CN109207475A - A kind of Rapid nucleic acid extracting method - Google Patents
A kind of Rapid nucleic acid extracting method Download PDFInfo
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- CN109207475A CN109207475A CN201811227897.6A CN201811227897A CN109207475A CN 109207475 A CN109207475 A CN 109207475A CN 201811227897 A CN201811227897 A CN 201811227897A CN 109207475 A CN109207475 A CN 109207475A
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- nucleic acid
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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
Abstract
The invention discloses a kind of Rapid nucleic acid extracting methods, method includes the following steps: Step 1: cracking nucleic acid samples: the sample containing nucleic acid being added in lysate, nucleic acid cleavage liquid is formed；Step 2: absorption nucleic acid: carrier being added in the nucleic acid cleavage liquid, the carrier of absorption nucleic acid is obtained；Step 3: removing impurity: the carrier for having adsorbed nucleic acid being rinsed in rinsing liquid, except the epinucleic impurity of removal；Step 4: amplified reaction: the carrier for being adsorbed with nucleic acid after rinsing being directly added into nucleic acid amplification system and is carried out amplification reaction；The carrier is cellulosic filter paper；The present invention provide it is a kind of simple, quickly, based on cellulosic filter paper be to extract nucleic acid in carrier, suitable for the nucleic acid amplification technologies such as PCR amplification and recombinase polymerase constant-temperature amplification, it is useful in room temperature-operating, biological sample suitable for various sources, the cellulosic filter paper for being loaded with nucleic acid can be directly added into nucleic acid amplification system and be carried out amplification reaction, whole process time-consuming is few.
Description
Technical field
The present invention relates to nucleic acid extraction field, in particular to a kind of Rapid nucleic acid extracting method.
Background technique
It is the pretreatment containing nucleic acid material before expanding for the significant challenge for carrying out molecular diagnosis using nucleic acid amplification technologies
And many and diverse nucleic acid extraction step.It is now widely used to be lacked based on silica and based on the technology of magnetic bead there is many
It falls into, including efficiency is too low (≤10%)；It is also in need to be optimized for particular target type, cause a kind of mentioning for sample type
Take process usually incompatible with other sample types；Although and utilize the method rate of recovery of phenol chloroform high, due to extracting solution
The toxicity of component is high and limits its use scope；Method course of dissolution based on detergent is simple, but needs to carry out high temperature change
Property (95 DEG C) processing, be not heat-staple additionally, due to reverse transcriptase, therefore be not suitable for single stage RT-PCR.Importantly,
Existing all methods are required to carry out centrifugation repeatedly, and extraction step is many and diverse, consuming time is long, needs specific equipment.
We describe a kind of method for avoiding this extraction process complexity in the application, it is simple, quick, be based on fiber
Enough nucleic acid is extracted in the system of plain filter paper, suitable for the use of the nucleic acid amplification technologies such as PCR, RPA, the system is in room temperature
Operation, suitable for the biological sample in various sources, and can be directly added into nucleic acid amplification for the cellulosic filter paper for being loaded with nucleic acid
It is carried out amplification reaction in system, whole process was less than 1 minute.
Summary of the invention
Invention is designed to provide a kind of Rapid nucleic acid extracting method, solves that extraction process is complicated, consuming time is long
And the problem of needing particular device.
The invention is realized in this way a kind of Rapid nucleic acid extracting method, method includes the following steps:
Step 1: cracking nucleic acid samples: the sample containing nucleic acid being added in lysate, nucleic acid cleavage liquid is formed；
Step 2: absorption nucleic acid: carrier being added in the nucleic acid cleavage liquid, the carrier of absorption nucleic acid is obtained；
Step 3: removing impurity: the carrier for having adsorbed nucleic acid being rinsed in rinsing liquid, except the epinucleic impurity of removal；
Step 4: amplified reaction: the carrier for being adsorbed with nucleic acid after rinsing being directly added into nucleic acid amplification system and is carried out
Amplified reaction；
The carrier is cellulosic filter paper.
