CN105441318B - It is configured with the nucleic acid product extraction equipment of barriers - Google Patents
It is configured with the nucleic acid product extraction equipment of barriers Download PDFInfo
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- CN105441318B CN105441318B CN201410403037.9A CN201410403037A CN105441318B CN 105441318 B CN105441318 B CN 105441318B CN 201410403037 A CN201410403037 A CN 201410403037A CN 105441318 B CN105441318 B CN 105441318B
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Abstract
The invention discloses a kind of nucleic acid product extraction equipment for being configured with barriers, comprising：Tubing string, with first and second tube body, tubing string is hollow and both ends open, make to form runner in tubing string, and first the open ora terminalis of one of tube body form the first opening, the open ora terminalis of one of second tube body forms the second opening, wherein, the internal diameter of tubing string is open along an axial direction from first to be gradually reduced to the second opening；Lid, it is to be coaxially disposed in the first opening, to connect the first tube body, lid forms top cover portion in one end and forms a hollow interior bolt in the other end of relative top cover portion, and hollow interior bolt is coaxially inserted in the first tube body by the first opening；Barriers, it is coaxially disposed in hollow interior bolt；And a bracket, this is used for the biological specimen of holding one；Wherein, bracket has plural through hole should.The nucleic acid product extraction equipment disclosed in this invention for being configured with barriers extracts suitable for automatic nucleic acid, to improve overall efficiency and quality.
Description
Technical field
The present invention is used for nucleic acid extraction about a kind of nucleic acid product extraction equipment for being configured with barriers especially with respect to one kind
The nucleic acid product extraction equipment for being configured with barriers.
Background technology
The application of the technologies such as identification, detection, diagnosis with gene flourishes and popularization, and numerous technical fields are for nucleic acid
The demand of abstraction technique increasingly increases；And be developed so far in response to a large amount of and Rapid Screening demand, nucleic acid extraction technology, progressively
Automation is moved towards by manual operation.The fields such as current either clinical treatment, basic research or legal medical expert's application, automatic nucleic acid
Abstraction technique already becomes indispensable instrument.
At present, the method species of conventional nucleic acid extraction is various and respectively has advantage and disadvantage, such as：Organic solvent (phenol and chloroform)
The conventional method of extraction, it is obtained, and nucleic acid purity, quality are good, but step is complicated tediously long and causes asking for organic solvent murder by poisoning
Topic；Resin process for sequestration (such as Chelex-100 resins), can quickly obtain nucleic acid but purity is not high, limit follow-up answer
With；Affine in immunity adsorption method, it is reached effect of extracting using monoclonal antibody absorption nucleic acid, is related to the preparation of antibody, and cost is high,
More difficult popularization；Adsorption column method (column-based method), centrifugation behaviour is mainly coordinated with silicon matrix adsorption column
Make extracting nucleic acid, it is simple and effective, turn into one of method of current main flow；Magnetic bead extracting process, its using small magnetic bead and
Magnetic fields, nucleic acid is adsorbed or be desorbed under high and low salting liquid environment, reaches nucleic acid extraction effect, simple and efficiency high, nucleic acid
Purity Coriolis mass is also good, is also increasingly becoming one of method of main flow.Wherein, " adsorption column method " and " magnetic bead extracting process " is because of it
The simple characteristic of operation, is easily substituted manually by plant equipment, and then turns into the main flow side for being applied to automatic nucleic acid extraction now
Method.
For the demand of high flux, Rapid Screening, automatic nucleic acid extracts the very hot network of development of platform, especially with " tubing string
Adsorption method " is large with " magnetic bead extracting process ".Wherein " magnetic bead extracting process " because it is superior for DNA effect of extracting,
Extensive use in terms of DNA extraction field occupies first place, but the application power of the extraction for RNA is slightly weak；The opposing party
Face, " adsorption column method " is preferable for RNA extracting power, but due to needing to coordinate centrifugation or vacuum attraction to take method to make
With process is comparatively complicated and time-consuming, and centrifugal device or vacuum suction device must be installed in platform, causes equipment body
Product is huge, therefore in terms of automation, still with volume light and handy " magnetic bead extracting process " still relatively takes advantage and it is universal.
However, no matter which kind of method used, at present in the construction and optimization of automatic nucleic acid extraction platform, to the greatest extent may be used
It is target that manually-operated " full automation ", which can be reduced, and specific demand includes：Reduction personnel contact with sample or a corpse or other object for laboratory examination and chemical testing
With operating, preventing aerosol (aerosol) from being formed, preventing sample cross contamination, improving the stability of platform in operation and accuracy, carry
Rise the efficiency of extraction and the quality of nucleic acid product etc.；Especially for the biological specimen that may have biohazard doubt, or method
Appreciation biological specimen is cured, the demand seems increasingly important.
For present situation, a certain degree of artificial participation is stilled need in the handling process of nucleic acid extraction and is operated, its
In be still difficult to be applied to automated system completely, it is necessary to manually handle with " pre-treatment " step of biological specimen.This is so-called
The pre-treatment step of biological specimen is mainly comprising the collection and cracking for biological source tissue.
For example, the pre-treatment program of traditional biological specimen, it is that biological specimen (biological tissue, a corpse or other object for laboratory examination and chemical testing) is inserted into examination
Pipe, adds reagent treatment, reagent treatment is sufficiently mixed with biological specimen, be used for follow-up extraction procedure.Traditionally, to add
The pre-treatment program of fast biological specimen, is inserted in test tube container frequently with solid plunger, promotes sample the means such as to stir or stamp
This reagent treatment between mixing, lytic effect, if however, such gimmick careless manipulation, or the configuration of plunger and test tube
Relative position or configuration design are improper, easily cause content (liquid) and overflow or even produce the significant problems such as splash；The opposing party
Face, biological specimen chip may also block or the collection of each stage corpse or other object for laboratory examination and chemical testing of RNA extraction procedure, therefore, the technology of this type
Still it is difficult to the effect for reaching quick mixing sample and reagent treatment, is also unfavorable for follow-up collection, is not suitable for being applied to automation
Nucleic acid extraction platform.
On the other hand, foregoing stirring or stamp the technology of formula and be also difficult to be applied to " solid-state biological specimen ".This meaning " solid-state
Biological specimen " refers to the biological specimen of non-liquid, includes extensively：Soft or solid-state animal vegetable tissue；Biology may have been attached
The solid matter of tissue (blood, scurf, body fluid, secretion etc.) such as samples swab stick, the biological organization sample of FFPE, cigarette
The mark of the base of a fruit and various criminal sides card or a corpse or other object for laboratory examination and chemical testing etc..By the biological tissue of institute's appendix on solid-state biological sample is mostly with micro-
Amount, the scattered or characteristic such as broken, therefore, pretreatment process, which concerns, obtains the content and quality of a corpse or other object for laboratory examination and chemical testing.More specifically, in Qian Chu
During reason, solid-state biological specimen suitable with reagent treatment must be sufficiently mixed, can be intactly by biological group of its appendix
Knit and fully disengage into reagent treatment environment, for further nucleic acid extraction, produced with obtaining the preferable nucleic acid of concentration and quality
Thing.Therefore, for foregoing conventional method, during biological specimen for liquid, had that liquid is overflowed, chip interference etc. is asked
Topic, if the missing of aforementioned techniques may be aggravated using to solid-state biological specimen, its interference effect that can trigger.
