CN104059849B - Device and method used for rapid extraction of nucleic acid - Google Patents
Device and method used for rapid extraction of nucleic acid Download PDFInfo
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- CN104059849B CN104059849B CN201410333236.7A CN201410333236A CN104059849B CN 104059849 B CN104059849 B CN 104059849B CN 201410333236 A CN201410333236 A CN 201410333236A CN 104059849 B CN104059849 B CN 104059849B
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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
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
Abstract
The invention relates to a device and method used for rapid extraction of nucleic acid which are mainly used for rapidly extracting nucleic acid (including RNA and DNA) from each clinical sample. The device mainly comprises an injector, a hollow adaptor, a nucleic acid adsorption membrane tube and a sampling probe, after the steps of splitting a clinical sample, sucking by virtue of an injector, adsorbing nucleic acid by virtue of a nucleic acid adsorption film, rinsing the nucleic acid adsorption film with rinsing liquid and eluting nucleic acid with eluant are carried out, a pure nucleic acid solution can be obtained to be used in a downstream experiment.
Description
Technical field
The present invention relates to biological technical field and in particular to a kind of can from various clinical samples rapid extraction nucleic acid
Device and method.
Background technology
(1) nucleic acid extraction
Nucleic acid extraction has had become as most important, the most basic operation in molecular biology experiment technology, but at present
The extraction scheme of nucleic acid is many, and complicated and simple differs.The related experiment such as molecular diagnosis, nucleic acid sequencing, functional genome
Complete all to rely on the successful extraction of nucleic acid, the own warp of traditional operating process can realize whole automatizatioies, but these are expensive
Instrument can not be promoted the use of on a large scale, China is still developing country at present, and vast different medical unit is simultaneously
The instrument of complexity can not be equipped with, therefore it provides a kind of nucleic acid-extracting apparatus of good economy performance are necessary.
Nucleic acid includes two kinds of molecules of dna, rna, is all to be existed with the state being combined with protein in cell, nucleic acid extraction
Mainly comprise the following steps: one, cell lysis, remove the protein that is combined with nucleic acid and the biomacromolecule such as polysaccharide, lipid, removal
Other unwanted nucleic acid molecules, when such as extracting dna molecule, should remove rna, vice versa, if extracting rna and dna altogether
If extraction, then it is not required to remove any one；2nd, precipitate nucleic acids or absorption nucleic acid；3rd, purification of nucleic acid, removes salt, organic
The impurity such as agent；4th, eluting or dissolving nucleic acid, using eluent or lysate by Nucleic Acid Elution or dissolving.
(2) clinical sample pre-treatment
Due to the process of sample and to preserve impact to dna and rna (especially rna) larger, therefore in nucleic acid determination
The process of specimen and suitable preservation are extremely important to the accurate and effective of measurement result.Clinical common specimen has serum (slurry), entirely
Blood, secretions, cotton swab, puss, body fluid, flesh tissue, paraffin section etc., the processing method of these clinical samples respectively has not
With.
1. serum (slurry) specimen
Plus appropriate erythrocyte cracked liquid (rupture of membranes liquid), the erythrocyte in cracking whole blood；Again plus appropriate nucleus split liquid
(broken karyolymph), the nucleus in cracking leukocyte, so that dna in core is discharged；Then add sds (sodium lauryl sulphate) etc. again
Detergent, dissolves lipoprotein, depolymerization nucleoprotein；Sds can form complex with protein, makes protein denaturation precipitation, but sds exists
Must be driven off during later nucleic acid extraction.
2. sputum specimen
Expectorant belongs to secretions, is clinically commonly used for tubercule bacillus dna and measures sample, due to containing a large amount of mucin in sputum specimen
And impurity, therefore in nucleic acid extraction, pre-treatment must be carried out to specimen, liquefied with 4%naoh, remove mucin, then
With boil or the classical approach such as protease, phenol/chloroform extract dna.
3. cotton swab
With (chlamydia, mycoplasma, gonococcuss, treponema pallidum, people papilloma during pcr method detection sexual reverse
Virus etc.), clinical samples are generally cotton swab.Specimen sampling is crucial, and the pathogenic infection epithelial cell such as chlamydia therefore takes
Material must get cell.Concrete grammar:
Plus 1ml physiological saline solution, fully shaking mixing, 12000rpm, it is centrifuged 5 minutes
Take urethral secretionies or cervical secretionses (being gathered by medical personnel) with cotton swab, put in sterile test tube or ep pipe and abandon
Supernatant, precipitation plus physiological saline solution 1ml, beat, 12000rpm, are centrifuged 5 minutes
4. body fluid
Humoral specimen, including hydrothorax, ascites, cerebrospinal fluid, urine etc., can directly be centrifuged, and taking precipitate extracts nucleic acid.Courageous and upright
Ascites pleural fluid, can be processed using erythrocyte cracked liquid:
5. puss
Sticky puss: process with sputum specimen, first liquefied with 4%naoh, then centrifuging and taking precipitation extracts dna.
Water sample puss: be directly centrifuged, precipitation physiology salt is washed 23 times and is used for dna extraction afterwards.
6. organize
Group is woven with flesh tissue and paraffin section.The process step of flesh tissue: first washed with physiology salt secondary, then will
It is smashed to pieces or shreds (using eye scissorss), plus extracts nucleic acid after Proteinase K digestion；Paraffin section is used for nucleic acid extraction, need to be first with two
Toluene dewaxes, then with carrying out dna extraction after Proteinase K digestion.
