CN107460109A - A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument - Google Patents
A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument Download PDFInfo
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- CN107460109A CN107460109A CN201710603541.7A CN201710603541A CN107460109A CN 107460109 A CN107460109 A CN 107460109A CN 201710603541 A CN201710603541 A CN 201710603541A CN 107460109 A CN107460109 A CN 107460109A
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- China
- Prior art keywords
- pcr
- bacterium colony
- mixing chamber
- sampling device
- reaction
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
- B01L7/5255—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones by moving sample containers
Abstract
The present invention relates to a kind of reaction system preparation for bacterium colony PCR and sampling device, it includes a reactive component offer portion and multiple mixing chambers and sample feeding pipe, the opening for allowing bacterium colony picking device to pass through is provided with the top of mixing chamber, the outlet in reactive component offer portion connects with each mixing chamber, the outlet of each mixing chamber is connected with a sample feeding pipe, and the exit of mixing chamber is provided with outlet valve.By using the reaction system preparation and sampling device for bacterium colony PCR, setting system is only needed to prepare parameter, pass through control system, the dosing pump set in Indicator Reaction component offer portion adds the respective components of set amount into mixing chamber, then one end for being stained with single bacterium colony of bacterium colony picking device is impregnated in reactive component mixture by opening, PCR system preparation can be completed, prepare obtained PCR reaction systems to be pumped directly in tubular type temperature regulating device by sample feeding pipe, you can complete PCR reactions.
Description
Technical field
The present invention relates to molecular biology equipment, more specifically it relates to a kind of reaction system for bacterium colony PCR prepare and
Sampling device and full-automatic PCR instrument.
Background technology
PCR (PCR) is the underlying technology of molecular biology, and its principle is, using DNA at 95 DEG C or so
High temperature double-stranded DNA is unwind, after cooling, annealed between the primer of high concentration and the DNA profiling to unwind, then by reaction system
Temperature adjustment to polymerase activity temperature, make polymerase complementary along the direction composition along phosphoric acid to pentose (5 ' -3 ')
Chain.Actual PCR instrument based on polymerase manufacture is exactly a temperature control device, can be in denaturation temperature, renaturation temperature, elongating temperature it
Between be controlled well.
Since perkin-elmercetus companies First PCR instrument comes out, tens different producers are there is now in state
Inside and outside production and selling PCR instrument.Between short several years, PCR instrument passes through the development of several generations, constantly employs new technology, and enter
One step develops towards the direction of convenient, practical, high-intelligentization and automation.
Although round pcr obtained very big development, but all PCR instruments are all to control PCR by the following method
Reaction：PCR pipe equipped with PCR reaction systems is placed in the temperature-control hole of fixed temperature control base, and is close to the interior of temperature-control hole
Wall, the temperature of PCR pipe and its internal reaction system filled is controlled by controlling the temperature of temperature control hole wall.Although this kind of device
Generalized, however it there is some it is unconquerable the shortcomings that.
It is one of main shortcoming that time-consuming.Normal PCR reactions generally comprise 30 circulations, wherein denaturation 30s, annealing
30s, extension 30s/kb.Plus denaturation 5min, last eventually to extend 10min, expanding 1kb fragment only needs one and a half hours left side
The right time.However, when being reacted in PCR instrument, it has been found that the fragment for expanding 1kb was generally required at least over 2 hours
Time.The reason is that the position of sample is fixed in PCR instrument, therefore it is necessary to the temperature for manipulating temperature control base change to
React the temperature required for the stage proceeded to.30 circulations, every time circulation have 3 alternating temperatures, that is to say, that need to carry out 90 times
The alternating temperature of left and right.This needs to consume the substantial amounts of time close to the heating and cooling process of 100 times.