For the problem of aforementioned conventional technology, TaiWan, China patent application case discloses to be carried in No. 201311885 specification
Gone out a kind of plunger assembly, it has been used in test tube, liquid handling is carried out to biological specimen.Referring to Fig. 1, this plunger 1 is hollow circle
Post, bottom are provided with hole 3, some long and narrow holes 4 are also formed on cylinder, by containing the test tube 2 of biological specimen 5 and treatment fluid
It is interior to move back and forth up and down, biological specimen 5 is sufficiently mixed with treatment fluid；And after mixing, it is not necessary to remove plunger 1 and allow liquid relief
Pipe 6 draws chip of the mixing liquid 7 without being drawn into biological specimen 5 in test tube 2 via the opening at the top of plunger 1, with profit
Extracting nucleic acid.Although the design of this plunger assembly in invisible spectro pre-treatment program has the problem of, carries for biological specimen
A solution is gone out, in practice, when this plunger assembly is applied to automatic nucleic acid extraction platform, has still had multinomial
Missing, in detail as following.
First, due to when carrying out nucleic acid extraction used treatment fluid (such as lysate) often high level salt solution and often to add
Added with compositions such as enzyme, interfacial agents, to promote biological specimen to disengage, therefore viscosity is generally higher；Therefore, when using plunger
When 1, after moving up and down extruding of the treatment fluid by plunger 1 that plunger 1 makes in test tube 2, pass through hole 3 and long and narrow hole 4 or flowing
In by the gap between test tube and plunger, although stirring effect can be produced effectively, due to the more viscid characteristic for the treatment of fluid, together
When have impact on fluency of the treatment fluid by hole 3 and long and narrow hole 4, produce for the liquid in the test tube of narrow space and substantially disturb
It is dynamic, increase the risk of liquid splash；Especially when force not at that time, it is possible to cause liquid to spill outside test tube 2.Second, when place
When managing in liquid containing adding ingredients such as enzyme, interfacial agents, the overall surface tension for the treatment of fluid is have impact on, thus treatment fluid is existed
By, there may be the phenomenon for attaching to peritreme or plunger wall, blocking the flowing for the treatment of fluid, or even draw when hole 3 and long and narrow hole 4
The problem of lotion body drips.Third, in mixed biologic sample 5 and treatment fluid, the problem of foam is common is produced, however, working as
During using the mixed biologic sample 5 of foregoing plunger 1 with treatment fluid, hole 3, narrow is circulated in because treatment fluid is frequently extruded
Gap between elongated hole 4 and test tube 2 and plunger 1, the problem of aggravation foam is produced, interference operation.Fourth, according to plunger 1 it
It design, must be moved up and down when operating plunger 1, make the plunger 1 of part must still be exposed to outside test tube；And moved simultaneously in plunger 1,
Invisible spectro liquid is also at the state of flowing, forms an extremely unstable system, plunger 1 is dripped treatment fluid possibility and increases
It is high.Fifth, according to above-mentioned potential misgivings, this plunger assembly is not suitable for rapidly moving up and down, therefore is difficult to lift nucleic acid extraction effect
Rate.In addition, foregoing each problem may cause the ill effects such as aerosol (aerosol) formation, cross pollution, infection harm.
In summary, the biological specimen pretreatment technology of platform is extracted currently for automatic nucleic acid, still there is multinomial shortcoming
Urgently overcome.
The content of the invention
For above-mentioned technical problem and demand, the present invention provides a kind of nucleic acid product extraction equipment for being configured with barriers,
It is characterized in that：One tubing string, there is one first tube body and one second tube body, the tubing string is hollow and both ends open, is made
A runner is formed in the tubing string, and the open ora terminalis of one of described first tube body forms one first opening, second body
The open ora terminalis in one of portion forms one second opening, wherein, the internal diameter of the tubing string along an axial direction from described first be open to
Second opening gradually reduces, and the mean inside diameter of first tube body is more than the mean inside diameter of second tube body；
One lid, it is coaxially disposed in the described first opening, to connect first tube body, the lid forms a top cover in one end
Portion simultaneously forms a hollow interior bolt in the other end of relatively described top cover portion, and the hollow interior bolt is coaxially inserted by the described first opening
In first tube body；One barriers, it is coaxially disposed in the hollow interior bolt；And a bracket, it is coaxially disposed the runner
In, for the biological specimen of holding one；Wherein, the bracket has plural through hole, for a reagent by the through hole, makes the examination
Agent in the runner in back and forth circulating.
Wherein, the center of the top cover portion forms a top cover hole, and hollow interior bolt forms an air flue, and the gas with top cover hole
Road is interconnected with the runner.
Wherein, it is with screw thread, trip between the lid and the first tube body or engages connection.
Wherein, the material of the barriers is including sponge, filter pulp, dust-proof cotton, ventilative filter membrane by being selected in following combination
Etc. material.
Wherein, the through hole on the bracket is geometry.
Wherein, the bracket is a discoid bracket (607) and forms upward shoulder in the bracket periphery.
Wherein, the bracket is that the bracket side wall of a discoid bracket (608) and the bracket is arc bracket side wall.
Wherein, a sealing ring is further configured in the arc bracket side wall of the bracket.
Therefore, the main object of the present invention is to provide a kind of nucleic acid product extraction equipment for being configured with barriers, can be for
Gas circulates, and has gas flow guiding function, also to avoid reagent liquid from splashing in operating process, causes to be infected with liquid shifting equipment
The problem of polluting.
Brief description of the drawings
Fig. 1, it is the nucleic acid product extraction equipment schematic diagram for being configured with barriers of prior art.
Fig. 2, it is the perspective view according to the nucleic acid product extraction equipment for being configured with barriers of the present invention.
Fig. 3 A, it is the stereo decomposing according to the nucleic acid product extraction equipment for being configured with barriers of one embodiment of the invention
Figure.
Fig. 3 B, it is the solid point of the nucleic acid product extraction equipment for being configured with barriers according to another embodiment of the present invention
Xie Tu.
Fig. 4 A, it is the sectional view according to the nucleic acid product extraction equipment for being configured with barriers of one embodiment of the invention.
Fig. 4 B, it is the sectional view according to the nucleic acid product extraction equipment for being configured with barriers of one embodiment of the invention.
Fig. 5, it is the close-up schematic view according to the nucleic acid product extraction equipment for being configured with barriers of the present invention.
Fig. 6 A, 6B, 6C, 6D, 6E, 6F, it is the support according to the nucleic acid product extraction equipment for being configured with barriers of the present invention
Seat schematic diagram.
Fig. 7 A, 7B, 7C, 7D, be according to the present invention be according to the present invention be configured with barriers nucleic acid product extract
The bracket schematic diagram of device.
Fig. 8, it is the schematic flow sheet of the manual operation biological specimen pre-treatment of prior art.
Fig. 9, it is schematic flow sheet of the manual operation biological specimen pre-treatment for automation nucleic acid extraction of prior art.
Figure 10, located before the nucleic acid product extraction equipment for being configured with barriers of the present invention is used in automatic nucleic acid extraction
Manage the schematic flow sheet of program.
Figure 11, it is the nucleic acid colloid electrophoresis figure according to the experimental example 1 and the gained of reference examples 1 of the present invention.
Figure 12, it is the nucleic acid colloid electrophoresis figure according to the experimental example 2 and the gained of reference examples 2 of the present invention.