(3) nucleic acid-extracting apparatus
Cn101684463a proposes a kind of trace clinical sample nucleic acid rapid extraction device, by sharp-crested suction pipe, the mistake with film
Filter and aspirator (syringe or plastic blister) three part composition, it is completed nucleic acid absorption, is cleaned and wash by three suctions
De- process, fast and convenient can extract nucleic acid from sample, so only drawback is that, sharp-crested suction pipe, the filter with film and suction
Device three part compound mode single and for fixed installation, (Fig. 2 of cn101684463a, figure when making aspirator with plastic blister
3) third time suction carries out that Nucleic Acid Elution is actual can not to complete that (the front waste liquid aspirating twice cannot be discharged, with the eluent of nucleic acid
It is mixed in waste liquid), and when making aspirator using syringe, the eluent with nucleic acid enters syringe and not only can be infected with syringe
Form biologic garbage, also result in the contamination of trace sample amplifying nucleic acid remarks emitter and lose and cause detection inaccurate.
Content of the invention
For overcoming problems of the prior art, present invention aim at provide a kind of simple, practical, efficient for
The device of nucleic acid rapid extraction and a kind of nucleic acid rapid extracting method.
The device for nucleic acid rapid extraction of the present invention, including syringe, adsorbed film and sample needle, also includes a hollow
Adapter, described adsorbed film is arranged on the bottom in an absorption membrane tube, and adapter is connected in the middle of syringe and absorption membrane tube
And syringe, adapter connect with absorption membrane tube fluid path.
Absorption membrane tube is the body of both ends open, and one end is the opening end of this body, and the other end fixes an osculum connecting tube,
Adsorbed film is arranged on this osculum connecting tube one end；Absorption membrane tube opening end size is mated with the outside dimension of adapter.
Syringe include cylinder, the tubulose connecting seat being placed in barrel front end and be placed in cylinder can push-and-pull push rod, institute
The external diameter stating osculum connecting tube is equal with the connecting seat outside dimension of syringe, and described adapter sets intermediate throughholes, intermediate throughholes
Size is mated with the connecting seat outside dimension of syringe.
Described sample needle includes needle stand and syringe needle, the external diameter of described osculum connecting tube and the needle stand internal diameter of described sample needle
Join.
Adapter one end connects syringe, the osculum connecting tube of other end connection absorption membrane tube, forms the drawing liquid of this device
Mode.
Or, adapter one end connects syringe, the other end is socketed in the opening end of absorption membrane tube, the osculum of absorption membrane tube
Connecting tube connects sample needle, and form this device pushes away liquid mode.
Described adapter profile is round table-like, the opening end of the little head end outside dimension of round table-like adapter and absorption membrane tube
Coupling, this opening end is from the little head end socket of round table-like adapter.
Another object of the present invention is to provide a kind of method of nucleic acid rapid extraction.
The method of the nucleic acid rapid extraction that the present invention provides, using aforementioned nucleic acid rapid extraction device, through sample cracking
After process, adsorbed film absorption sample amplifying nucleic acid, rinsing liquid rinse the step such as adsorbed film remove impurity and elution nucleic acid, obtain
Obtain pure nucleic acid solution.
The method can be " two take out one pushes away " method: " one takes out " makes the nucleic acid absorption in lysate on filter membrane；" two take out " makes drift
Washing liquid cleans impurity by filter membrane；" one pushes away " will adsorb the nucleic acid compositions eluting on filter membrane, thus completing biological specimen
The extraction of amplifying nucleic acid composition；Specifically include following steps:
A) reagent sign subpackage:
By the lysate that may relate in nucleic acid extraction, rna adsorption liquid, rna enzyme inhibitor, 95% ethanol, 75% ethanol,
Each person-portion subpackage such as rinsing liquid, eluent is managed to 1.5ep, and each pipe is indicated；
B) clinical sample is added in cracking liquid pipe and (if be related to rna extracted, add rna adsorption liquid and the suppression of rna enzyme
Preparation), and mix；
C) suction: using the drawing liquid form of nucleic acid-extracting apparatus, crack mixed liquor with the opening end adsorbing membrane tube from sample
Middle suction, the suction being produced using syringe pump, the nucleic acid compositions in sample flow through during adsorbed film because of the specific adsorption of film
And adsorb on adsorbed film, filtrate is the waste liquid having the impurity such as cell debris and protein, and waste liquid enters syringe cavity and waste liquid
Merely through this adsorbed film once；
D) two suction: using the drawing liquid form of nucleic acid-extracting apparatus, aspirated from rinsing liquid pipe with the opening end adsorbing membrane tube,
The suction being produced using syringe pump, cleans adsorbed film with rinsing liquid, and cleaning remains in protein or other on adsorbed film
Composition, filtrate enters syringe cavity and this filtrate merely through adsorbed film once；
E) one push away: change the clean syringe of another, first suck eluent, then push away liquid using nucleic acid-extracting apparatus
Form (membrane tube will be adsorbed reversely, make adsorbed film in front, opening end socket adapter one end of absorption membrane tube, absorption membrane tube
Osculum connecting tube connects the needle stand of sample needle), the eluent in syringe cavity is added in absorption membrane tube, is pushed away using syringe
Eluent is flowed through adsorbed film by the pressure that pressure produces, and the nucleic acid compositions eluting on film is pushed to the collection of follow-up nucleic acid by absorption
In container or detection means, complete the extraction process of sample amplifying nucleic acid.