In addition to expending the time in heating and cooling process, existing PCR also have one it is huge the shortcomings that be, be not suitable for complete
Automation mechanized operation.Because circular response is carried out in fixed position, it is necessary to prepare reaction system in PCR pipe, then will be equipped with
The PCR pipe of reaction system is transferred in the reacting hole of PCR instrument.Due to being frequently necessary to prepare multiple reaction systems (for example, template is not
With, primer difference etc.), no matter system is prepared and the transfer of PCR pipe, is required for spending a large amount of manpowers, can not be accomplished full-automatic.Mesh
Before untill, also without any instrument can accomplish from PCR system be formulated into PCR reaction complete full-automation.Also have in PCR reactions
A kind of more special PCR is reacted compared with bacterium colony PCR, i.e. after the reactive component beyond removing template is prepared, with toothpick etc.
Bacterium colony picking device picking single bacterium colony, and that end for being stained with bacterium colony is impregnated in reactive component mixed liquor, with this to reactive component
Template is added in mixing, so as to be configured to PCR reaction systems.
The content of the invention
Applicants have invented a kind of tubular type temperature regulating device, reaction system is entered from reaction tube inlet, by pipe shaft, reaction
The temperature of system and identical with the pipe shaft temperature of present position, and is reacted at such a temperature by heat transfer.
Based on the device, the invention provides a kind of reaction system preparation for bacterium colony PCR and sampling device, it includes
One reactive component offer portion and multiple mixing chambers and sample feeding pipe, being provided with the top of the mixing chamber allows bacterium colony picking device
The opening passed through, the outlet in the reactive component offer portion connect with each mixing chamber, and each mixing chamber goes out
Mouth is connected with a sample feeding pipe, and the exit of the mixing chamber is provided with outlet valve.
Prepared by using this for bacterium colony PCR reaction system and sampling device, user can be not subjected to prepare PCR
The hard work of system, especially when up to tens, sample is even hundreds of.Only need setting system to prepare parameter, pass through control
System processed, the dosing pump set in Indicator Reaction component offer portion add the respective components of set amount into mixing chamber, then will
One end for being stained with single bacterium colony of bacterium colony picking device is impregnated in reactive component mixture by the opening, you can completes PCR bodies
System is prepared, and the PCR reaction systems for preparing to obtain are pumped directly in tubular type temperature regulating device by sample feeding pipe, you can it is anti-to complete PCR
Should.
In one embodiment, the reactive component offer portion is the device equipped with hybrid reaction component.
Specifically, the every other component being equipped with the reactive component offer portion in PCR reaction systems outside removing template
Mixed liquor.That is, in described reactive component offer portion be equipped with containing buffer solution, enzyme, dNTP, primer pair mixed liquor.
In one embodiment, the reactive component offer portion includes multiple component provider and a premixer, often
Entrance of the outlet of the individual component provider with the premixer connects, outlet and the mixing chamber of the premixer
Entrance connects.Component provider is respectively provided with each component, and is input to according to the value of setting by measuring pump in premixer, so as to
Obtain the mixture available for multiple PCR reacting doses, the measuring pump in premixer is by the mixture input of PCR amounts react
In mixing chamber, then into mixing chamber, insertion is stained with the bacterium colony picking device of single bacterium colony, so as to be configured to PCR reaction systems.
Preferably, the multiple component provider is supplied PCR reaction buffers, nucleotides to the premixer respectively, gathered
Synthase and primer pair.
Preferably, the reaction system for bacterium colony PCR is prepared and sampling device is also provided with manipulator.Manipulator
Bacterium colony picking device picking single bacterium colony can be clamped automatically, and is impregnated in mixing chamber, PCR reaction systems are configured to, so as to realize bacterium
Fall PCR from the preparation of reaction system and the full-automation of loading.
Present invention also offers a kind of full-automatic PCR instrument, it include above-mentioned PCR reaction systems prepare and sampling device with
And tubular type temperature regulating device, the PCR reaction systems are prepared and sampling device passes through the sample feeding pipe with the tubular type temperature regulating device
Connection.
