CN106906208A - A kind of method of high efficiency extraction nucleic acid - Google Patents
A kind of method of high efficiency extraction nucleic acid Download PDFInfo
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Abstract
The invention discloses a kind of method of high efficiency extraction nucleic acid, it is related to biological technical field.Methods described includes：Sample to be tested and high rigidity microballoon are placed in nucleic acid Extract, are shaken, obtain first treatment fluid；The first supernatant after first treatment fluid centrifugation is extracted, then mixes first supernatant with acidic buffer, obtain after-treatment liquid；The centrifugation of after-treatment liquid is obtained into secondary supernatant, secondary supernatant is mixed with the buffer solution with broken cell and nucleic acid absorption dual-use function, after centrifugation, remove supernatant, be precipitated；The precipitation is cleaned, nucleic acid is obtained after drying at room temperature.Present invention application acidic buffer, than the impurity for more thoroughly removing influence PCR amplification efficiencies, improves amplification efficiency.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of method of high efficiency extraction nucleic acid.
Background technology
Molecular biology is the biological study method that developed recently gets up.The first step of molecular biology is that nucleic acid is carried
Take.
In practice, method for extracting nucleic acid mainly has PC methods (phenol/chloroform), extracts kit method etc., extracts kit one
As release reagent, the impurity scavenger reagent such as albumen and washing reagent, elution reagent composition including nucleic acid, extracting nucleic acid needs three
Step or FOUR EASY STEPS：I.e. broken cell free nucleic acid, removal interfering material and/or washing, wash-out nucleic acid.But, for complicated sample
This, the pick-up rate for extracting nucleic acid nucleic acid using existing method is not high, and the field trash mistake of PCR amplifications is influenceed in the nucleic acid after extraction
It is many, influence follow-up study or diagnosis.
In order to improve the nucleic acid extraction efficiency of complex samples, the excrement for improving the research and development of the companies such as amplification efficiency, Qiagen is special
With extracting special extracts kit of box/soil etc., but, special extracts kit poor universality, and due to using counter-infiltration
The technologies such as film, it is with high costs, meanwhile, special extracts kit can not meet clinical molecular Biological Detection to Special Bacteria-
Such as clostridium difficile brood cell detection demand.
Need the fast and convenient reliable method for extracting nucleic acid of exploitation and reagent badly in order to solve the above problems.
The content of the invention
It is an object of the invention to provide a kind of method of high efficiency extraction nucleic acid, so that before solving present in prior art
State problem.
To achieve these goals, the method for high efficiency extraction nucleic acid of the present invention, methods described includes：
S1, sample to be tested and high rigidity microballoon are placed in nucleic acid Extract, are shaken, and obtain first treatment fluid；
S2, extracts the first supernatant after first treatment fluid centrifugation, then mixes first supernatant with acidic buffer,
Obtain after-treatment liquid；
S3, secondary supernatant is obtained by the centrifugation of after-treatment liquid, by secondary supernatant and with broken cell and nucleic acid absorption
The buffer solution mixing of dual-use function, after centrifugation, removes supernatant, is precipitated；
S4, cleans the precipitation, and nucleic acid is obtained after drying at room temperature；
The nucleic acid Extract includes 50~1000mmol/L LiCl, 50~250mmol/L Tris-HCl and SDS；
The SDS is 0.5~2.5% with the w/v of the nucleic acid Extract；
The acidic buffer includes sodium acetate, acetic acid and distilled water, the weighing body of sodium acetate and the acidic buffer
Product is than being (0.5~5) g：100mL；Acetic acid is (5~20) with the volume ratio of the acidic buffer：100；The acidic buffer
Liquid pH value is less than 4；
It is described to include that 4~8mol/L guanidinesalts class and volume accounting are containing broken cell-nucleic acid absorption dual-use function buffer solution
50% isopropanol.
Preferably, in step S1, the time of concussion is 30s~120s.
Preferably, the high rigidity microballoon and the volume ratio of the sample to be tested are (0.5~1.0)：10；The nucleic acid is taken out
It is (5~10) to go out liquid with the volume ratio of the sample to be tested：10.
Preferably, the high rigidity microballoon is zirconium oxide microballoons.
Preferably, a diameter of 0.2mm~1.0mm of the high rigidity microballoon.