The method also can be " three push away " method: " one pushes away " makes the nucleic acid absorption in lysate on filter membrane；" two push away " makes rinsing
Liquid cleans impurity by filter membrane；" three push away " will adsorb the nucleic acid compositions eluting on filter membrane, thus completing biological specimen center
The extraction of sour composition；Specifically include following steps:
A) reagent sign subpackage:
By the lysate that may relate in nucleic acid extraction, rna adsorption liquid, rna enzyme inhibitor, 95% ethanol, 75% ethanol,
Each person-portion subpackage such as rinsing liquid, eluent is managed to 1.5ep, and each pipe is indicated (using different letters and chromatic zones
Point)；
B) clinical sample is added in cracking liquid pipe and (if be related to rna extracted, add rna adsorption liquid and the suppression of rna enzyme
Preparation), and mix.
C) first suck sample cracking mixed liquor with syringe, the liquid form that pushes away using nucleic acid-extracting apparatus (adsorbs membrane tube anti-
To not setting sample needle), pushing, using syringe, the pressure producing makes cracking mixed liquor flow through filter membrane, and the nucleic acid in sample becomes
Adsorb on adsorbed film because of the specific adsorption of film when shunting is through adsorbed film, filtrate is to have the impurity such as cell debris and protein
Waste liquid, waste liquid be discharged and waste liquid merely through adsorbed film once；
D) resorb rinsing liquid with syringe, the liquid form that pushes away using nucleic acid-extracting apparatus (is adsorbed membrane tube reversely, do not set and add
Sample pin), push, using syringe, the pressure producing and rinsing liquid is added to cleaning adsorbed film in adsorption tube, clearly remain in absorption
Protein on film or other composition, filtrate be discharged and this filtrate merely through adsorbed film once；
E) change the clean syringe of another and suck eluent, push away liquid form (adsorbed film using nucleic acid-extracting apparatus
Pipe reversely, adds sample needle), the eluent in syringe cavity is added in absorption membrane tube, pushes generation using syringe
Eluent is flowed through adsorbed film by pressure, and the nucleic acid compositions eluting on film is pushed to follow-up nucleic acid collecting container or inspection by absorption
Survey in device, complete the extraction process of sample amplifying nucleic acid.
Using above scheme, nucleic acid-extracting apparatus of the present invention adopt bulk-breaking mode, are used by assembling collocation, design is skilful
Wonderful, structure is simple, practical.Wherein, adapter two ends can be simultaneously connected with different parts, and absorption membrane tube detachably and reversely fills
Join, with hollow and size coincide adapter use cooperatively, complete nucleic acid extraction from absorption washing taking to eluting different phase
Join, make nucleic acid extraction simple, quick, efficiently.Meanwhile, the present invention instead of high speed centrifuge using syringe, using extraction
Device just can reach detached effect in liquid phase heterogeneous system.
The present invention can extract as a kind of simple and direct means that before nucleic acid amplification, biological specimen is carried out with nucleic acid extraction
Sample purity meets the requirement of next step nucleic acid amplification enough, without the need for any instrument, safe operation and simple, disposable
Use, thus pathogen or host nucleic acids extraction can be widely used in, for Clinical detection and diagnosis.The present invention extracts
Installation cost is cheap, simple to operate need any specific apparatus simultaneously and not, the operating time is short, about only needs to 10 15 minutes,
Thus the present invention can be widely used in the relatively poor basic hospital of some experiment conditions and economically less developed region or
Country；It is also possible to meet the nucleic acid quick diagnosis of the demand of high-level hospital, particularly emergency room and bedside well.
Brief description
Fig. 1 is used for the device exploded view of nucleic acid rapid extraction for the present invention；
Fig. 2 device exercises decomposition and constitutional diagram during pumping function；
Fig. 3 device exercises decomposition and constitutional diagram when pushing function；
The Salmonella dna pcr augmentation detection result that Fig. 4 present example 1 obtains；
The trichinella dna amplification of nucleic acid testing result that Fig. 5 embodiment of the present invention 2 obtains；
The h1n1rna rt lamp augmentation detection result that Fig. 6 embodiment of the present invention 3 obtains.
Specific embodiment
The present invention is provided to the method for the device of nucleic acid rapid extraction and nucleic acid extraction, for from various clinical samples
Rapid extraction nucleic acid (includes rna and dna).
Provided by the present invention for nucleic acid rapid extraction device referring to shown in Fig. 1 Fig. 3, main include syringe 1, inhale
Membrane pipe 2, adapter 3 and 4 four parts of sample needle, wherein:
Syringe 1 is common tubular disposable syringe or the similar device that can exercise syringe function, and it has cylinder
Body 11, it is placed in cylinder 11 front end for connecting the tubulose connecting seat 12 of syringe needle and be placed in cylinder 11 can push-and-pull
Push rod 13；Sample needle 4 is common injection needle, including needle stand 41 and syringe needle 42.
