polidocanol and derivatives | |
| CN105247065A (en) | Compositions and methods for nucleic acid extraction | |
| CN113151397B (en) | Nucleic acid extraction kit for extracting virus sample based on magnetic bead method | |
| CN105112400A (en) | Kit for extracting free DNA | |
| CN104962553A (en) | Using method for extracting virus DNA by using micro-nucleic acid releasing agent | |
| DE60109272T2 (en) | PROCESS FOR CLEANING PROTEINS OR MIXTURES OF PROTEINS AND NUCLEIC ACIDS AND SUBSEQUENT COMPOUNDS OF PARTICLES AND PROTEINS | |
| CN105734044A (en) | Rinsing liquid for nucleic acid extraction purification | |
| EP2115123A2 (en) | Selective lysis of sperm cells | |
| CN102146112B (en) | Method for extracting desoxyribonucleic acid from formalin fixed and paraffin embedded tissues | |
| Laurin et al. | New incompatibilities uncovered using the Promega DNA IQ™ chemistry | |
| EP3904532A1 (en) | Nucleic acid extraction composition, reagent and kit containing the same and use thereof | |
| CN103789277A (en) | Preparation method of artificial dual false virus particle comprising HCV (hepatitis C virus) and HIV (human immunodeficiency virus) nucleic acid fragments | |
| CN105950610A (en) | Method for rapidly extracting fecal bacterial genome DNA and virus RNA through paramagnetic particle method | |
| CN103710338A (en) | Kit for extracting DNA (deoxyribonucleic acid) from white cells in human whole blood | |
| CN112391445A (en) | Sample releasing agent, kit and pretreatment method for virus nucleic acid detection | |
| CN109913445B (en) | One-step washing magnetic bead method blood DNA extraction kit | |
| CN101629217B (en) | RT-LAMP detection kit and detection method of swine influenza virus | |
| CN103184214A (en) | Hepatitis B virus nucleic acid rapid extraction reagent | |
| Lee et al. | DNA extraction methods in forensic analysis | |
| CN108676794A (en) | A kind of rapid extracting method for animal tissue's pathological material of disease nucleic acid | |
| Aguado et al. | VirWaTest, a point-of-use method for the detection of viruses in water samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20181019 |