A further technical solution of the present invention is: the shape of the cellulosic filter paper is round or/and polygon, the fibre
The size for tieing up plain filter paper is no more than 9mm2.Its as nucleic acid absorption carrier without it is any processing and modification, this with it is other
Nucleic acid absorption carrier is usually passed through into a variety of different processing in the invention announced and modification is completely different and excessive
Area nucleic acid release efficiency can be greatly reduced instead, to influence subsequent nucleic acid amplification effect.
A further technical solution of the present invention is: the lysate include guanidine hydrochloride, guanidinium isothiocyanate, Tris,
At least 3 in Tween20, NaCl, TritonX-100, EDTA, PVP, sodium citrate, laurel creatine sodium and β mercaptoethanol
Kind, wherein the pH value of Tris is 8.0.Guarantee that DNA, RNA are discharged into solution.
A further technical solution of the present invention is: the lysate includes 1.5M guanidine hydrochloride, 4M guanidinium isothiocyanate, 50mM
Tris, 1%Tween20,100mM-150mM NaCl, 0.5%TritonX-100,5mM EDTA, 2%PVP, 25mM citric acid
It is at least three kinds of in sodium, 0.5% laurel creatine sodium and 0.1mM β mercaptoethanol.
A further technical solution of the present invention is: the lysate is divided into DNA lysate and RNA lysate.
A further technical solution of the present invention is: the DNA lysate be guanidine hydrochloride, Tris, Tween20, NaCl,
At least three kinds of in TritonX-100, EDTA and PVP, wherein the pH value of Tris is 8.0.
A further technical solution of the present invention is: the RNA lysate be guanidinium isothiocyanate, Tris, Tween20, NaCl,
It is at least three kinds of in TritonX-100, EDTA, PVP, sodium citrate, laurel creatine sodium and β mercaptoethanol, the wherein PH of Tris
Value is 8.0.
A further technical solution of the present invention is: the high sample of protein content adds cracking after first being handled with Proteinase K
Liquid increases the release efficiency of nucleic acid, the high sample of content such as animal tissue, blood.
A further technical solution of the present invention is: the rinsing liquid includes 5-20mM Tris and 0.05-0.2%Tween-
20, Tris pH value is 8.0.Main purpose is the impurity removed in lysate, and guarantees the ingredient not and influence that subsequent nucleic acid expands
Increase reaction.
A further technical solution of the present invention is: the amplified reaction includes PCR amplification and recombination enzymatic polymerization
Enzyme constant-temperature amplification.
Beneficial effects of the present invention: it is provided herein it is a kind of it is simple, quickly, based on cellulosic filter paper be to be mentioned in carrier
Nucleic acid is taken, the nucleic acid amplification technologies such as PCR amplification (PCR) and recombinase polymerase constant-temperature amplification (RPA) are suitable for
It uses, method is useful in room temperature-operating, suitable for the biological sample in various sources, and the cellulose for being loaded with nucleic acid can be filtered
Paper is directly added into nucleic acid amplification system and carries out amplification reaction, and whole process was less than 1 minute.
Detailed description of the invention
Fig. 1 is a kind of Rapid nucleic acid extracting method flow chart provided by the invention；
Fig. 2 is the Nasopharyngeal swabs sample provided by the invention for showing and handling from DNA lysate, with cellulosic filter paper nucleic acid
Extract the PCR result of analyte detection mycoplasma；
Fig. 3 is the Nasopharyngeal swabs sample provided by the invention for showing and handling from RNA lysate, with cellulosic filter paper nucleic acid
Extract the RPA result of analyte detection Flu-A；
Fig. 4 is the blood serum sample provided by the invention for showing and handling from RNA lysate, with cellulosic filter paper nucleic acid extraction
The RPA result of analyte detection dengue fever；
Fig. 5 is the arabidopsis thaliana sample provided by the invention for showing and handling from DNA lysate, with cellulosic filter paper core
Acid extracts the RPA result of analyte detection pseudomonas syringae；
Fig. 6 is the whole blood sample provided by the invention for showing and handling from DNA lysate, with cellulosic filter paper nucleic acid extraction
The PCR result of analyte detection BRAF gene V600E mutation；
Fig. 7 is the cucumber plant sample provided by the invention for showing and handling from RNA lysate, with cellulosic filter paper nucleic acid
Extract the RPA result of the cucumber sample of analyte detection infection cucumber mosaic virus (CMV).