Adapter 3 is cylinder or the round platform of hollow, its intermediate throughholes 31 size and syringe 1 tubulose connecting seat 12 outer diameter ruler
Very little coupling, enables this tubulose connecting seat 12 to be inserted in adapter 3 intermediate throughholes 31；Separately, adapter 3 appearance is cylindric or circle
Mesa-shaped, its outside dimension is mated with absorption membrane tube 2, described below；Adapter 3 can have resilient material such as rubber, plastics
Make.
Absorption membrane tube 2 is the body of both ends open, and available plastic material is made, and its one end is the opening end 21 of this body,
The other end fixes an osculum connecting tube 22, and absorption membrane tube 2 inner bottom part (connecting tube 22 one end is referred to as bottom) sets adsorbed film 23 (commercially available business
Product, such as pellosil silica membrane)；The both ends open of absorption membrane tube 2 all can be connected with adapter 3, wherein opening end 21
Size is mated with the outside dimension of cylindric adapter 3, or mates with the little head end outside dimension of round table-like adapter so that turning
Connect device 3 to be socketed with opening end 21；Absorption membrane tube 2 osculum connecting tube 22 external diameter identical with syringe 1 connecting seat 12, both with add
The needle stand 41 internal diameter coupling of sample pin 4, and mate with adapter 3 intermediate throughholes 31 size, so that this osculum connecting tube 22 can be inserted
Connect sample needle 4 in needle stand 41, connection adapter 3 in intermediate throughholes 31 can be inserted into again.
In the present invention, when extracting solution, connect the connecting seat 12 of syringe 1 with adapter 3 one end, the other end connects absorption
The osculum connecting tube 22 of membrane tube 2, makes adsorbed film 23 (before and after defining with syringe use direction in the present invention, that is, fill syringe needle rear
One end is defined as " front ", and when push rod extracts, the direction of motion is defined as " afterwards "), that is, combine the drawing liquid shape of nucleic acid rapid extraction device
Formula (as shown in Figure 2).When releasing solution, connect the connecting seat 12 of syringe 1 with adapter 3 one end, the other end stretches into adsorbed film
The opening end 21 of pipe 2, and the osculum connecting tube 22 adsorbing membrane tube 2 reconnects the needle stand 41 of sample needle 4, that is, combine nucleic acid quick
Extraction element push away liquid form (as shown in Figure 3).
It is used for the device of nucleic acid rapid extraction using the present invention, through clinical sample cracking process, syringe pump cracking
After the steps such as liquid, nucleic acid absorption film absorption nucleic acid, rinsing liquid rinsing nucleic acid absorption film, elution nucleic acid, it is possible to obtain pure
Nucleic acid solution be used for downstream test.
The ultimate principle of nucleic acid extraction of the present invention, is that the adsorbed film (alternatively referred to as filter membrane) in adsorption tube has specifically to nucleic acid
Adsorption, the nucleic acid absorption in sample and cracking mixed liquor is then passed through rinsing purifying and reaches the purpose of purification, finally
It is eluted out from adsorbed film, to realize nucleic acid and separate with sample ensureing that the maximum of a small amount of or high value sample amplifying nucleic acid is returned again
Receipts amount.Under normal circumstances, the nucleic acid extraction after cracking needs in liquid phase heterogeneous system, and the centrifugal force using centrifuge reaches
To Liquid liquid Separation, the effect of solid-liquor separation.But apparatus of the present invention only need to " two take out once push away " or " three push away " and can complete institute
There is operation." two take out one pushes away " is: " one takes out ": make the nucleic acid absorption in lysate on filter membrane；" two take out ": make rinsing liquid pass through to filter
Film completes to clean the purpose of impurity；" one pushes away ": by nucleic acid compositions eluting on filter membrane for the absorption, thus completing in biological specimen
The extraction of nucleic acid compositions." three push away ": " one pushes away ": make the nucleic acid absorption in lysate on filter membrane；" two push away ": so that rinsing liquid is passed through
Filter membrane completes to clean the purpose of impurity；" three push away ": by nucleic acid compositions eluting on filter membrane for the absorption, thus completing biological specimen
The extraction of amplifying nucleic acid composition.
For this reason, the method taking nucleic acid that the present invention provides uses above nucleic acid-extracting apparatus, a kind of method is referred to as " two
Take out one to push away " method, comprise the following steps:
A) reagent sign subpackage:
By the lysate that may relate in nucleic acid extraction, rna adsorption liquid, rna enzyme inhibitor, 95% ethanol, 75% ethanol,
Each person-portion subpackage such as rinsing liquid, eluent is managed to 1.5ep, and each pipe is indicated (using different letters and chromatic zones
Point)；
B) clinical sample is added in cracking liquid pipe and (if be related to rna extracted, add rna adsorption liquid and the suppression of rna enzyme
Preparation), and mix；
C) suction: using the drawing liquid form of nucleic acid-extracting apparatus, crack mixed liquor with the opening end adsorbing membrane tube from sample
Middle suction, the suction being produced using syringe pump, the nucleic acid compositions in sample flow through during adsorbed film because of the specific adsorption of film
And adsorb on adsorbed film, filtrate is the waste liquid having the impurity such as cell debris and protein, and waste liquid enters syringe cavity and waste liquid
Merely through this adsorbed film once；
D) two suction: using the drawing liquid form of nucleic acid-extracting apparatus, aspirated from rinsing liquid pipe with the opening end adsorbing membrane tube,
The suction being produced using syringe pump, cleans adsorbed film with rinsing liquid, and cleaning remains in protein or other on adsorbed film
Composition, filtrate enters syringe cavity and this filtrate merely through adsorbed film once；
E) one push away: change the clean syringe of another, first suck eluent, then push away liquid using nucleic acid-extracting apparatus
Form (membrane tube will be adsorbed reversely, make adsorbed film in front, opening end socket adapter one end of absorption membrane tube, absorption membrane tube
Osculum connecting tube connects the needle stand of sample needle), the eluent in syringe cavity is added in absorption membrane tube, is pushed away using syringe
Eluent is flowed through adsorbed film by the pressure that pressure produces, and the nucleic acid compositions eluting on film is pushed to the collection of follow-up nucleic acid by absorption
In container or detection means, complete the extraction process of sample amplifying nucleic acid.The so obtained nucleic acid water for not containing other impurities
Solution, can be used for nucleic acid amplification.