【Primary clustering symbol description】
Axial direction A
Central shaft C
Radial direction R
Driving direction T
Plunger 1
Test tube 2
Hole 3
Long and narrow hole 4
Biological specimen 5
Pipette 6,80
Mixing liquid 7
First tube body 10
First opening 11
Lid 20
Second tube body 30
Second opening 33
Barriers 40
Solid-state sample 50
FFPE samples 52,56
Liquid storage container 70
Reagent 90,92,96
It is configured with the nucleic acid product extraction equipment 100,200 of barriers
First pipeline 101
First screw thread 110
Hollow interior bolt 201
Top cover portion 203
Air flue 204
Second screw thread 210
Second pipe 301
Microcentrifugal tube 500
Crack a corpse or other object for laboratory examination and chemical testing 521,522,523,561,562,563
Corpse or other object for laboratory examination and chemical testing chip 524
Nucleic acid 564
Bracket 60,601,602,603,604,605,606,607,608,60
9、610
Filter plug 801
Water layer 901
Organic layer 902
Tubing string 1030
Tube wall 1032
Bottom plate 2010
Through hole 2011
Interior bolt flow channel 2020
Top cover hole 2030
Through hole 6010,6020,6030,6050,6060,6070,6,090 60
10、6020、6030、6050、6060、6070、6080、6090、6100
Filter screen 6040
Bracket side wall 6071,6081
Bracket face 6072,6082,6092,6102
Shoulder 6074,6084
Nucleic acid product PCR testing results 11a, 11b, 12a, 12b
Thick extract 901 ', 92 ', 96 '
Electrophoresis result A1, A2, A3, A4, A5, A6, A7, M1, M2, M3, M4, M5,
M6、M7
Embodiment
To make the purpose of the present invention, technical characteristic and advantage, can more correlative technology field personnel understood, and be able to reality
The present invention is applied, coordinates appended schema herein, specifically illustrates the technical characteristic and embodiment of the present invention, and enumerate preferable implementation
Example further illustrates.It is the expression signal relevant with feature of present invention with the schema hereinafter compareed, also need not be according to
Completely drawn according to practical situation；And it is related in the explanation on this case embodiment in technology well known to those skilled in the art
Hold, be also not repeated here, conjunction is first chatted bright.
One of for the benefit of explanation present invention, the present invention shown in Figure 2 embodiment, it is by configuration to define the present invention in advance
There is the length direction of tubing string 1030 (in appearance to be cylindric) in the nucleic acid product extraction equipment 100 of barriers, be defined as " axle
To direction " A, and the nucleic acid product extraction equipment 100 for being configured with barriers has the central shaft C of an imagination along this axial direction A；
And the horizontal direction (i.e. the cross-sectional direction of cylinder) of tubing string 1030 is pressed, it is defined as " radial direction " R.In addition, based on foregoing
Central shaft C, when each component of the present invention is set, put and set such as centered on this central shaft C, then with the term of " coaxial "
It is described.
Referring to Fig. 2, the nucleic acid product extraction equipment for being configured with barriers of the present invention includes tubing string 1030, lid 20 and support
Seat 60.Embodiments thereof is sequentially described in detail below.
First, the tubing string 1030 of the present invention is that in axial direction A sequentially forms the first tube body 10 and the second tube body 30.
And tubing string 1030 is hollow and both ends open, make to form runner in the tubing string 1030, and this runner is according to the first tube body 10, the
Two tube bodies 30 further can sequentially divide into the first pipeline 101 and second pipe 301.Wherein, the first tube body 10 is vertically
Opening ora terminalis of the direction A in the open end of neighbouring tubing string 1030 forms the first opening 11, the second tube body 30 in axial direction A in
The ora terminalis of the open end of neighbouring tubing string 1030 forms the second opening 33.Wherein, because tubing string 1030 is hollow, thus pipeline is made
1030 have particular inside diameters to R directions, and runner radially has specific caliber (diameter i.e. radially), and
When tubing string 1030 is located at same cross-section location with runner, the internal diameter of tubing string 1030 and the caliber of runner are substantially the same.Tubing string
The thickness for the tube wall 1032 that 1030 internal diameter can be adjusted by tubing string 1030 with the pipe diameter size of runner is reached, and the present invention is unlimited
The thickness distribution of tubulation wall 1032, if when implementing in response to current technology the manufacture and use present invention normality, the present invention is with pipe
The integral thickness of wall 1032 is unanimously preferred embodiment.Tube wall 1032 only can be adjusted on demand in tubing string 1030 not according to this user
Thickness, configuration with position to reach the effect of difference, such as：It is to save material cost or riser wall to reduce its thickness
Lightness, increase its thickness with reinforced structure or adjustment mechanical performance, adjust its configuration and optimized with sharp fluid environment etc., all should cover
In in the category of the present invention.
Secondly, according to the design of the nucleic acid product extraction equipment 100 for being configured with barriers of the present invention, within tubing string 1030
In axial direction A 33 gradually reduces from the first opening 11 to second opening in footpath, make the first tube body 10 mean inside diameter (according to
Preferable embodiment, the visual average caliber for the first pipeline 101) it is more than the mean inside diameter of the second tube body 30
(according to preferable embodiment, the visual average caliber for second pipe 301)；And in a preferred embodiment,
The diameter of second opening 33 is generally between 0~3mm, and for preferable diameter between 1~3mm, optimal diameter is big
It is small between 1~1.5mm.According to the design of this tubing string 1030, make the nucleic acid product extraction equipment 100 for being configured with barriers overall
Funnel-form is presented, the tip of a pipe shape is made up of less second tube body 30 of mean inside diameter, liquid is used as by the second opening 33
Body reagent gateway, help to carry out drawing and discharging for liquid reagent in biological specimen pre-treatment program.
The present invention is configured with the lid 20 of the nucleic acid product extraction equipment 100 of barriers, is coaxially disposed in the first opening
On 11, to connect the first tube body 10.According to preferable embodiment, lid 20 has linking tubing string 1030 with moving
The function of liquid equipment (driving force for providing liquid relief), the nucleic acid product extraction equipment 100 for enabling to be configured with barriers is by lid
20 are connected to liquid shifting equipment, to drive the movement of the reagent liquid used in biological specimen pre-treatment program；And according to existing
For technology, for liquid shifting equipment mainly still using the means of intake-gas as the power resources for driving liquid movement, this is this area
Technology can will readily appreciate that, therefore no longer row repeats.
To reach above-mentioned purpose and effect, referring to Fig. 2,3A, 3B, wherein Fig. 3 A are matching somebody with somebody according to one of present invention embodiment
The three-dimensional exploded view of the nucleic acid product extraction equipment of barriers is equipped with, and Fig. 3 B are the configurations according to another embodiment of the present invention
There is the three-dimensional exploded view of the nucleic acid product extraction equipment of barriers.As shown in Figure 3A, lid 20 is further provided with a top cover portion 203
With the hollow interior bolt 201 extended from the one end of lid 20；In operation, hollow interior bolt 201 can coaxially be put by the first opening 11
Enter in the first pipeline 101；And the center of top of top cover portion 203 forms top cover hole 2030, makes hollow interior bolt 201 and the shape of top cover hole 2030
Runner into air flue 204, and air flue 204 and tubing string 1030 is interconnected, and when liquid shifting equipment intake-gas, this air flue 204 is
For the passage of gas flowing.In addition, when implementing the lid 20 of the manufacture present invention, top cover portion 203 can use with hollow interior bolt 201
Integrally formed manufacture production system is (as shown in Figure 3A)；Also after can be using manufacture top cover portion 203 respectively and hollow interior bolt 201
It is assembled into lid 20 (as shown in Figure 3 B).
It is with screw thread, trip between the tube body 10 of lid portion 203 and first in addition, according to the preferred embodiments of the invention
Or engagement connection.Screw thread, trip or engagement connection be for component connection means commonly used in the art, its principle with mode of operation not
It is specified in this again.On the implementation, user can reach connection lid 20 and the using modes such as screw thread, trip, engagements on demand
One tube body 10；Such as：Threaded connection is with the characteristic that easily assembles and can dismantle once again；And trip connection is equally easy to group
Dress, also in manufacture, and its fastening effect is good；Engagement connection, design are simple with manufacture.According to preferable embodiment,
Lid 20 reaches the movement that airtight effect is advantageous to liquid shifting equipment driving reagent liquid, therefore this hair after being connected with the first tube body 10
It is bright be with the tube body 10 of lid 20 and first be to be threaded to preferable embodiment aspect, with simultaneously reach be fastenedly connected with it is airtight
Effect；Similarly, when other connection means of use, the present invention also covers the various sealing rings of use (O-ring) or other are auxiliary
Component is helped, the tube body 10 of lid 20 and first is reached the aspect of airtight effect.