Specific embodiment
A kind of Rapid nucleic acid extracting method, method includes the following steps:
Step 1: cracking nucleic acid samples: the sample containing nucleic acid being added in lysate, nucleic acid cleavage liquid is formed；It is described
Sample includes free nucleic acid material in zooblast material or plant cell materials or bacterial cell material or viral material or body fluid
Material；
The lysate include guanidine hydrochloride, guanidinium isothiocyanate, Tris, Tween20, NaCl, TritonX-100, EDTA,
At least three kinds of in PVP, sodium citrate, laurel creatine sodium and β mercaptoethanol, wherein the pH value of Tris is 8.0；The cracking
Liquid includes 1.5M guanidine hydrochloride, 4M guanidinium isothiocyanate, 50mM Tris, 1%Tween20,100mM-150mM NaCl, 0.5%
In TritonX-100,5mM EDTA, 2%PVP, 25mM sodium citrate, 0.5% laurel creatine sodium and 0.1mM β mercaptoethanol
It is at least three kinds of；The lysate is divided into DNA lysate and RNA lysate；The DNA lysate be guanidine hydrochloride, Tris,
At least three kinds of in Tween20, NaCl, TritonX-100, EDTA and PVP, wherein the pH value of Tris is 8.0；The RNA is split
Solution liquid be guanidinium isothiocyanate, Tris, Tween20, NaCl, TritonX-100, EDTA, PVP, sodium citrate, laurel creatine sodium with
And it is at least three kinds of in β mercaptoethanol, wherein the pH value of Tris is 8.0；After the high sample of protein content is first handled with Proteinase K
Add lysate, the high sample of content such as animal tissue, blood；
Step 2: absorption nucleic acid: carrier being added in the nucleic acid cleavage liquid, the carrier of absorption nucleic acid is obtained；
The carrier is cellulosic filter paper；The shape of the cellulosic filter paper is round or/and polygon, the cellulose
The size of filter paper is no more than 9mm2；
Step 3: removing impurity: the carrier for having adsorbed nucleic acid being rinsed in rinsing liquid, except the epinucleic impurity of removal；
The rinsing liquid includes 5-20mM Tris and 0.05-0.2%Tween-20, and the pH value of Tris is 8.0；
Step 4: amplified reaction: the carrier for being adsorbed with nucleic acid after rinsing being directly added into nucleic acid amplification system and is carried out
Amplified reaction；
The amplified reaction includes PCR amplification and recombinase polymerase constant-temperature amplification.
Embodiment one:
Detect the PCR result of the mycoplasma pneumoniae in the Nasopharyngeal swabs sample that DNA lysate is handled.Steps are as follows:
1, the Nasopharyngeal swabs with sample is directly immersed in 1ml DNA lysate, 1ml DNA lysate is 1.5M hydrochloric acid
Guanidine, 50mM Tris (PH=8.0), 100mM NaCl, 5mM EDTA and 1%Tween20 are handled 10 seconds.
2, the DNA lysate that cellulosic filter paper immerses the step 1 is handled 10 seconds.
3, the cellulosic filter paper obtained the step 2 immerses rinsing liquid, rinsing liquid be 10mM Tris pH=8.0 and
0.1%Tween-20 is handled 10 seconds.
4, the cellulosic filter paper for obtaining the step 3 immerses PCR reaction system 10 seconds, or is retained in reaction system
In.
5, what PCR amplification 90 minutes, standard items plasmid and bacterial genomes DNA extraction kit (Beijing Tiangeng) were extracted
Same sample is positive control, and the no sample Nasopharyngeal swabs of parallel processing is negative control.
As a result as shown in Fig. 2, equally being expanded using PCR method, the result of the purifying of filter paper microplate and kits is without aobvious
Write sex differernce.Pillar location is consistent with standard items.