What the present invention provided takes the method for nucleic acid to use above nucleic acid-extracting apparatus, and another kind of method is referred to as " three push away "
Method, comprises the following steps:
A) reagent sign subpackage:
By the lysate that may relate in nucleic acid extraction, rna adsorption liquid, rna enzyme inhibitor, 95% ethanol, 75% ethanol,
Each person-portion subpackage such as rinsing liquid, eluent is managed to 1.5ep, and each pipe is indicated (using different letters and chromatic zones
Point)；
B) clinical sample is added in cracking liquid pipe and (if be related to rna extracted, add rna adsorption liquid and the suppression of rna enzyme
Preparation), and mix.
C) first suck sample cracking mixed liquor with syringe, the liquid form that pushes away using nucleic acid-extracting apparatus (adsorbs membrane tube anti-
To not setting sample needle), pushing, using syringe, the pressure producing makes cracking mixed liquor flow through filter membrane, and the nucleic acid in sample becomes
Adsorb on adsorbed film because of the specific adsorption of film when shunting is through adsorbed film, filtrate is to have the impurity such as cell debris and protein
Waste liquid, waste liquid be discharged and waste liquid merely through adsorbed film once；
D) resorb rinsing liquid with syringe, the liquid form that pushes away using nucleic acid-extracting apparatus (is adsorbed membrane tube reversely, do not set
Sample needle), push, using syringe, the pressure producing and rinsing liquid is added to cleaning adsorbed film in adsorption tube, clearly remain in suction
Protein on membrane or other composition, filtrate be discharged and this filtrate merely through adsorbed film once；
E) change the clean syringe of another and suck eluent, push away liquid form (adsorbed film using nucleic acid-extracting apparatus
Pipe reversely, adds sample needle), the eluent in syringe cavity is added in absorption membrane tube, pushes generation using syringe
Eluent is flowed through adsorbed film by pressure, and the nucleic acid compositions eluting on film is pushed to follow-up nucleic acid collecting container or inspection by absorption
Survey in device, complete the extraction process of sample amplifying nucleic acid.The so obtained aqueous solution nucleate for not containing other impurities, can use
In nucleic acid amplification.
The nucleic acid extracting through the present invention can apply to microorganism detection, detection in Gene Mutation, single nucleotide polymorphism
Detection etc. is related to the downstream experiment of nucleic acid.Nucleic acid is can be dna or rna.
It is that the isothiocyanic acid flesh solution clinically commonly used or chelex lysate etc. can for the lysate in the present invention
Solution with cell lysis；Rinsing liquid is the cleanout fluid of ph < 7, such as 70% ethanol；Eluent be 10mm tris-hcl or
Distilled water；Adsorbed film is the various film that can adsorb nucleic acid, such as pellosil (silica membrane).Agents useful for same in the present invention
All commercially available with material.
The application of the present invention is described with example below.
Example 1: the extraction of Salmonella dna
This example taking the extraction of Salmonella dna as a example, verifies whether nucleic acid-extracting apparatus of the present invention and method are effective.This reality
Example extracts totally 4 parts of sample (1 is negative control, and 24 is Salmonella), proceeds as follows respectively:
1. crack
Take the bacterium solution of 500 microlitres of Salmonella incubated overnight, add the lysate (composition: naoh, 25mmol of equivalent；
Tris hcl, 10mmol；Triton x 100,1%；Np 40,1%；Edta, 0.1mmol；Chelex 100,2%.)；Mix
Rear chamber is gentle and quiet to put 5 minutes, using the drawing liquid form of nucleic acid-extracting apparatus, mixed liquor is pumped to absorption membrane tube, makes mixed liquor warp
Cross adsorbed film, the waste liquid without nucleic acid enters syringe cavity.
2. clean
Using the drawing liquid form of nucleic acid-extracting apparatus, rinsing liquid is drawn in absorption membrane tube, ibid step operation makes rinsing
Liquid cleans the impurity on adsorbed film, and waste liquid enters into syringe cavity.
3.dna eluting
Change a clean syringe, 120 microlitres of eluents are sucked into syringe cavity, pushing away using nucleic acid-extracting apparatus
Liquid form, pushes, with syringe, the power producing and eluent is pushed into absorption membrane tube, eluent flows through adsorbed film, and will adsorb in film
On nucleic acid compositions pushed to a new centrifuge tube by sample needle together with eluent, standby.