The beneficial effects of the invention are as follows：
Carry out cell wall damage using zirconium oxide in reagent of the invention, because zirconium oxide is nonmagnetic, inconductivity, without surface
Functional group and extreme hardness, may apply to the pre-treatment of centrifugal column method or paramagnetic particle method nucleic acid extraction, without influence next step
Operation.Zirconium oxide microballoons and nucleic acid extraction liquid are acted on into sample simultaneously, then various lifes are acted on physics and chemical method
Thing cell or virus/bacteriophage outer wall, reach the purpose for improving extraction efficiency.Present invention application acidic buffer, than more thoroughly
The impurity of removal influence PCR amplification efficiencies, improves amplification efficiency.Present invention application broken cell-nucleic acid absorption in precipitate nucleic acids
Dual-use function buffer solution, carries out brokenly thin saturation absorption nucleic acid again, farthest ensure that nucleic acid pick-up rate, is complex samples
Molecular biology research high-quality nucleic acid is provided, the range of application of molecular biology research is improved in order to expand, especially
It is for grand genome research provides powerful.The present invention is tired to nucleic acid extractions such as the complex samples such as sputum, excrement and brood cells
The nucleic acid extraction of difficult sample provides powerful and new method.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of the method for high efficiency extraction nucleic acid.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with accompanying drawing, the present invention is entered
Row is further described.It should be appreciated that specific embodiment described herein is only used to explain the present invention, it is not used to
Limit the present invention.
1. on the explanation of high efficiency extraction nuclei aoid methods
It is method for extracting nucleic acid that complex environment sample is proposed that I, the method for the invention are, in order to from complex environment sample
The nucleic acid used for molecule diagnosis is proposed in this, the complex environment sample includes：Soil, excrement, sludge and including food
And/or the field trash of sputum and/or fester.
The method of the invention can also simply crush the sclereid wall of the bacterium specific forms such as gemma, high efficiency extraction very much
Nucleic acid, resulting nucleic acid is the full nucleic acid in sample, and full nucleic acid can be realized to the detection of nucleic acids of sample or carry out grand gene
Group analysis.
II, the method for the invention
In step S1, the high rigidity microballoon is nonmagnetic, inconductivity, the high rigidity microballoon without surface functional group；
Step S1 is by the purpose of concussion is afterwards in sample and high rigidity microballoon addition nucleic acid Extract：Fracturing cell walls, exine and
Fungal spore.
Strong concussion 30~120 seconds after high rigidity microballoon and sample, the mixing of nucleic acid Extract, to destroy cell membrane, cell
The structures such as film, Exosporium, with release intracellular nucleic acid or it is convenient after the step of release nucleic acid effect.
Explanation on zirconium oxide：The common metal particle phase such as zirconium oxide and steel ball is than nonmagnetic, inconductivity, without surface
Functional group, and its hardness is more much higher than glass microsphere, and strong concussion there will not be the phenomenon such as broken；Consider nucleic acid simultaneously
Physicochemical properties and field trash physicochemical properties, by many experiments, discovery can be reliable by acidic treatment
Removal of inclusions, both are combined together can increase substantially nucleic acid extraction and refining effect.
Explanation on nucleic acid Extract：The nucleic acid Extract include 50~1000mmol/L LiCl, 50~
250mmol/L Tris-HCl and 0.5~2.5% w/v SDS；LiCl is lithium chloride, Tris-HCI is trihydroxy methyl ammonia
Methylmethane-hydrochloric acid, SDS is lauryl sodium sulfate.
Explanation on acidic buffer：The acidic buffer include 0.5~5% w/v sodium acetate, 5~
The acetic acid of 20% volume ratio, the acidic buffer pH value is less than 4.
In step S2, first treatment fluid by first supernatant is moved into new centrifuge tube after low-speed centrifugal, then with acid
Property buffer solution mixing, to realize the removal influence polyphenol of pcr amplification reaction, polysaccharide, spoilage product, biological enzyme inhibitor, compost
In the impurity such as organic compound, phytochrome, fumaric acid, tannic acid purpose.