In addition, the product and mold design of the existing ready-made specification of screw thread, trip and the technological means of engagement can directly cover
With favorably manufacturing and be easy to user's flexible application.Therefore, the present invention does not limit the connection side of the tube body 10 of lid 20 and first
Formula, integrally formed manufacture method is also not excluded for, only lid 20 can reach the function of connecting tubing string 1030 and liquid shifting equipment with before
The every effect person stated, the category of the present invention all should be covered by.For the benefit of concise description, hereafter only said with the means of threaded connection
The bright present invention is configured with the embodiment of the nucleic acid product extraction equipment 100 of barriers.
Fig. 3 A and Fig. 4 A are referred to, wherein Fig. 4 A are produced according to the nucleic acid for being configured with barriers of one of present invention embodiment
The sectional view of thing extraction equipment.The nucleic acid product extraction equipment 100 for being configured with barriers of the present invention, it is using threaded connection
Means lid 20 is connected to the first tube body 10；Specifically, the ora terminalis of the first tube body 10 adjacent to first opening 11 it
Outer wall forms the first screw thread 110, and the inner edge of top cover portion 203 is accordingly provided with the second screw thread 210, makes hollow interior bolt 201 by the
After one opening 11 coaxially inserts the first pipeline 101, the second screw thread 210 spins to the first screw thread 110, reaches threaded connection, with tight
It is affixed to close the first tube body 10 and lid 20；Similarly, when there is the first tube body of dismounting 10 with 20 demand of lid, user
The first tube body 10 and lid 20 can be reversely outwarded winding easily, release both engagements.
Please with further reference to Fig. 4 B, be the nucleic acid product extraction equipment 100 that illustrative configurations have barriers set-up mode and
It is used in relative position during liquid shifting equipment.Wherein, Bio-Pretreatment program reagent used is mainly liquid, by liquid storage container
70 are held, and liquid storage container 70 is stretched into by the open end of the second tube body 30, and draw or discharge reagent liquid by the second opening 33, are made
Reagent liquid can be in the first pipeline 101 with being flowed in second pipe 301.
Referring again to Fig. 2,4A, 4B, due to manually operated or non-full-automatic biological specimen pre-treatment program there may be
The problem of cross pollution, therefore the present invention is further in top cover portion 203 and in forming a bottom plate on the other end of relative top cover
2010 and a through hole 2011 is configured on bottom plate 2010；Wherein, through hole 2011 can be circulated with supplied gas, have gas flow guiding
Function, also to avoid reagent liquid from splashing in operating process, cause to be infected with the problem of liquid shifting equipment pollutes.In addition,
The diameter of this through hole 2011 and the second 33 diameters of opening of tubing string 1030 coordinate, less than or equal to the second opening 33
Diameter, diameter is generally between 0~3mm, and preferable diameter is between 1~3mm, optimal diameter
Between 1~1.5mm.The principal element that through hole 2011 coordinates with the second 33 diameters of opening is：When through hole 2011
It is too small, bubble can be produced because of extruding when reagent liquid is pressed through through hole 2011 by operating process, and then by interference experiment
Operation；And if through hole 2011 is excessive, be able to can cause biological specimen obstruction through hole 2011 problem, therefore the present invention and then
Limiting the size of through hole 2011 needs the second 33 sizes of opening of tubing string 1030 to coordinate, big less than or equal to the second opening 33
It is small.The problem of to avoid cross pollution in biological specimen pre-treatment program, according to being configured with for preferable embodiment
The nucleic acid product extraction equipment 100 of barriers, it, in being further provided with a barriers 40 in air flue 204, is same with tubing string 1030
Axle is set, and the first opening 11 is flowed to close and obstruct reagent.
Therefore, when reagent liquid flows in the first pipeline 101 and second pipe 301, on the one hand controllable liquid relief is set
Standby air-flow (force size) is to avoid reagent liquid from rushing at the first opening 11；On the other hand, interior bolt 204 towards second opening 33 it
Direction is the semi-enclosed function of both having may achieve gas flow guiding with the design of through hole 2011, also can effectively stop reagent liquid mistake
Degree flows to liquid shifting equipment；In addition, referring to Fig. 5, signal gas bolt flow channel 2020 and gas edge within being formed in interior bolt 204
The situation of driving direction T flowings, it can be seen that, the design of barriers 40 is also overall to enhance the effect for taking precautions against reagent liquid backflow
Fruit.According to above-mentioned purpose and function, the materials of barriers 40 to have gas permeability concurrently with water proofing property as preferable embodiment aspect,
This so-called water proofing property is to broadly refer to the effect to barrier liquid flowing, in addition to ventilative spill resistant material, is had specific
The material of the liquid absorption capacity of volume can also achieve water proofing property.Specifically, the material of barriers 40 be preferably sponge, filter pulp,
The materials such as dust-proof cotton, ventilative filter membrane.
Followed by referring to Fig. 2,3A, 3B, 4A, 4B.The nucleic acid product extraction equipment bag for being configured with barriers of the present invention
It is to be coaxially disposed in the first pipeline 101, for holding biological specimen containing bracket 60；Wherein, there is bracket 60 plural through hole to make examination
Agent makes reagent liquid can be in back and forth being circulated in the runner of tubing string 1030 by these through holes.According to the concept of the present invention, bracket 60
Be holding biological specimen, reagent liquid is fully contacted biological specimen by through hole thereon, and by driving reagent liquid in
Back and forth circulated in pipeline, reach and wash away biological specimen, promote the biological tissue with nucleic acid compositions to disengage.Purpose accordingly, this hair
Bright bracket 60 is using the stable runner for being fixedly arranged on tubing string 1030 as preferable embodiment aspect.
According to one of present invention embodiment, an oblate column is substantially presented in the outward appearance of bracket 60, and the external diameter of bracket 60 is substantial
It is more than or equal to the internal diameter of second pipe 30 less than the internal diameter of the first pipeline 10；Therefore, based on aforementioned biological pretreating device
100th, the mean inside diameter of 200 first tube bodies 10 is allowed to configuration more than the mean inside diameter of the second tube body 30, when bracket 60 and pipe
After post 1030 is separately manufactured again row assembling when, bracket 60 is after coaxially inserting the first pipeline 101 in advance, is less than the by mean inside diameter
The second pipe 301 of one pipeline 101 props up bracket 60 and reaches fixed effect.It is same to reach according to another embodiment of the present invention
Axle sets the effect of bracket 60, can construct component simultaneously in being provided with engaging tooth, prominent rib etc. in the first pipeline 101 or second pipe 301
The appearance design of corresponding adjustment bracket 60, make the construction components such as engaging tooth, prominent rib are corresponding to fasten or abut bracket 60, reach fixed
The effect of bracket 60.According to another embodiment of the present invention, bracket 60 can also be reached with tubing string 1030 by integrally formed manufacture
Into process that is fixed, and then eliminating assembling bracket 60.