4. nucleic acid amplification
Application Salmonella pcr amplifing reagent (2 × tap mix test kit, the limited public affairs of Tiangeng biochemical technology (Beijing)
Department), expand the dna extracting respectively, concrete reaction is as follows: 20 microlitres of Salmonella amplification reaction solution, extracts in centrifuge tube
5 microlitres of nucleic acid (dna) solution, cumulative volume is 25 microlitres, response procedures: 95 DEG C, circulation in 5 minutes；94 DEG C, 30 seconds, 58 DEG C,
30 seconds, 72 DEG C, totally 35 circulations in 30 seconds；72 DEG C, circulation in 7 minutes；4 DEG C of preservations.
5. result detection
Amplified production is detected by electrophoresis, and result such as accompanying drawing 4 (wherein m is nda marker, and 1 is negative control, 2
4 is Salmonella).The testing result that can be seen that Salmonella sample from the result of Fig. 4 is the positive, shows that the present invention is husky
Door Salmonella dna extracts highly effective.
Example 2: loop-mediated isothermal amplification method detects the trichinella in blood
This example by loop-mediated isothermal amplification method detect blood in trichinella as a example, illustrate nucleic acid-extracting apparatus of the present invention and
The application of method.Detection totally 8 parts of sample (1 6 samples be positive infection blood sample, 78 samples be negative control), carry out respectively as
Lower detection operation:
1. crack
Take 500 microlitres of blood sample, add the lysate (composition: naoh, 25mmol of equivalent；Tris hcl,
10mmol；Triton x 100,1%；Np 40,1%；Edta, 0.1mmol；Chelex 100,2%.)；Mix rear chamber gentle and quiet
Put 5 minutes, using the drawing liquid form of nucleic acid-extracting apparatus, mixed liquor is pumped to absorption membrane tube, make mixed liquor through adsorbed film, no
Waste liquid containing nucleic acid enters syringe cavity.
2. clean
Using the drawing liquid form of nucleic acid-extracting apparatus, rinsing liquid is drawn in absorption membrane tube, ibid step operation makes rinsing
Liquid cleans the impurity on adsorbed film, and waste liquid enters into syringe cavity.
3.dna eluting
Change a clean syringe, 120 microlitres of eluents are sucked into syringe cavity, pushing away using nucleic acid-extracting apparatus
Liquid form, pushes, with syringe, the power producing and eluent is pushed into absorption membrane tube, eluent flows through adsorbed film, and will adsorb in film
On nucleic acid compositions pushed to a new centrifuge tube by sample needle together with eluent, standby.
4. nucleic acid amplification
Application trichinella loop-mediated isothermal amplification reagent (lamp method dna amplification kit (dna amplification
Kit), Japanese Eiken Chemical (eiken chemical co., ltd, tochigi, japan)), amplification extracts
Trichinella dna.Concrete reaction is as follows: 20 microlitres of trichinella amplification reaction solution, nucleic acid (dna) solution 5 extracting in centrifuge tube is micro-
Rise, cumulative volume is 25 microlitres, reaction temperature and time: 65 °, react 60 minutes.
5. result detection
With real-time transmissometer, amplified production is detected.Result such as Fig. 5.The result of Fig. 5 shows, the blood sample of positive infection
(sample 1 6) detection of nucleic acids result after amplification is the positive.Show that this example is extracted trichinella dna in blood sample and be can be used for detecting
In.
Example 3: the extraction of influenza A viruss h1n1rna and detection in throat swab
This example taking the extraction of influenza A viruss h1n1rna in throat swab and detection as a example, illustrates nucleic acid extraction of the present invention
Device and method is also suitably for extracting rna.Totally 8 parts of sample of detection (1 be negative control, 28 are positive sample), carry out respectively as
Lower extraction is operated with detection:
1. crack
Take throat swab sample 200u1, add appropriate lysate (composition: naoh, 25mmol；Tris hcl, 10mmol；
Triton x 100,1%；Np 40,1%；Edta, 0.1mmol；Chelex 100,2%.), rna adsorption liquid (composition: cracking
Equivalent 99% ethanol is added in liquid) and rna enzyme inhibitor (composition: carrier rna), fully mix, room temperature stands 5 minutes；
Using the drawing liquid form of nucleic acid-extracting apparatus, mixed liquor is pumped to absorption membrane tube, makes mixed liquor through adsorbed film, without nucleic acid
Waste liquid enters syringe cavity.
2. clean
Using the drawing liquid form of nucleic acid-extracting apparatus, rinsing liquid is drawn in absorption membrane tube, ibid step operation makes rinsing
Liquid cleans the impurity on adsorbed film, and waste liquid enters into syringe cavity.
3.rna eluting
Change a clean syringe, 120 microlitres of eluents are sucked into syringe cavity, pushing away using nucleic acid-extracting apparatus
Liquid form, pushes, with syringe, the power producing and eluent is pushed into absorption membrane tube, eluent flows through adsorbed film, and will adsorb in film
On nucleic acid compositions pushed to a new centrifuge tube by sample needle together with eluent, standby.
4. nucleic acid amplification
Application rt lamp constant-temperature amplification reagent (lamp method rna amplification kit (rna amplification kit), day
This Eiken Chemical (eiken chemical co., ltd, tochigi, japan)), amplification extracts
h1n1rna.Concrete reaction is as follows: 20 microlitres of rt lamp amplification reaction solution, nucleic acid (rna) solution 5 extracting in centrifuge tube is micro-
Rise, cumulative volume is 25 microlitres, reaction temperature and time: 60 °, 60 minutes.