Processed by acidic buffer, polyphenol, polysaccharide, spoilage product, biological enzyme inhibitor in first supernatant, compost
In the impurity such as organic compound, phytochrome, fumaric acid, tannic acid form agglomerate, then by centrifuged deposit to lower floor, reach
To the purpose for removing influence PCR reactive materials.
By can be with low-speed centrifugal after concussion, solid constituent and the liquid such as the larger soil block of separated volume, excrement, swill
Nucleic acid and microbe granular in phase are separated.
It is in step S3, after-treatment liquid is by the secondary supernatant after centrifugation and dual with broken cell-nucleic acid absorption
Function buffer solution mixing purpose be：Broken cell, adsorbs more polynucleotide again, reaches high efficiency extraction efficiency.
Explanation on the buffer solution with broken cell and nucleic acid absorption dual-use function：It is described to be inhaled with broken cell and nucleic acid
The buffer solution of attached dual-use function includes guanidinesalt class and isopropanol.
III, the method for the invention avoid the poison of the phenol/chloroform of traditional method for extracting nucleic acid during nucleic acid is extracted
Property, realizing during complicated sample nucleic acid extraction, efficient rapid extraction obtains can be used for detection of nucleic acids, grand genome analysis
High-quality sample nucleic acid needed for equimolecular biological study and application.
Using having arrived nucleic acid Extract, acidic buffer, double with broken cell-nucleic acid absorption in the method for the invention
Weight function buffer solution, cleaning solution and eluent, during a nucleic acid extraction, carry out breaking-wall cell twice and nucleic acid are carried twice
Take, meanwhile, the broken cell Combination of Methods of methods described physical/chemical method, the hard guarantor on physical method directed toward bacteria surface
Sheath, chemical method is directed to and grinds off skelteon or unprotected vegetative cell, to improve nucleic acid extraction efficiency.
Embodiment 1
Checking zirconium oxide：Weigh 1.0g zirconium oxides (diameter 1.0mm) and 1.0g steel balls (diameter 1.0mm) and 1.0g glass
Pearl (diameter 1.0mm), is placed on 1ml centrifuge tubes, and visual estimation volume, zirconium oxide is minimum, and steel ball takes second place, and bead volume is maximum；1
Minute, 300rpm shook 3 minutes strongly, poured out observation, it is found that bead is shatter.Zirconium oxide and steel ball have no fragment, are repeated 10 times
Experiment, it is as a result identical.
Rinse zirconium oxide and steel ball repeatedly using sterile distilled water, load high temperature under 1ml 121 degree of 1.2 atmospheric pressure of centrifuge tube
Bacillus subtilis spore (1*10 is added after 15 points of sterilizing in centrifuge tube4CFU/ml) 1ml, centrifuging and taking supernatant after shaking 3 minutes strongly
Liquid, fluorescent PCR is carried out after extracting nucleic acid using Qiagen companies QIAamp DNA Mini Kit, observes Ct values, zirconium oxide treatment
Sample Ct values are forward compared with steel ball treatment sample, it is considered to which zirconium oxide molecular weight (123.22) is bigger (55.85) than iron molecule amount, proportion
Corresponding high, every physical impact Li Genggao of particle, destruction brood cell's effect is better than steel ball.
Embodiment 2 prepares nucleic acid extracting reagent
1st, nucleic acid Extract (following A liquid) is prepared
1) 121.14g Tris are weighed and is dissolved in 1L pure water, it is molten to adjust pH8.3 preparation 1mol/L Tris-HCl with concentrated hydrochloric acid
Liquid.2) weigh 42.9gLiCl and be dissolved in 1L pure water, prepare 1mol/LLiCl solution.3) 250ml 1mol/L Tris-HCl are taken molten
Liquid and 750ml 1mol/L LiCl solution, then add 20mlSDS be sufficiently mixed preparation nucleic acid Extract.
2nd, acidic buffer (following B liquid) is prepared
Sodium acetate 20g is taken, is dissolved with 100ml glacial acetic acid, then add sterile distilled water to final volume 1L, surveying pH value is
3.6。
3rd, preparing has broken thin full-nucleic acid absorption dual-use function buffer solution (following C liquid):
Weigh 764.24g guanidine hydrochlorides and be dissolved in 1L distilled water, prepare 8mol/L concentration guanidine hydrochloride solutions；When using and isopropanol
Isometric mixing.