Embodiment two:
Detect the RPA result of the Flu-A in the Nasopharyngeal swabs sample that RNA lysate is handled.Steps are as follows:
6, the Nasopharyngeal swabs with sample is directly immersed in 1ml RNA lysate, 1ml RNA lysate is the different sulphur cyanogen of 4.0M
Sour guanidine, 25mM sodium citrate, 0.5% (m/V) laurel creatine sodium, 0.1mM β mercaptoethanol are handled 10 seconds.
7, cellulosic filter paper the RNA lysate that the step 6 obtains is immersed to handle 10 seconds.
8, the cellulosic filter paper obtained the step 7 immerses rinsing liquid, rinsing liquid be 10mM Tris (pH=8.0) and
0.1%Tween-20 is handled 10 seconds.
9, the cellulosic filter paper for obtaining the step 8 immerses RPA reaction system 10 seconds, or is retained in reaction system
In.
10, RPA amplification 15 minutes, standard items Strain and virus RNA extraction kit (the raw work in Shanghai) are extracted same
A sample is positive control, and the no sample Nasopharyngeal swabs of parallel processing is negative control.
As a result as shown in figure 3, equally being expanded using RPA method, the result of the purifying of filter paper microplate and kits is without aobvious
Write sex differernce.Pillar location is consistent with standard items.
Embodiment three:
Detect the RPA result of dengue fever in the blood serum sample of RNA lysate processing.Steps are as follows:
11,1.2ml RNA lysate is added in dengue fever patients serum's sample 300ul, RNA lysate is 50 μ g/ml
Proteinase K, 25mM sodium citrate, 0.5% (m/V) laurel creatine sodium, 4.0M guanidinium isothiocyanate and 0.1mM β sulfydryl second
Alcohol,
12, first with 50 μ g/ml Proteinase K, 25mM sodium citrate, 0.5%m/V laurel creatine sodium and 0.1mM β
Mercaptoethanol is handled 1 hour, is added 4.0M guanidinium isothiocyanate and is handled 10 seconds.
13, cellulosic filter paper the RNA lysate that the step 12 obtains is immersed to handle 10 seconds.
14, the cellulosic filter paper for obtaining the step 13 immerses wash buffer, and rinsing liquid is 10mM Tris (pH=
8.0) it is handled 10 seconds with 0.1%Tween-20.
15, the cellulosic filter paper for obtaining the step 14 immerses RPA reaction system 10 seconds, or is retained in reaction system
In.
16, RPA is expanded 15 minutes, and standard items plasmid is positive control, and the no sample lysate of parallel processing is negative right
According to.
As a result as shown in figure 4, equally being expanded using RPA method, the result of the purifying of filter paper microplate and kits is without aobvious
Write sex differernce.Pillar location is consistent with standard items.
Example IV:
Detect the RPA result of the pseudomonas syringae in the arabidopsis thaliana sample that DNA lysate is handled.Step is such as
Under:
17, by 10mg sample be added 100ul DNA of plants lysate, the DNA lysate be 50mM Tris 8.0,
150mM NaCL, 2%PVP and 1%Tween20, pulverize, and handle 10 seconds.
18, cellulosic filter paper the DNA of plants extracting solution that the step 17 obtains is immersed to handle 10 seconds.
19, the cellulosic filter paper for obtaining the step 18 immerses rinsing liquid, and rinsing liquid is 10mM Tris (pH=8.0)
It is handled 10 seconds with 0.1%Tween-20.
20, the cellulosic filter paper for obtaining the step 19 immerses RPA reaction system 10 seconds, or is retained in reaction system
In.
21, RPA is expanded 15 minutes, and standard items plasmid is positive control, and the no sample extracting solution of parallel processing is negative right
According to.
Structure using RPA method as shown in figure 5, equally expanded, and the result of the purifying of filter paper microplate and kits is without aobvious
Write sex differernce.Pillar location is consistent with standard items.