On the bracket of the present invention, the present invention is not particularly limited the design of its through hole, can be the through hole of various geometries,
And through hole footpath (it essentially corresponds to the sectional area of through hole) includes extensive Size Distribution.For example, referring to Fig. 6 A, 6B, 6C,
6D, it is a variety of aspects of the through hole of the bracket 60 of the citing signal present invention：Fig. 6 A are that signal bracket 601 is logical provided with multiple rectangles
Hole 6010, and through hole 6010 is regularly arranged, wherein, the diameter of through hole 6010 is generally between 0~3mm, preferable diameter
Size is between 1~3mm, and optimal diameter is between 1~1.5mm.Fig. 6 B are that signal bracket 602 is provided with multiple circles
Or the through hole 6020 of ellipse, and through hole 6020 is arranged with irregular mode, wherein, the diameter of through hole 6020 is generally 0
Between~3mm, preferable diameter is between 1~3mm, and optimal diameter is between 1~1.5mm.Fig. 6 C are signals
Bracket 603 is provided with the through hole 6030 of multiple rhombuses, and through hole 6030 is arranged with regular fashion, wherein, the diameter of through hole 6030 is big
Between small generally 0~3mm, preferable diameter is between 1~3mm, and optimal diameter is between 1~1.5mm.Figure
6D is the function for the through hole for illustrating bracket 603, can be by equally having the filter screen 6040 of through hole or penetrating grid to substitute implementation, equally
It may achieve foregoing holding biological specimen and make the functions such as reagent liquid circulation.Fig. 6 E are signal brackets 605 provided with multiple by small shape
The through hole 6050 that forms of slit, wherein, the diameter of through hole 6050 is generally between 0~3mm, preferable diameter
Between 1~3mm, optimal diameter is between 1~1.5mm.6F figures be signal bracket 606 be provided with it is multiple by small shape and
The through hole 6060 that the slit of bending is formed, wherein, the diameter of through hole 6060 is generally between 0~3mm, preferable diameter
Size is between 1~3mm, and optimal diameter is between 1~1.5mm.In 6A, 6B, 6C, 6D figure through hole 6010,
6020th, 6030,6040,6050 diameter is coordinated with the second 33 diameters of opening of tubing string 1030, is less than or equal to
The diameter of second opening 33, it is too small when through hole 6010,6020,6030,6040,6050, is operated in reagent liquid
Journey can be because extruding produces bubble when being pressed through through hole 2011, and then interference experiment is operated；And through hole 6010,6020,
If the 6030,6040,6050 is excessive, can cause the problem of biological specimen obstruction, therefore, through hole 6010 of the present invention, 6020,
6030th, 6040,6050 diameter needs the second 33 diameters of opening of tubing string 1030 to coordinate, less than or equal to the second opening
33 size.With avoid reagent liquid from producing bubble in operation and cause biological specimen blocking via hole 6010,6020,
6030th, 6040,6050 problem.
On the bracket of the present invention, the present invention is not particularly limited the design of its configuration.For example, referring to Fig. 7 A, 7B, 7C,
7D is the bracket 60 for illustrating the present invention in response to different demands.The embodiment aspect for now only enumerating four kinds of brackets is illustrated：Fig. 7 A are
Illustrate a discoid bracket 607, its bracket face 6072 is provided with multiple circular or ellipse through holes 6070, along discoid
The periphery of bracket 607 form upward shoulder 6074, the bracket side wall 6071 of bracket 607 is straight wall, makes bracket 607 overall
Present to referred to as discoid, being designed with for this disk contains biological specimen beneficial to stable, and its peripheral side keep away with pipe string internal wall it
Friction area is big, can strengthen the effect of fixed bracket 607.Fig. 7 B are to illustrate another discoid bracket 608, its bracket face 6082
Multiple circular or ellipse through hole 6080 is provided with, upward shoulder is formed along the discoid periphery of bracket 608
6084, make bracket 608 is overall to present to referred to as discoid；Palpus expositor, the bracket side wall 6081 of bracket 608 is arc bracket side
Wall 6081, the application timing of this design are included when bracket 608 is made up of elastomers such as rubber, high polymer elastic plastics, can
Fixed effect is reached in design by arc bracket side wall 6081；And when needing to strengthen the fixed effect of bracket 608 of hard,
The present invention also includes to be reached using the means of the accessory parts such as sealing ring (O-ring), and now sealing ring can be arranged at arc support
In the space that the bracket side wall of seat side wall 6081 or other configurations is formed, this aspect contributes to the fluid for optimizing reagent liquid steady
It is qualitative, it is more suitable for automating.Fig. 7 C are another discoid brackets 609 of signal, and its bracket face 6092 is provided with multiple rectangles
Through hole 6090, upward shoulder 6094 is formed along the discoid periphery of bracket 609, make bracket 609 is overall to present symmetrically
It is discoid, this aspect illustrates that the present invention is not particularly limited the configuration of through hole set by bracket.Fig. 7 D be one configuration of signal it is simple and
Easily fabricated bracket 610, multiple through holes 6100 are which is provided with, but the flat no shoulder in its bracket face 6102 designs, this is simple
Configuration can reduce production and be made this, the sufficient a large amount of, Rapid Screening of the biological specimen that is particularly suitable for use in automatic nucleic acid extraction put down
Platform.
Must expositor, the present invention is not intended to limit the species of external liquid shifting equipment, and sets up to exempt or significantly adjust shifting
Liquid equipment and it is derivative the problem of, the present invention be to coordinate the conventional device specification of existing liquid shifting equipment as preferable embodiment aspect,
And the principle of device on automating liquid shifting equipment with regular size is as it is known to those skilled in the art that therefore no longer being gone to live in the household of one's in-laws on getting married
State.However, when following liquid shifting equipment is progressively improved, according to the design of the present invention, the configuration in lid portion 203 can be also adjusted by
Design and reach, not only lower production and be made this, also can flexible application to various liquid shifting equipments, and then make the present invention easily general
And.
To be easier to understand the specific implementation content of the nucleic acid product extraction equipment for being configured with barriers of the present invention, can solve
Technical problem certainly and the effect reached, are exemplified below experimental example, and coordinate related signal to be illustrated with data schema.
First, the flow of summary explanation Traditional Man operation biological specimen pre-treatment.Because automatic nucleic acid extracts platform
Applicable biological specimen kind is various, wherein, this meaning solid-state biological specimen higher with the difficulty of the pre-treatment of solid-state biological specimen
Including but not limited to：Swab stick, blood card, stub, areca dregs, chewing gum slag, hair, adhesive tape etc.) and formalin fixation paraffin bag
Biological organization sample (hereinafter referred to as FFPE samples) is buried, being easier to generation technology missing influences nucleic acid extraction performance；Therefore, below
Explanation be respectively using " solid-state biological specimen " and " FFPE samples " as it is of the invention to implement biological specimen pre-treatment program it
Aspect, and enumerate experimental example and reference examples according to it.
The present invention is applied to the biological specimen pre-treatment program of various automatic nucleic acids extraction platform (equipment), such as foregoing
Platform is extracted using automatic nucleic acids such as adsorption column method, magnetic bead extracting process；However, for the benefit of succinct signal is with illustrating this
The implementation result of invention, the experimental example of the explanation manually operated alternative mentioned with reference examples is to use commercially available reagent set below
Group (Genomic DNA Tissue Kit) the commercially available automatic nucleic acid extraction equipment of cooperation (Automated Nucleic Acid Extractor) operated, its principle is detailed with details of operation
It is exposed in the description of product, is that those skilled in the art can be easilyUnderstandAnd operation is followed, therefore be not described in detail then at this, conjunction is first chatted
It is bright.
It is the schematic flow sheet that prior art operates solid-state Sample pretreatment with Traditional Man referring to Fig. 8.In short, will
After solid-state sample 50 inserts microcentrifugal tube 500, reagent 90 is drawn with pipette 80 and added in microcentrifugal tube 500, will be solid
Aspect sheet 50 is sufficiently mixed with reagent 90, is heated 1 hour with 90 DEG C again after first being heated 1 hour with 56 DEG C, is promoted biological tissue to split
Explain.Wherein, because the composition of reagent 90 contains oil-phase solution (or organic solution) liquid compatible with water, through foregoing heating with splitting
Layering is produced after solving program, lower floor is water layer 901, and upper strata is organic layer 902.Then, the aqueous phase of lower floor is drawn with pipette 80
Layer liquid removes water layer 901, that is, obtains thick extract 901 ', this thick extract 901 ' is to be used for follow-up nucleic acid extraction program.This
During conventional method manual operation, pipette 80 from the pipette 80 for being provided with filter plug 801 to be preferred, to avoid manual operation from being spread out
Raw cross-contamination issue.