Brief description of the drawings
Fig. 1 is embodiment 1 for bacterium colony PCR reaction system preparation and the schematic diagram of sampling device；
Fig. 2 is embodiment 2 for bacterium colony PCR reaction system preparation and the schematic diagram of sampling device；
Fig. 3 is the schematic diagram of an example of temperature regulating device；
Fig. 4 is the viewgraph of cross-section of one example of temperature regulating device；
Fig. 5 is the viewgraph of cross-section of an example of temperature regulating device；
Fig. 6 is the schematic diagram of an example of temperature regulating device；
In accompanying drawing, the list of parts representated by each label is as follows：
1st, reactive component offer portion, 101, component provider, 102, premixer, 2, opening, 3, mixing chamber, 4, sample feeding pipe,
5th, temperature control post, 6, reaction tube pipe shaft, 7, entrance, 8, outlet, 9, reaction product discharge pipe, 501, degeneration area, 502, annealed zone,
503rd, extension area, 601, denaturation section, 602, annealing section, 603, extension of section, 604, insulation short tube, 701, outlet valve,
801st, valve.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
Embodiment 1
As shown in figure 1, the reaction system of the embodiment prepare and sampling device include reactive component offer portion (1) with
And mixing chamber (3) and sample feeding pipe (4), the opening (2) for allowing bacterium colony picking device to pass through is provided with the top of the mixing chamber (3),
The outlet of the reactive component offer portion (1) connects with each mixing chamber (3), the outlet of each mixing chamber (3)
It is connected with a sample feeding pipe (4), the exit of the mixing chamber (3) is provided with outlet valve.The reactive component offer portion 1 is
Device equipped with hybrid reaction component, be equipped with the reactive component offer portion 1 in PCR reaction systems except can be in addition to template it is all
The mixture of other components.
Prepared by using the PCR reaction systems and sampling device, user can be not subjected to prepare the heavy of PCR system
Work, especially when up to tens, sample is even hundreds of.Only need setting system to prepare parameter, by control system, refer to
Show that the dosing pump in reactive component offer portion 1 adds the respective components of set amount into mixing chamber 3, then by bacterium colony picking device
The one end for being stained with single bacterium colony is impregnated in reactive component mixture by the opening, you can realization is automatically finished PCR system
Prepare, after the completion of preparation, outlet valve is opened, and PCR reaction systems are pumped directly in tubular type temperature regulating device by sample feeding pipe 4, i.e.,
PCR reactions can be completed.In the present embodiment, it is necessary to first prepare reactive component mixture, reactive component is added mixture to
In offer portion 1.
Embodiment 2
As shown in Fig. 2 the reaction system of the embodiment prepare and sampling device include reactive component offer portion (1) with
And mixing chamber (3) and sample feeding pipe (4), the opening (2) for allowing bacterium colony picking device to pass through is provided with the top of the mixing chamber (3),
The outlet of the reactive component offer portion (1) connects with each mixing chamber (3), the outlet of each mixing chamber (3)
It is connected with a sample feeding pipe (4), the exit of the mixing chamber (3) is provided with outlet valve.
The reactive component offer portion 1 includes multiple component provider 101 and a premixer 102, each component
Entrance of the outlet of provider 101 with the premixer 102 connects, outlet and the mixing chamber 3 of the premixer 102
Entrance connects.The multiple reactive component provider 101 supplies PCR reaction buffers, nucleosides to the premixer 102 respectively
Acid, polymerase and primer pair.Reactive component provider 101 is respectively provided with each component, and defeated by measuring pump according to the value of setting
Enter into premixer 102, so as to obtain the mixture available for multiple PCR reacting doses, the measuring pump in premixer 102 is by one
In the mixture input mixing chamber of the amount of PCR reactions, then one end for being stained with single bacterium colony of bacterium colony picking device is opened by described
Mouth is impregnated in reactive component mixture, so as to be configured to PCR reaction systems.
In a preferred embodiment, the reaction system for bacterium colony PCR is prepared and sampling device is also settable
There is manipulator.Manipulator can clamp bacterium colony picking device picking single bacterium colony automatically, and be impregnated in mixing chamber, be configured to PCR reactions
System, so as to realize bacterium colony PCR from the preparation of reaction system and the full-automation of loading.