Embodiment five:
Detect the PCR knot of BRAF gene V600E mutation in the malignant mela noma patient blood sample of DNA lysate processing
Fruit.Steps are as follows:
22,1.2ml DNA lysate is added in whole blood sample 300ul, the lysate is 50 μ g/ml Proteinase
K, 50mM Tris (PH 8.0), 100mM NaCl, 0.5%TritonX-100,1%Tween20 and 0.8M guanidine hydrochloride.
23, the sample of the step 22 first uses 50 μ g/ml Proteinase K, 50mM Tris (PH8.0), 100mM
NaCl, 0.5%TritonX100,1%Tween20 are handled 1 hour, are added 0.8M guanidine hydrochloride and are handled 10 seconds.
24, cellulosic filter paper the RNA lysate that the step 23 obtains is immersed to handle 10 seconds.
25, the cellulosic filter paper for obtaining the step 24 immerses rinsing liquid, and rinsing liquid is 10mM Tris (pH=8.0)
It is handled 10 seconds with 0.1%Tween-20.
26, the cellulosic filter paper for obtaining the step 25 immerses RPCR reaction system 10 seconds, or is retained in reactant
In system.
27, PCR amplification 90 minutes, standard items plasmid are positive control, and the no sample lysate of parallel processing is negative right
According to.
Structure using PCR method as shown in fig. 6, equally expanded, and the result of the purifying of filter paper microplate and kits is without aobvious
Write sex differernce.Pillar location is consistent with standard items.
Embodiment six:
Detect the RPA result of the cucumber sample of the infection cucumber mosaic virus CMV of RNA lysate processing.Steps are as follows:
28, by 10mg sample be added 100ul plant RNA lysate, the RNA lysate be 4.0M guanidinium isothiocyanate,
50mM Tris (PH 8.0), 100mM NaCl, 0.5%TritonX-100,1%Tween20, pulverize, and handle 10 seconds.
29, cellulosic filter paper the plant RNA extraction liquid that the step 28 obtains is immersed to handle 10 seconds.
30, the cellulosic filter paper for obtaining the step 29 immerses rinsing liquid, and rinsing liquid is 10mM Tris (pH=8.0)
It is handled 10 seconds with 0.1%Tween-20.
31, the cellulosic filter paper for obtaining the step 30 immerses RT-RPA reaction system 10 seconds, or is retained in reaction
In system.
32, RPA is expanded 15 minutes, and standard items plasmid is positive control, and the no sample extracting solution of parallel processing is negative right
According to.
Structure using RPA method as shown in fig. 7, equally expanded, and the result of the purifying of filter paper microplate and kits is without aobvious
Write sex differernce.Pillar location is consistent with standard items.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of Rapid nucleic acid extracting method, it is characterised in that: method includes the following steps:
Step 1: cracking nucleic acid samples: the sample containing nucleic acid being added in lysate, nucleic acid cleavage liquid is formed；
Step 2: absorption nucleic acid: carrier being added in the nucleic acid cleavage liquid, the carrier of absorption nucleic acid is obtained；
Step 3: removing impurity: the carrier for having adsorbed nucleic acid being rinsed in rinsing liquid, except the epinucleic impurity of removal；
Step 4: amplified reaction: the carrier for being adsorbed with nucleic acid after rinsing being directly added into nucleic acid amplification system and is expanded
Reaction；
The carrier is cellulosic filter paper.
2. a kind of Rapid nucleic acid extracting method according to claim 1, it is characterised in that: the shape of the cellulosic filter paper
Size for round or/and polygon, the cellulosic filter paper is no more than 9mm2。
3. a kind of Rapid nucleic acid extracting method according to claim 1, it is characterised in that: the lysate includes hydrochloric acid
Guanidine, guanidinium isothiocyanate, Tris, Tween20, NaCl, TritonX-100, EDTA, PVP, sodium citrate, laurel creatine sodium and β
At least three kinds of in mercaptoethanol, wherein the pH value of Tris is 8.0.