5. result detection
Amplification is detected with real-time transmissometer, result such as Fig. 6 (1 is negative control, and 28 are positive sample).
The testing result that the result of Fig. 6 can be seen that all positive sample is the positive.Show that this example can be extracted h1n1rna and be used in combination
In detection.
Claims (8)
1. it is used for the device of nucleic acid rapid extraction, including syringe, adsorbed film and sample needle it is characterised in that also including in one
Empty adapter, described adsorbed film is arranged on the bottom in an absorption membrane tube, and adapter is connected in syringe and absorption membrane tube
Between and syringe, adapter with absorption membrane tube fluid path connect；
Absorption membrane tube is the body of both ends open, and one end is the opening end of this body, and the other end fixes an osculum connecting tube, referred to as
Bottom, adsorbed film is arranged on this osculum connecting tube one end；Absorption membrane tube opening end size is mated with the outside dimension of adapter；
Syringe include cylinder, the tubulose connecting seat being placed in barrel front end and be placed in cylinder can push-and-pull push rod, described little
The external diameter of mouth connecting tube is equal with the connecting seat outside dimension of syringe, and described adapter sets intermediate throughholes, intermediate throughholes size
Mate with the connecting seat outside dimension of syringe.
2. according to claim 1 be used for nucleic acid rapid extraction device it is characterised in that described sample needle include needle stand and
Syringe needle, the external diameter of described osculum connecting tube is mated with the needle stand internal diameter of described sample needle.
3. it is used for the device of nucleic acid rapid extraction according to claim 1 it is characterised in that the connection of adapter one end is injected
Device, the osculum connecting tube of other end connection absorption membrane tube, form the drawing liquid mode of this device.
4. the device for nucleic acid rapid extraction according to claim 1 or claim 2 is it is characterised in that the connection of adapter one end is noted
Emitter, the other end is socketed in the opening end of absorption membrane tube, the osculum connecting tube connection sample needle of absorption membrane tube, forms this device
Push away liquid mode.
5. it is used for the device of nucleic acid rapid extraction according to claim 4 it is characterised in that described adapter profile is round platform
Shape, the little head end outside dimension of round table-like adapter is mated with the opening end of absorption membrane tube, and this opening end is from round table-like adapter
The socket of little head end.
6. a kind of method of nucleic acid rapid extraction, usage right requires the device of 1 to 5 arbitrary described nucleic acid rapid extraction, passes through
Sample cracking process, adsorbed film absorption sample amplifying nucleic acid, rinsing liquid rinsing adsorbed film remove impurity and elution nucleic acid step
Afterwards, obtain pure nucleic acid solution.
7. according to claim 6 method it is characterised in that be " two take out one pushes away " method: " one takes out " makes the nucleic acid in lysate
It is adsorbed on filter membrane；" two take out " makes rinsing liquid pass through filter membrane and cleans impurity；" one pushes away " will adsorb the nucleic acid compositions on filter membrane
Eluting, thus complete the extraction of biological specimen amplifying nucleic acid composition；Specifically include following steps:
A) reagent sign subpackage:
By the lysate that may relate in nucleic acid extraction, rna adsorption liquid, rna enzyme inhibitor, 95% ethanol, 75% ethanol, rinsing
Liquid, eluent each person-portion subpackage is managed to 1.5ep, and each pipe is indicated；
B) clinical sample being added in cracking liquid pipe, and mix, if be related to rna extracted, adding rna adsorption liquid and rna enzyme
Inhibitor；
C) suction: using the drawing liquid form of nucleic acid-extracting apparatus, taken out from sample cracking mixed liquor with the opening end adsorbing membrane tube
Inhale, the suction being produced using syringe pump, the nucleic acid compositions in sample flow through to be inhaled because of the specific adsorption of film during adsorbed film
Be attached on adsorbed film, filtrate is the waste liquid having cell debris and protein impurities, waste liquid enter syringe cavity and waste liquid merely through
This adsorbed film is once；
D) two suction: using the drawing liquid form of nucleic acid-extracting apparatus, aspirated from rinsing liquid pipe with the opening end adsorbing membrane tube, utilize
The suction that syringe pump produces, cleans adsorbed film with rinsing liquid, and cleaning remains in protein or other composition on adsorbed film,
Filtrate enters syringe cavity and this filtrate merely through adsorbed film once；
E) one push away: change the clean syringe of another, first suck eluent, then push away liquid shape using nucleic acid-extracting apparatus
Formula, will adsorb membrane tube reversely, make adsorbed film in front, opening end socket adapter one end of absorption membrane tube, adsorb the little of membrane tube
Mouth connecting tube connects the needle stand of sample needle, and dress syringe needle one end is front, and the eluent in syringe cavity is added in absorption membrane tube,
Push, using syringe, the pressure producing and eluent flowed through adsorbed film, and by absorption the nucleic acid compositions eluting on film push to
In follow-up nucleic acid collecting container or detection means, complete the extraction process of sample amplifying nucleic acid.