Embodiment 3
The full-automatic PCR instrument of the present invention includes above-mentioned PCR reaction systems preparation and sampling device and tubular type temperature control dress
Put, the PCR reaction systems are prepared and sampling device is connected with the tubular type temperature regulating device by the sample feeding pipe 4.
As shown in figure 3, the temperature regulating device includes temperature control post 5 and reaction tube, the reaction tube includes entrance 7, outlet 8 and pipe
Body 6, the pipe shaft 6 of the reaction tube are spirally coiled in 5 on the temperature control post, and the entrance 7 connects with sample feeding pipe 4, the outlet 8
Connected with reaction product discharge pipe 9.
In running, reaction system is entered by sample feeding pipe 4 from entrance 7, and by pipe shaft 6, the temperature of reaction system is led to
Cross heat transfer and identical with the temperature of pipe shaft 6 of present position, and reacted at such a temperature, completed after reaction from reaction product
Discharge pipe 9 comes out.Such tubular type temperature regulating device can be used for the reaction environment for building isothermal reaction and temperature cycles reacting condition.
In another embodiment, as shown in figure 4, the periphery of the cross section of temperature control post 5 of temperature regulating device is divided into three
Heated at constant temperature area, degeneration area 501, annealed zone 502 and extension area 503 being followed successively by, the temperature of the degeneration area 501 is 90-98 DEG C,
The temperature of the annealed zone 502 is 45-70 DEG C, and the temperature of the extension area 503 is 70-75 DEG C, the entrance of the reaction tube 7
In one end by the annealed zone 503 of the degeneration area 501, the outlet 8 of the reaction tube is located at the extension area 503 and leans on institute
State one end of degeneration area 501.The device can be used in PCR instrument, the temperature regulating device as PCR reactions.
The division in three heated at constant temperature areas to be formed the temperature needed for the three phases of a PCR cycle in reaction tube
Degree, and in the temperature regulating device, these three warm areas are all constant temperature, it is not necessary to denaturation temperature-annealing temperature-elongating temperature it
Between carry out cyclic switching.Reaction system flows successively through annealed zone, extension area since degeneration area, and the temperature for completing a PCR is followed
Ring, so as to often once complete the PCR reactions of a circulation around the operation of temperature control post.
In another embodiment, heated at constant temperature area may include multiple heated at constant temperature units, part heated at constant temperature unit
Degeneration area is formed, part forms annealed zone, and part forms extension area.Can be by changing the quantity of the heated at constant temperature unit in each area
To adjust the length in each area, so as to control the reaction time.
In another embodiment, temperature control post and pipe shaft can be divided into three sections, and one section is used for the initial denaturation stage, one section with
In the circular response stage, also one section is used for the last extension stage eventually.
In another embodiment, as shown in figure 5, the pipe shaft 6 of temperature regulating device reaction tube is according to residing heated at constant temperature area
Segmentation, often enclose spiral and correspondingly form three constant temperature sections, be followed successively by denaturation section 601, annealing section 602 and extension of section
603, and connected between adjacent section by short tube made of heat insulator 604.By being adopted between adjacent section
Take the short tube 604 of heat insulation to connect, preferably avoid the heat transfer between the different constant temperature sections of pipe shaft 6.In the present embodiment
So-called heat insulator refers to the relatively low material of pyroconductivity, rather than definitely athermanous material.
In a preferred scheme, the surface of temperature control post 5 has the bung flange to match with the reaction tube pipe shaft 6
Groove, the reaction tube pipe shaft 6 are embedded in the bung flange groove.The setting makes it possible to more preferably precisely control reaction tube pipe
The temperature of body 6.It is highly preferred that separated between heated at constant temperature area corresponding to the bung flange groove by heat insulator, preferably every
From the heat transfer between adjacent heated at constant temperature area.So-called heat insulator refers to the relatively low material of pyroconductivity in the present embodiment
Material, rather than definitely athermanous material.