It is the schematic flow sheet that prior art operates the pre-treatment of FFPE samples 52 with Traditional Man referring to Fig. 9.For FFPE
Sample 52, due to it is existing using reagent set group possessed the effects of good cracking FFPE samples, its operating process is generally same
In the 50 of aforementioned processing solid-state sample.In short, FFPE samples 52 are inserted in microcentrifugal tube 500, drawn with pipette 80
Reagent 92 is simultaneously added in microcentrifugal tube 500, and FFPE samples 52 and reagent 92 are sufficiently mixed, after first being heated 1 hour with 56 DEG C
Heated 1 hour with 90 DEG C again, paraffin contained in FFPE samples 52 is melted while is exposed and crack biological tissue wherein
A corpse or other object for laboratory examination and chemical testing, as cracked a corpse or other object for laboratory examination and chemical testing 521, a cracking corpse or other object for laboratory examination and chemical testing 522 in Fig. 9, cracking shown in a corpse or other object for laboratory examination and chemical testing 523.Then, the step of completing heating cracking
Afterwards, a corpse or other object for laboratory examination and chemical testing 521,522,523 is cracked via the abundant corpse or other object for laboratory examination and chemical testing chip 524 for cracking, forming thick extract 92 ' and residual, wherein, this
Thick extract 92 ' is be used for follow-up nucleic acid extraction program, only using above stilling need with centrifugation means removal corpse or other object for laboratory examination and chemical testing chip 524
Interference.
As can be seen here, above-mentioned conventional method must be before automatic nucleic acid extraction be performed, before manual operation biological specimen
The program of processing, it is difficult to avoid operating problems derived from complicated and manual operation, therefore, it is effective that the present invention now proposes one
Solution.
It is the nucleic acid product extraction equipment 100 for being configured with barriers with the present invention referring to Figure 10, coordinates automatic nucleic acid
Extraction equipment carries out the schematic flow sheet of the pre-treatment of full-automatic operation FFPE samples 56；In addition it is concise description, Figure 10 is only simple
It is single to illustrate automation mechanized operation device with being configured with the interface part that the nucleic acid product extraction equipment 100 of barriers contacts, therefore not
Show whole automation mechanized operation device.
Now equally by taking the pre-treatment of FFPE samples 56 as an example：First, FFPE samples 56 are inserted in tubing string, by the support of bracket 60
FFPE samples 56 are held, lid is assembled into tubing string afterwards completes to be configured with the nucleic acid product extraction equipment 100 of barriers, then fills
If to automatic nucleic acid extraction equipment (such as：ModelSerial equipment) carry out automation mechanized operation.It is loaded with FFPE samples
This 56 nucleic acid product extraction equipment 100 for being configured with barriers gos deep into liquid storage container 70, by the second opening of tubing string 1030
Drawn at 33 reagent 96 (such as：Lysate, cell pyrolysis liquid), reagent 96 is passed through the through hole on bracket 60 and FFPE samples 56
Mixing；Then, frequency and reagent volume are put by the flow velocity of acquiescence, suction, driving reagent 96 is reached fully in back and forth being circulated in runner
The effect of mix reagent 96 and FFPE samples 56；Meanwhile by the temperature control heating program preset, make reagent 96 and FFPE samples
56 mixture synchronization thermally equivalent when mixing, promotes lytic effect, the biological tissue's corpse or other object for laboratory examination and chemical testing for making FFPE samples 56 contained is released
Go out, as shown in a cracking corpse or other object for laboratory examination and chemical testing 561,562,563.FFPE samples 56 are via the sufficient mixing of pre-treatment program with after cracking, releasing
Go out contained nucleic acid 564 into thick extract 96 ', now thick extract can directly be obtained by automatic nucleic acid extraction equipment
96 ' use for follow-up nucleic acid extraction.And needed for the FFPE samples of different type (volume, biological tissue's species etc.) with every
Ask, user can inhale the scheduling for putting program with reagent by elasticity adjustment heating schedule in advance, further to improve the efficiency of pre-treatment.
The narration of following experimental example and reference examples is referred to, the effect that the present invention reaches further is presented.
Experimental example 1：
This experimental example uses solid-state biological specimen to adopt inspection swab stick used.In advance by swab stick pick quantitative blood (20 μ l,
15 μ l, 10 μ l, 5 μ l), then blood swab stick will be stained with and be respectively implanted being configured with the nucleic acid product extraction equipment of barriers of the present invention,
After screwing lid, automation magnetic bead extraction equipment (model is directly installed up to：Super；RBC
Bioscience after), automation carries out the pre-treatment program and nucleic acid extraction program of biological specimen, and each solid-state biological specimen is most
60 μ l nucleic acid product can be obtained eventually.
Reference examples 1：
This reference examples uses solid-state biological specimen to adopt inspection swab stick used.In advance by swab stick pick quantitative blood (20 μ l,
15 μ l, 10 μ l, 5 μ l), then blood swab stick will be stained with and be put into microcentrifugal tube, will be micro by manually adding lysate and Proteinase K
Centrifuge tube is placed on heater with 55 DEG C of continuous heating 30min to obtain pyrolysis product (lysate), that is, completes pre-treatment program；
Then, pyrolysis product is transferred in the reactive tank that automation magnetic bead extraction equipment uses with manual operation, subsequently with magnetic bead
Extracting process separating and extracting nucleic acid product.The present embodiment is that the complete solution of pre-treatment is applied directly into automation magnetic bead extraction to set
Standby (model：Super, RBC bioscience) carry out automatic nucleic acid extraction procedures, each solid-state biological specimen
60 μ l nucleic acid product can finally be obtained.
According to the nucleic acid product of above-mentioned experimental example 1 and the gained of reference examples 1, further with polymerase chain reaction (following letter
Claim PCR) method, expand stable performance gene " glyceraldehyde 3 phosphate dehydrogenase " (Glyceraldehyde 3-phosphate
dehydrogenase；Hereinafter referred to as GADPH) DNA, confirm whether its concentration and quality are applied to subsequent analysis, correlated results
As shown in figure 11.Referring to Figure 11, the nucleic acid product PCR testing results 11b of experimental example 1 is shown, is stained with the μ l of blood volume 20 electrophoresis result
A1, the electrophoresis result A2 for being stained with the μ l of blood volume 15, the electrophoresis result A3 for being stained with the μ l of blood volume 10, the electrophoresis result A4 that is stained with the μ l of blood volume 5 are shown
Obvious and clearly PCR primer, represent no matter blood corpse or other object for laboratory examination and chemical testing content number, its expanding effect is good, at the same reflect via
The acquired Nucleic acid quality of the method for experimental example 1 is preferable, suitable for follow-up genetic test.Review the nucleic acid product of reference examples 1
PCR testing result 11a, it is stained with the μ l of blood volume 20 electrophoresis result M1, the electrophoresis be stained with the μ l of blood volume 15 electrophoresis result M2, be stained with the μ l of blood volume 10
As a result M3, be stained with the μ l of blood volume 5 electrophoresis result M4, show that the brightness of its PCR primer is slightly weak, and PCR primer amount is unstable, is especially stained with
The μ l of blood volume 15 electrophoresis result M2 and be stained with the μ l of blood volume 5 electrophoresis result M4 product amount it is substantially less, reflect biological specimen
Pre-treatment program may not to the utmost ideal caused by result.