4. a kind of Rapid nucleic acid extracting method according to claim 3, it is characterised in that: the lysate includes 1.5M salt
Sour guanidine, 4M guanidinium isothiocyanate, 50mM Tris, 1%Tween20,100mM-150mM NaCl, 0.5%TritonX-100,5mM
It is at least three kinds of in EDTA, 2%PVP, 25mM sodium citrate, 0.5% laurel creatine sodium and 0.1mM β mercaptoethanol.
5. a kind of Rapid nucleic acid extracting method according to claim 3, it is characterised in that: the lysate is divided into DNA and splits
Solve liquid and RNA lysate.
6. a kind of Rapid nucleic acid extracting method according to claim 5, it is characterised in that: the DNA lysate is hydrochloric acid
At least three kinds of in guanidine, Tris, Tween20, NaCl, TritonX-100, EDTA and PVP, wherein the pH value of Tris is 8.0.
7. a kind of Rapid nucleic acid extracting method according to claim 5, it is characterised in that: the RNA lysate is different sulphur
Cyanic acid guanidine, Tris, Tween20, NaCl, TritonX-100, EDTA, PVP, sodium citrate, laurel creatine sodium and β sulfydryl second
At least three kinds of in alcohol, wherein the pH value of Tris is 8.0.
8. a kind of Rapid nucleic acid extracting method described in -7 any one according to claim 1, it is characterised in that: protein content is high
Sample first handled with Proteinase K after add lysate.
9. a kind of Rapid nucleic acid extracting method described in -7 any one according to claim 1, it is characterised in that: the rinsing liquid
Including 5-20mM Tris and 0.05-0.2%Tween-20, the pH value of Tris is 8.0.
10. a kind of Rapid nucleic acid extracting method described in -7 any one according to claim 1, it is characterised in that: the amplification
Reaction includes PCR amplification and recombinase polymerase constant-temperature amplification.
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| CN111073810A (en) * | 2019-12-20 | 2020-04-28 | 深圳市华迈生物医疗科技有限公司 | Microfluidic chip, system and method integrating nucleic acid extraction, amplification and detection |
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| CN111206030A (en) * | 2020-02-18 | 2020-05-29 | 南京农业大学 | Virus nucleic acid extraction method based on glass fiber filter paper |
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| CN112430687A (en) * | 2020-12-10 | 2021-03-02 | 广西大学 | Method for rapidly detecting potato virus based on RT-PCR (reverse transcription-polymerase chain reaction) of filter paper strip capture virus nucleic acid |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609496A (en) * | 2019-01-16 | 2019-04-12 | 浙江工商大学 | A kind of extracting method of the feces of livestock and poultry total DNA for PCR amplification |
| CN110938523A (en) * | 2019-12-20 | 2020-03-31 | 深圳市华迈生物医疗科技有限公司 | Centrifugal microfluidic chip, system and detection method for SAT |
| CN111073810A (en) * | 2019-12-20 | 2020-04-28 | 深圳市华迈生物医疗科技有限公司 | Microfluidic chip, system and method integrating nucleic acid extraction, amplification and detection |
| CN111139287A (en) * | 2020-01-20 | 2020-05-12 | 青岛农业大学 | Nucleic acid on-site extraction method applied to animal tissues |
| CN111206030A (en) * | 2020-02-18 | 2020-05-29 | 南京农业大学 | Virus nucleic acid extraction method based on glass fiber filter paper |
| CN112195177A (en) * | 2020-10-28 | 2021-01-08 | 上海慕柏生物医学科技有限公司 | Nucleic acid extraction method and kit |
| CN112501251A (en) * | 2020-11-02 | 2021-03-16 | 武汉轻工大学 | Method for efficiently extracting RNA of streptococcus suis |
| CN112430687A (en) * | 2020-12-10 | 2021-03-02 | 广西大学 | Method for rapidly detecting potato virus based on RT-PCR (reverse transcription-polymerase chain reaction) of filter paper strip capture virus nucleic acid |
| CN113337500A (en) * | 2021-05-26 | 2021-09-03 | 中国检验检疫科学研究院 | RT-RPA method-based trace nucleic acid releasing agent, and preparation method and application thereof |
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