In another embodiment, as shown in fig. 6, the pipe shaft has multiple outlets 8, it is located at respectively away from the entrance
At 7 different bung flange numbers, and each outlet 8 is both provided with outlet valve 801, and the outlet 8 farthest from entrance 7 can be not provided with exporting
Valve 801.The outlet valve 801 causes residing exit to have two states, and a state is reaction system to next spiral shell
Circle movement, another state is the pipe shaft 6 that reaction system leaves reaction tube, is come out from outlet.For example, reaction tube pipe shaft 6 can be
30-60 circles are coiled on temperature control post 1, and outlet 8 is set at the number of turns different away from entrance, setting the state of outlet valve 801 makes instead
System is answered to be come out by the specific number of turns and from corresponding outlet 8 by the reaction product discharge pipe 9 with the outlet, so as to
Carry out specific PCR reaction cycle numbers.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (7)
1. a kind of reaction system for bacterium colony PCR is prepared and sampling device, it is characterised in that is provided including a reactive component
Portion (1) and mixing chamber (3) and sample feeding pipe (4), opening of allowing bacterium colony picking device to pass through is provided with the top of the mixing chamber (3)
Mouth (2), the outlet of the reactive component offer portion (1) connect with each mixing chamber (3), each mixing chamber (3)
Outlet be connected with a sample feeding pipe (4), the exit of the mixing chamber (3) is provided with outlet valve.
2. the reaction system according to claim 1 for bacterium colony PCR is prepared and sampling device, it is characterised in that described
Reactive component offer portion (1) is the device equipped with hybrid reaction component.
3. the reaction system according to claim 2 for bacterium colony PCR is prepared and sampling device, it is characterised in that described
The mixture for the every other component being equipped with reactive component offer portion (1) in PCR reaction systems outside removing template.
4. the reaction system according to claim 1 for bacterium colony PCR is prepared and sampling device, it is characterised in that described
Reactive component offer portion (1) includes multiple component provider (101) and a premixer (102), each component provider
(101) entrance of the outlet with the premixer (102) connects, outlet and the mixing chamber (3) of the premixer (102)
Entrance connection.
5. the reaction system according to claim 4 for bacterium colony PCR is prepared and sampling device, it is characterised in that described
Multiple component provider (101) supply PCR reaction buffers, nucleotides, polymerase and primer to the premixer (102) respectively
It is right.
6. the reaction system for bacterium colony PCR according to any one of claim 1-5 is prepared and sampling device, its feature
It is, in addition to manipulator.
7. a kind of full-automatic PCR instrument, it is characterised in that including being used for the anti-of bacterium colony PCR any one of claim 1-6
System preparation and sampling device and tubular type temperature regulating device are answered, the PCR reaction systems are prepared and sampling device and the tubular type
Temperature regulating device is connected by the sample feeding pipe (4).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710603541.7A CN107460109A (en) | 2017-07-23 | 2017-07-23 | A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710603541.7A CN107460109A (en) | 2017-07-23 | 2017-07-23 | A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument |
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| CN107460109A true CN107460109A (en) | 2017-12-12 |
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| CN201710603541.7A Pending CN107460109A (en) | 2017-07-23 | 2017-07-23 | A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument |
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Effective date of registration: 20211201 Address after: 237012 room 323, engineering innovation center, Wanxi University, Gulou Street, Yu'an District, Lu'an City, Anhui Province Applicant after: Lu'an kuntairui Technology Development Co.,Ltd. Address before: 830000 room 425, incubation building, Xinjiang biomedical innovation and entrepreneurship Park, No. 181, Xicai Road, high tech Industrial Development Zone (new urban area), Urumqi, Xinjiang Uygur Autonomous Region Applicant before: XINJIANG QUINTARABIO BIOTECHNOLOGY CO.,LTD. |
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Application publication date: 20171212 |