4th, cleaning solution：70% concentration ethanol is routinely used.
5th, nucleic acid eluents：It is used conventionally TE buffer solutions (10mMTris-HCl, 1mM EDTA PH=8.0).
The checking of the nucleic acid extracting reagent of embodiment 3
1st, prepared by sample：Fertile soil 100g, excrement 100g, sludge 100g are taken, 1ml hay bacilluses are added in every kind of sample
Spore suspensions (concentration 106CFU/ml) are sufficiently stirred for；It is divided into every part of 1g amount, is divided into experimental group and control group, every kind of sample reality
Group 50 parts, 50 parts of control group are tested, it is standby.
2nd, experimental group reagent nucleic acid is extracted：
2.1 take three parts of sample addition A liquid (nucleic acid Extract) 1ml, 300rpm/1 shunt excitation violent shocks of every kind of sample swings 3 points of mixing
Clock, 3000rpm centrifugations take supernatant for 5 minutes and move to new 1ml centrifuge tubes；Fertile soil sample supernatant present brown it is slightly cloudy,
Light yellow slightly cloudy, the sludge sample supernatant dark brown of stool sample supernatant is muddy.
2.2 addition B liquid (acidic buffer) 200ul, the 3000rpm after 10 times that turns upside down is centrifuged 5 minutes, and supernatant is moved to
New 1ml centrifuge tubes, supernatant is limpid colourless.
2.3 will take 200ul after 8mol/Ltris-HCl solution and isopropanol in equal volume mixing is added in supernatant, runs up and down
Mix 10 times, place room temperature 5 minutes, 10000rpm is centrifuged 5 minutes, discards supernatant.
2.4 take 70% ethanol 1ml turns upside down 10 times and washs the nucleic acid of precipitation, 10000rpm centrifugations, discards supernatant, weight
Again twice, drying at room temperature 5 minutes.
2.5 take TE buffer solutions nucleic acid obtain 9 tube nucleus acid it is standby.
4 zirconium oxide microballoons of embodiment+experimental group extracts reagent nucleic acid extraction
Carried out while embodiment 3 checking of zirconium oxide microballoons+experimental group nucleic acid extracting reagent
4.1 take three parts of samples of every kind of sample, are added in the 2ml centrifuge tubes of prepackage 1.0g zirconium oxide microballoons (diameter 1.0mm),
Addition A liquid (nucleic acid Extract) 1ml, 300rpm/1 shunt excitation violent shock swings 3 minutes, and 3000rpm centrifugations take supernatant in 5 minutes and move to newly
1ml centrifuge tubes；Fertile soil sample supernatant presentation brown is slightly cloudy, light yellow slightly cloudy, the sludge mark of stool sample supernatant
This supernatant dark brown is muddy.
4.2 addition B liquid (acidic buffer) 200ul, the 3000rpm after 10 times that turns upside down is centrifuged 5 minutes, and supernatant is moved to
New 1ml centrifuge tubes, supernatant is limpid colourless.
4.3 will take 200ul after 8mol/Ltris-HCl solution and isopropanol in equal volume mixing is added in supernatant, runs up and down
Mix 10 times, place room temperature 5 minutes, 10000rpm is centrifuged 5 minutes, discards supernatant.
4.4 take 70% ethanol 1ml turns upside down 10 times and washs the nucleic acid of precipitation, 10000rpm centrifugations, discards supernatant, weight
Again twice, drying at room temperature 5 minutes.
4.5 take TE buffer solution nucleic acid, obtain 9 tube nucleus acid standby.
The control group extracts reagent nucleic acid extraction of embodiment 5
Three parts of samples of every kind of sample are taken, nucleic acid extraction is carried out using control group reagent to specifications；Control group reagent is adopted
With the special extracts reagent QIAamp DNA Stool Mini Kit of Qiagen companies excrement nucleic acid, extract to specifications, send out
All some muddy, the final acquisition 9 tube nucleus acid of existing each step supernatant.