Experimental example 2：
This experimental example uses solid-state biological specimen as FFPE samples, and divided into greatly according further to the volume of FFPE samples
Type, medium-sized, small samples.Need expositor, due to this experimental example be intended to verify different pre-treatments mode whether can influence it is not androgynous
The nucleic acid extraction effect of the FFPE samples of product, therefore be not specifically defined for volume size, when only performing, still follow experimental example
The principle that 2 is large-scale with reference examples 2, medium-sized, small samples volume is identical, with Libiee compared with.Further, since large-scale FFPE samples may
For strip, to accelerate pre-treatment efficiency, after FFPE samples can be similarly bent into several equal portions (every section≤0.5CM is preferable)
Insert and be configured with the nucleic acid product extraction equipment of barriers again.
Large-scale, medium-sized, small samples are inserted into being configured with the nucleic acid product extraction equipment of barriers of the present invention respectively,
Add lysate reagent.This lysate reagent is by 400 μ l paraffin solvents, 180 μ l cracked solutions (1%SDS；30mM Tris-
Hcl；10mM EDTA) mixed with 20 μ l Proteinase Ks (proteinase K).After lid is screwed, directly it is installed up to automatic
Change magnetic bead extraction equipment (model：Super；RBC bioscience)；Start simultaneously at and heated under 56 DEG C of environment
And carry out compressing and mix 1 hour, then heating and compressing mix 1 hour under 90 DEG C of environment, afterwards draw mixed liquor to matching somebody with somebody
It is equipped with the nucleic acid product extraction equipment of barriers and stands 10 minutes at room temperature, mixed liquor can be observed and start to produce layering now
As；After the μ l of thick extract about 190 in the water layer of lower floor to be moved into new cuvette groove, residual tissue then stays in and is configured with barriers
Nucleic acid product extraction equipment in.Then, 200 μ l buffer As L (Buffer AL) are added to aqueous layer liquid, and are mixed
It is even, used for follow-up nucleic acid extraction program.This experimental example is to carry out nucleic acid point with adsorption column method (chromatography tubing string separation method)
From：Add 200 μ l 100%EtOH into thick extract be well mixed after, by all liq add chromatography tubing string in, with chromatography
Tubing string separation method carries out nucleic acid separation, is to use QIAGEN nucleic acid purification set groups (QIAGEN；QIAamp DNA FFPE
Tissue Kit；Cat.No.56404 chromatography tubing string)；Its operating procedure is to follow its product operation instruction, therefore not detailed herein
State, final step is to be purged with respectively with 60 μ l ATE buffer solutions and obtain large-scale, medium-sized, small samples nucleic acid products.
Reference examples 2：
Large-scale, medium-sized, small samples are inserted in microcentrifugal tube respectively, add lysate reagent.This lysate reagent
It is by 400 μ l paraffin solvents, 180 μ l cracked solutions (1%SDS；30mM Tris-Hcl；10mM EDTA) and 20 μ l Proteinase Ks
(proteinase K) is mixed.In being shaken mixing 10 seconds, mixed liquor is obtained.Then, with manually to foregoing each mixed
Close liquid to be heated, be after being heated 1 hour under 56 DEG C of environment, slightly to stand at room temperature, while heater can be heated up
To 90 DEG C, device temperature to be heated is completed, and is heated 1 hour under 90 DEG C of environment；During be moderately subject to oscillation on small scale mixing promote
Cracking.During due to heating, aqueous vapor can be produced in microcentrifugal tube, therefore somewhat of short duration can centrifuge aqueous vapor from lower to mixed liquor
In.When this step, mixed liquor can be observed and start to produce lamination；Then, with manual operation pipette, extract lower floor it
The μ l of thick extract about 190 in water layer move into new microcentrifugal tube.200 μ l buffer As L (Buffer AL) are added to aqueous phase
Layer liquid, and be well mixed, used for follow-up nucleic acid extraction program.This reference examples is with adsorption column method (chromatography tubing string point
From method) carry out nucleic acid separation：Add 200 μ l 100%EtOH into thick extract be well mixed after, all liq is added
Chromatograph in tubing string, carry out nucleic acid separation to chromatograph tubing string separation method, be to use QIAGEN nucleic acid purification set groups (QIAGEN；
QIAamp DNA FFPE Tissue Kit；Cat.No.56404 chromatography tubing string)；Its operating procedure is to follow its product to use
Illustrate, therefore be not described here in detail, final step is to be purged with respectively with 60 μ l ATE buffer solutions and obtain large-scale, medium-sized, small samples
Nucleic acid product.
According to the nucleic acid product of above-mentioned experimental example 2 and the gained of reference examples 2, its concentration and Nucleic acid quality are further detected, its
As a result it is shown in table 1.As shown in Table 1, either large-scale, medium-sized, small samples, the core for being configured with barriers of the present invention is used
The nucleic acid product concentration of acid product extraction equipment gained is above artificial pre-treatment；With regard to specific wavelength light absorption value (OD) ratio
For (OD 260/280), nucleic acid product (DNA) quality is same as the group (small samples) of control or is slightly better than the group of control
(large-scale, medium-sized sample)；For specific wavelength light absorption value (OD) ratio (OD 260/230), automation pre-treatment group it
The residual situation of organic solvent is similar to artificial pre-treatment group.Data above shows the barriers of being configured with using the present invention
Nucleic acid product extraction equipment, it is applied to automatic nucleic acid really and extracts, and can effectively obtains that concentration is high and quality also good nucleic acid
Product.
The nucleic acid product testing result of table 1.
According to the nucleic acid product of above-mentioned experimental example 2 and the gained of reference examples 2, further with polymerase chain reaction (following letter
Claim PCR) method, stable performance gene GADPH DNA is expanded, confirms whether its quality is applied to subsequent analysis, correlated results is such as
Shown in Figure 12.Referring to Figure 12, according to experimental example 2 and reference examples 2：Knot is detected to the nucleic acid product PCR of large-scale sample separation acquirement
Fruit 12a is shown；The nucleic acid product PCR testing results 12b of centering pattern this separation acquirement is shown；It is obtained to small samples separation
Nucleic acid product PCR testing results 12b is shown.Wherein, for large-scale sample, with the electrophoresis result M5 of artificial pre-treatment and automation
The electrophoresis result A5 performances of pre-treatment are similar, and only the former is slightly weaker than the latter；For medium-sized sample, with the electrophoresis knot of artificial pre-treatment
Fruit M6 shows its non-output PCR primer, and the electrophoresis result A6 for automating pre-treatment then shows its output PCR primer；For small
Pattern sheet, its non-output PCR primer is shown with the electrophoresis result M7 of artificial pre-treatment, and automate the electrophoresis result A7 of pre-treatment
Then show its output PCR primer.As can be seen here, make the Nucleic acid quality by the automation pre-treating method gained of the present invention good,
Be enough to be directly used in follow-up analysis, although and the group of artificial pre-treatment can measure nucleic acid concentration, can not smooth output PCR
Product, show that its quality is still not enough to be used directly for foranalysis of nucleic acids, it is necessary to which additional processing or purifying can use.
In addition to foregoing extraction DNA implementation content, the present invention also be applied to extraction RNA, be exemplified below experimental example 3 with it is right
3 explanations extract RNA particular content using the nucleic acid product extraction equipment for being configured with barriers of the present invention as usual.Test below
Example 3 is to carry out RNA with adsorption column method (chromatography tubing string separation method) to separate with reference examples 3, is to use QIAGEN nucleic acid purifications
Set group (QIAGEN；RNeasy FFPE Kit；Cat.No.73504 chromatography tubing string)；Its operating procedure is to follow the description of product,
It is not specified in this.