8. according to claim 6 method it is characterised in that be " three push away " method: " one pushes away " makes the nucleic acid absorption in lysate
On filter membrane；" two push away " makes rinsing liquid clean impurity by filter membrane；" three push away " will adsorb the nucleic acid compositions eluting on filter membrane, from
And complete the extraction of biological specimen amplifying nucleic acid composition；Specifically include following steps:
A) reagent sign subpackage:
By the lysate that may relate in nucleic acid extraction, rna adsorption liquid, rna enzyme inhibitor, 95% ethanol, 75% ethanol, rinsing
Liquid, eluent each person-portion subpackage is managed to 1.5ep, and makes a distinction sign to each pipe using different letters and color；
B) clinical sample being added in cracking liquid pipe, and mix, if be related to rna extracted, adding rna adsorption liquid and rna enzyme
Inhibitor；
C) first suck sample cracking mixed liquor with syringe, push away liquid form using nucleic acid-extracting apparatus, absorption membrane tube reversely, no
If sample needle, pushing, using syringe, the pressure producing makes cracking mixed liquor flow through filter membrane, and the nucleic acid compositions in sample flow through
Adsorb on adsorbed film because of the specific adsorption of film during adsorbed film, filtrate is the waste liquid having cell debris and protein impurities,
Waste liquid be discharged and waste liquid merely through adsorbed film once；
D) resorb rinsing liquid with syringe, push away liquid form using nucleic acid-extracting apparatus, absorption membrane tube reversely, does not set sample-adding
Pin, pushes, using syringe, the pressure producing and rinsing liquid is added to cleaning adsorbed film in adsorption tube, cleaning remains in adsorbed film
On protein or other composition, filtrate be discharged and this filtrate merely through adsorbed film once；
E) change the clean syringe of another and suck eluent, push away liquid form using nucleic acid-extracting apparatus, absorption membrane tube is anti-
To, add sample needle, the eluent in syringe cavity is added in absorption membrane tube, will using the pressure that syringe pushing produces
Eluent flows through adsorbed film, and the nucleic acid compositions eluting on film is pushed to follow-up nucleic acid collecting container or detection means by absorption
In, complete the extraction process of sample amplifying nucleic acid.
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| CN104059849B (en) * | 2013-07-12 | 2017-01-18 | 中国人民解放军疾病预防控制所 | Device and method used for rapid extraction of nucleic acid |
| CN105567670A (en) * | 2014-10-11 | 2016-05-11 | 中国医学科学院基础医学研究所 | Method and apparatus for preserving nucleic acid in urine sample |
| CN104593252B (en) * | 2015-01-12 | 2017-02-22 | 国家纳米科学中心 | Sample pretreatment and amplification integrated nucleic acid analysis system based on pipette tip and application of system |
| CN105779277A (en) * | 2015-01-13 | 2016-07-20 | 温和心 | Nucleic acid extraction suction tube |
| JP2016158557A (en) * | 2015-03-02 | 2016-09-05 | セイコーエプソン株式会社 | Container, biological substance purification cartridge, and assembly kit for biological substance purification cartridge |
| CN105296327B (en) * | 2015-10-21 | 2017-10-27 | 陈辉 | A kind of nucleic acid-extracting apparatus and its extracting method |
| CN106047660A (en) * | 2016-07-07 | 2016-10-26 | 李丽倩 | Nucleic acid extraction device for nucleic acid isolation machine |
| CN106754343B (en) * | 2017-01-12 | 2017-10-31 | 武汉菲思特生物科技有限公司 | DNA sequencing apparatus and system based on pyrosequencing |
| CN106947683B (en) * | 2017-05-24 | 2024-02-20 | 苏州天隆生物科技有限公司 | Nucleic acid extraction and purification device and method |
| CN109207340B (en) * | 2017-06-30 | 2022-08-12 | 开启基因股份有限公司 | Nucleic acid extraction assembly |
| CN107574165A (en) * | 2017-09-27 | 2018-01-12 | 广州和实生物技术有限公司 | A kind of method for extracting nucleic acid and the extraction element for realizing this method |
| CN110272807B (en) * | 2018-03-13 | 2020-11-24 | 武汉医蒂生物科技有限公司 | Positive pressure device for nucleic acid extraction and purification |
| CN110437980B (en) * | 2019-08-28 | 2023-12-22 | 湖北微伞医疗科技有限公司 | Nucleic acid extraction adsorption column |
| CN113249188A (en) * | 2021-06-03 | 2021-08-13 | 宁波康程德诺生物医药有限公司 | Rapid nucleic acid extraction device and method for extracting nucleic acid |
| CN114045207B (en) * | 2021-09-26 | 2024-02-06 | 厦门健康工程与创新研究院 | Nucleic acid extraction detection system and method |
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| JP4102149B2 (en) * | 2002-09-25 | 2008-06-18 | 富士フイルム株式会社 | Nucleic acid separation and purification equipment |
| CN101684463A (en) * | 2008-09-28 | 2010-03-31 | 杭州优思达生物技术有限公司 | Method and kit for rapidly extracting nucleic acids from trace clinical samples |
| CN102409079A (en) * | 2010-08-26 | 2012-04-11 | 杭州优思达生物技术有限公司 | Novel infectious disease nucleic acid rapid detection kit and its detection method |
| CN104059849B (en) * | 2013-07-12 | 2017-01-18 | 中国人民解放军疾病预防控制所 | Device and method used for rapid extraction of nucleic acid |
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