6 zirconium oxide microballoons of embodiment+control group extracts reagent nucleic acid extraction
Three parts of samples of every kind of sample are taken, is added in the 2ml centrifuge tubes of prepackage 1.0g zirconium oxide microballoons (diameter 1.0mm), added
Plus after physiological saline 1ml, 300rpm/1 shunt excitation violent shock swings 3 minutes, 3000rpm centrifugation take within 5 minutes supernatant move to new 1ml from
Heart pipe, operates same as Example 5 afterwards；Using control group reagent to specifications, nucleic acid extraction is carried out；Control group reagent is adopted
With the special extracts reagent QIAamp DNA Stool Mini Kit of Qiagen companies excrement nucleic acid, extract to specifications, send out
Existing each step supernatant all some muddinesses.
The hay bacillus fluorescent PCR of embodiment 7 is verified
Enter performing PCR using the hay bacillus 16s rRNA gene primers and probe in embodiment 1 to verify.Its result such as table 1.
Table 1 enters performing PCR the result using the hay bacillus 16s rRNA gene primers and probe in embodiment 1
Visible method for extracting nucleic acid of the present invention and reagent be compared with the high reagent of confidence level in industry from the above, its
Nucleic acid extraction efficiency substantially increases, and the Nucleic acid quality for being obtained is good, and harvest yield is big, while suppressing in being not readily susceptible to complicated sample
The material interference of PCR amplifications, its reason considers that the effect for removing interfering material is good compared with contrast agents, during nucleic acid extraction,
In addition to supernatant some muddinesses after first step A is also processed, heterochromatic and muddiness is not observed in other steps, also illustrates
Elimination effect of the reagent to impurity in this method.
By using above-mentioned technical proposal disclosed by the invention, following beneficial effect has been obtained：Simultaneously with physics and change
Method acts on various biological cells or virus/bacteriophage outer wall, and the purpose ratio for reaching raising extraction efficiency is more thoroughly removed
The impurity of PCR amplification efficiencies is influenceed, amplification efficiency is improved；For the molecular biology research to complicated sample provides high-quality core
Acid, improves the range of application of molecular biology research, powerful is provided especially for grand genome research, while to phlegm
The nucleic acid extraction of the difficult sample of complexity sample and brood cell etc. the nucleic acid extraction such as liquid, excrement provides powerful and new method.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
Depending on protection scope of the present invention.
Claims (5)
1. a kind of method of high efficiency extraction nucleic acid, it is characterised in that methods described includes：
S1, sample to be tested and high rigidity microballoon are placed in nucleic acid Extract, are shaken, and obtain first treatment fluid；
S2, extracts the first supernatant after first treatment fluid centrifugation, then mixes first supernatant with acidic buffer, obtains
After-treatment liquid；
S3, secondary supernatant is obtained by the centrifugation of after-treatment liquid, and secondary supernatant is dual with broken cell and nucleic acid absorption
The buffer solution mixing of function, after centrifugation, removes supernatant, is precipitated；
S4, cleans the precipitation, and nucleic acid is obtained after drying at room temperature；
The nucleic acid Extract includes 50~1000mmol/L LiCl, 50~250mmol/L Tris-HCl and SDS；It is described
SDS is 0.5~2.5% with the w/v of the nucleic acid Extract；
The acidic buffer includes sodium acetate, acetic acid and distilled water, the w/v of sodium acetate and the acidic buffer
It is (0.5~5) g：100mL；Acetic acid is (5~20) with the volume ratio of the acidic buffer：100；The acidic buffer pH
Value is less than 4；
It is described to include that 4~8mol/L guanidinesalts class and volume accounting are 50% containing broken cell-nucleic acid absorption dual-use function buffer solution
Isopropanol.
2. the method for high efficiency extraction nucleic acid according to claim 1, it is characterised in that in step S1, the time of concussion is 30s
~120s.
3. the method for high efficiency extraction nucleic acid according to claim 1, it is characterised in that the high rigidity microballoon is to be measured with described
The volume ratio of sample is (0.5~1.0)：10；The nucleic acid Extract is (5~10) with the volume ratio of the sample to be tested：10.
4. the method for high efficiency extraction nucleic acid according to claim 1, it is characterised in that the high rigidity microballoon is that zirconium oxide is micro-
Ball.
5. the method for high efficiency extraction nucleic acid according to claim 1, it is characterised in that the high rigidity microballoon it is a diameter of
0.2mm~1.0mm.
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Application publication date: 20170630 |