Experimental example 3：
First, FFPE samples are converted into several deciles (every section is not more than 0.5 centimeter) not fractureing insert the present invention's as far as possible
It is configured with the nucleic acid product extraction equipment of barriers, screws lid, lysate reagent is drawn by the pipette tip of the present apparatus.This splits
Solving liquid reagent is mixed by 400 μ l dimethylbenzene, 150 μ l PKD buffer solutions and 10 μ l Proteinase Ks (proteinase K).Connect
, heated under 56 DEG C of environment and carry out compressing mixing 1 hour, followed by it is small to mix 1 for heating and compressing under 90 DEG C of environment
When, all liq is drawn to afterwards in the nucleic acid product extraction equipment for be configured with barriers in being stored at room temperature 10 minutes, observable
Start to produce lamination to mixed liquor, moved into the μ l of thick extract about 140 in the water layer of lower floor by the pipette tip of the present apparatus
After new cuvette groove, residual tissue, which then stays in, to be configured with the nucleic acid product extraction equipment of barriers.Then, in standing 3 on ice
Minute, the DNA enzymatic buffer solution of the volume of water layer cumulative volume 1/10th and 10 μ l DNA enzymatic stoste are subsequently added into, is spun upside down
Microcentrifugal tube is with well mixed, in being stored at room temperature 15 points.Add 320 μ l RBC buffer solutions it is well mixed after, add 1200 μ
L 100%EtOH, and all sample mixed liquors are added in chromatography tubing string, final step is pure with 60 μ l RNase-free
Water purges with acquirement nucleic acid product.
Reference examples 3：
First, FFPE samples will be converted into several deciles (every section be not more than 0.5 centimeter) do not fracture as far as possible insert it is micro from
Heart pipe, add lysate.This lysate reagent is by 400 μ l dimethylbenzene, 150 μ l PKD buffer solutions and 10 μ l Proteinase Ks
(proteinase K) is mixed.Then, mixing 10 seconds is shaken, obtains a mixed liquor.Heating stepses are carried out afterwards,
Be foregoing mixed liquor heats to 15 under 56 DEG C of environment/after, slightly stand at room temperature, while heater can be warming up to
80 DEG C, device temperature to be heated is completed, and mixed liquor is heated under 80 DEG C of environment up to 15 points.During heating, aqueous vapor can be produced in tubing string, because
This can somewhat it is of short duration centrifugation by aqueous vapor from lower into mixed liquor.When this step, mixed liquor can be observed and start to produce layering now
As upper strata is organic layer, and lower floor is water layer, and residual tissue is then stayed among water layer.Then, with the water of pipette, extract lower floor
After the μ l of thick extract about 140 in layer move into new microcentrifugal tube, 3 minutes are stood on ice, is subsequently added into total aqueous phase liquid layer
The DNA enzymatic stoste of the DNA enzymatic buffer solution of the volume of cumulative volume 1/10th and 10 μ l, microcentrifugal tube is spun upside down to mix
It is even, in being stored at room temperature 15 points.Add 320 μ l RBC solution it is well mixed after, add 1200 μ l 100%EtOH, and will be all
Sample mixed liquor add chromatography tubing string in, final step is to purge with acquirement nucleic acid product with 60 μ l RNase-free pure water.
According to the nucleic acid product extraction equipment for being configured with barriers of the foregoing present invention, there is provided one kind may be directly applied to
The nucleic acid product extraction equipment for being configured with barriers of existing automatic nucleic acid extraction equipment, except can effectively lift efficiency, and
It more effectively can significantly lower aerosol (aerosol) formation, cross pollution, infection that existing technology and manual operation may derive
The ill effects such as harm.
Although the present invention various preferred embodiments have been disclosed it has been observed that so its be not limited to the present invention, it is any ripe
This area those skilled in the art is practised, without departing from the spirit and scope of the invention, when a little changes and retouching can be made, thus it is of the invention
Scope of patent protection must be defined depending on the appended claim institute defender of this specification.
Claims (8)
- A kind of 1. nucleic acid product extraction equipment (100) for being configured with barriers, it is characterised in that：One tubing string (1030), has one first tube body (10) and one second tube body (30), and the tubing string (1030) is hollow And both ends open, make to form a runner in the tubing string (1030), and the open ora terminalis of one of described first tube body (10) is formed The open ora terminalis of one first opening one of (11), second tube body (30) forms one second opening (33), wherein, the tubing string (1030) internal diameter gradually reduces along an axial direction from the described first opening (11) to the described second opening (33), makes described the The mean inside diameter of one tube body (10) is more than the mean inside diameter of second tube body (30)；One lid (20), it is coaxially disposed and is open in described first on (11), to connect first tube body (10), the lid (20) form a top cover portion (203) in one end and form a hollow interior bolt in the other end of relatively described top cover portion (203) (201), the hollow interior bolt (201) is coaxially inserted in first tube body (10) by the described first opening (11)；One barriers (40), it is coaxially disposed in the hollow interior bolt (201)；AndOne bracket (60), is coaxially disposed in the runner, for the biological specimen of holding one；Wherein, the bracket (60) has multiple Number through holes (6010), for a reagent by the through hole (6010), make the reagent in back and forth being circulated in the runner.
- 2. the nucleic acid product extraction equipment (100) of barriers is configured with according to claim 1, it is characterised in that described The center of top cover portion (203) forms a top cover hole (2030), and the hollow interior bolt (201) forms one with the top cover hole (2030) Air flue (204), and the air flue (204) is interconnected with the runner.
- 3. the nucleic acid product extraction equipment (100) of barriers is configured with according to claim 1, it is characterised in that described It is with screw thread, trip between lid (20) and first tube body (10) or engages connection.
- 4. the nucleic acid product extraction equipment of barriers is configured with according to claim 1, it is characterised in that the barriers (40) material is including sponge, filter pulp, dust-proof cotton, ventilative filter membrane by being selected in following combination.
- 5. the nucleic acid product extraction equipment (100) of barriers is configured with according to claim 1, it is characterised in that described The through hole (6010) on bracket (60) is geometry.
- 6. the nucleic acid product extraction equipment (100) of barriers is configured with according to claim 1, it is characterised in that described Bracket (60) is one discoid, and forms upward shoulder (6074) in the bracket (60) periphery.
- 7. the nucleic acid product extraction equipment (100) of barriers is configured with according to claim 1, it is characterised in that described Bracket (60) is one discoid, and the bracket side wall (6081) of the bracket (60) is arc bracket side wall (6081).
- 8. the nucleic acid product extraction equipment (100) of barriers is configured with according to claim 7, it is characterised in that described A sealing ring is further configured on the arc bracket side wall (6081) of bracket (60).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410403037.9A CN105441318B (en) | 2014-08-15 | 2014-08-15 | It is configured with the nucleic acid product extraction equipment of barriers |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410403037.9A CN105441318B (en) | 2014-08-15 | 2014-08-15 | It is configured with the nucleic acid product extraction equipment of barriers |
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| CN105441318A CN105441318A (en) | 2016-03-30 |
| CN105441318B true CN105441318B (en) | 2018-02-13 |
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| CN201410403037.9A Expired - Fee Related CN105441318B (en) | 2014-08-15 | 2014-08-15 | It is configured with the nucleic acid product extraction equipment of barriers |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109207340B (en) * | 2017-06-30 | 2022-08-12 | 开启基因股份有限公司 | Nucleic acid extraction assembly |
| TWI742905B (en) * | 2019-12-10 | 2021-10-11 | 財團法人工業技術研究院 | Nucleic acid extracting device |
| CN112940061B (en) | 2019-12-10 | 2024-05-03 | 财团法人工业技术研究院 | Nucleic acid extraction device |
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| JP3630493B2 (en) * | 1995-03-20 | 2005-03-16 | プレシジョン・システム・サイエンス株式会社 | Liquid processing method and apparatus using dispenser |
| CN201648403U (en) * | 2009-10-19 | 2010-11-24 | 杭州优思达生物技术有限公司 | Nucleic acid extraction apparatus without instruments |
| CN203663494U (en) * | 2014-01-14 | 2014-06-25 | 精专生医股份有限公司 | Sample extraction device, suction component thereof, fluid control device and accommodating suite |
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