CN112063496A

CN112063496A - Closed PCR (polymerase chain reaction) instrument suitable for full-automatic pipetting workstation and application

Info

Publication number: CN112063496A
Application number: CN202011250220.1A
Authority: CN
Inventors: 刘蕊; 吴卓
Original assignee: Singlera Genomics Inc
Current assignee: Jiangsu Huayuan Biotechnology Co ltd
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2020-12-11
Anticipated expiration: 2040-11-11
Also published as: CN112063496B

Abstract

The invention provides a closed PCR reactor capable of being embedded into a full-automatic pipetting workstation, which at least comprises: a PCR component comprising a PCR reaction zone and a heat conducting plate; the heat conducting plate is arranged above the PCR reaction area; the heat conducting plate is divided into a membrane sealing component placing area and a window for exposing the PCR reaction area; a first temperature regulator is arranged below the PCR reaction zone; the membrane sealing assembly is arranged above the heat conducting plate and can horizontally move from the membrane sealing assembly placing area to a window for exposing the PCR reaction area; the membrane sealing assembly comprises a second temperature regulator, and the second temperature regulator can cover the PCR reaction area; a power assembly; and a controller. The full-automatic liquid transfer workstation of PCR reaction appearance adaptation of this application possesses miniaturization, occupation space is few, control by temperature change precision is high, the integrated level is high, very is applicable to various molecular experiments. The membrane can be automatically sealed, so that the pollution problem can be solved under the condition of ensuring cost economy and preventing the experimental result from being polluted.

Description

Closed PCR (polymerase chain reaction) instrument suitable for full-automatic pipetting workstation and application

Technical Field

The invention relates to the field of biological detection instruments, in particular to a closed PCR reactor capable of being embedded into a full-automatic pipetting workstation and application thereof.

Background

The Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying and amplifying specific DNA fragments, and can be regarded as special DNA replication in vitro, and the biggest characteristic of the PCR is that trace amount of DNA can be greatly increased. PCR is carried out by using DNA which is denatured at 95 ℃ in vitro to become single-stranded, combining primer and single-stranded at low temperature (usually about 60 ℃) according to the principle of base complementary pairing, adjusting the temperature to the optimum reaction temperature (about 72 ℃) of DNA polymerase, and synthesizing the complementary strand by DNA polymerase along the direction from phosphoric acid to pentose (5 '-3'). The PCR instrument manufactured based on polymerase is actually a temperature control device, and can be well controlled among denaturation temperature, renaturation temperature and extension temperature.

According to the basic principles of molecular diagnosis and the like, a plurality of automatic pipetting workstations have been developed at home and abroad, and a plurality of mature PCR instruments are also available on the market. But often involve in the reality experiment and need will move liquid workstation and PCR appearance cooperation scene of using, because of present external PCR appearance equipment volume is great, can't directly integrate in the application of sample workstation. On the other hand, PCR experiments are very easy to be polluted by aerosol, and the existing platform pollution prevention measures in the domestic market are not satisfactory at present.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention is directed to a closed PCR reaction apparatus capable of being embedded in a fully automatic pipetting station and applications thereof.

To achieve the above and other related objects, the present invention provides a closed PCR reactor capable of being embedded in a fully automatic pipetting station, wherein the PCR reactor at least comprises:

a PCR component comprising a PCR reaction zone and a heat conducting plate; the heat conducting plate is arranged above the PCR reaction area; the heat conducting plate is divided into a membrane sealing component placing area and a window for exposing the PCR reaction area; a first temperature regulator is arranged below the PCR reaction zone;

the membrane sealing assembly is arranged above the heat conducting plate and can horizontally move from the membrane sealing assembly placing area to a window for exposing the PCR reaction area; the membrane sealing assembly comprises a second temperature regulator, and the second temperature regulator can cover the PCR reaction area;

the power assembly is connected with the membrane sealing assembly and used for providing power for the movement of the membrane sealing assembly;

and the controller is electrically connected with the power assembly, the first temperature regulator and the second temperature regulator and is used for controlling the first temperature regulator, the second temperature regulator and the power assembly.

The invention also provides the application of the closed PCR reaction instrument, which is suitable for a full-automatic pipetting workstation.

As mentioned above, the enclosed PCR reaction instrument and the application thereof of the invention have the following beneficial effects:

the full-automatic liquid workstation that moves of PCR reaction appearance adaptation of this application can be embedded into inside the full-automatic liquid workstation that moves, possesses characteristics miniaturized, occupation space is few, the control by temperature change precision is high, the integrated level is high, very is applicable to various molecular experiment. Meanwhile, PCR experiments are extremely easy to be polluted by aerosol, and the existing platform pollution prevention measures in the domestic market are not satisfactory at present. The invention adopts the design idea of automatic film sealing. The aluminum film can be automatically sealed, so that the pollution problem is solved under the conditions of ensuring cost economy and preventing the experimental result from being polluted.

Drawings

FIG. 1 is an exploded view of a closed PCR reaction apparatus according to an embodiment of the present invention.

FIG. 2a is an exploded view of the PCR assembly and the controller of the enclosed PCR reaction apparatus according to one embodiment of the present invention.

FIG. 2b is an exploded view of a second temperature regulator of the enclosed PCR reaction apparatus according to one embodiment of the present invention.

FIG. 3 is a front view of a closed type PCR reactor according to another embodiment of the present invention.

FIG. 4 is a plan view of a closed type PCR reaction apparatus according to another embodiment of the present invention.

FIG. 5 is a side view (in the case of sealing a film) of a closed type PCR reaction apparatus according to another embodiment of the present invention.

FIG. 6 is a side view of a closed PCR reaction apparatus (when not sealed) according to another embodiment of the present invention.

Description of the element reference numerals

1 PCR Assembly

11 PCR reaction zone

11196 orifice plate adapter

112 fixed upper cover

113 fixed chassis

12 Heat-conducting plate

121 seal membrane module placing area

122 PCR reaction zone exposure window

13 first temperature regulator

131 heat conducting fin

2 seal membrane module

21 second temperature regulator

22 Heat cover plate

3 power assembly

31 electric machine

32 rotating shaft

33 drive wheel

34 transmission shaft clamping groove

35 driving rod

36 guide rod

4 controller

5 temperature adjusting sheet

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1-6. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.

As shown in fig. 1, fig. 3 and fig. 4, the enclosed PCR reactor capable of being embedded in a fully automatic pipetting station according to an embodiment of the present invention at least includes:

a PCR assembly 1 including a PCR reaction region 11 and a heat conducting plate 12; the heat conducting plate 12 is arranged above the PCR reaction area 11; the heat conducting plate 12 is divided into a membrane sealing component placing area 121 and a PCR reaction area exposing window 122; a first temperature regulator 13 is arranged below the PCR reaction zone;

a sealing membrane assembly 2 disposed above the heat conducting plate 12, as shown in fig. 5 and 6, the sealing membrane assembly 2 can horizontally move from the sealing membrane assembly placing region 121 to the PCR reaction region exposing window 122; the membrane sealing assembly 2 comprises a second temperature regulator 21 which can cover the PCR reaction zone 11;

the power component 3 is connected with the film sealing component 2 and used for providing power for the movement of the film sealing component 2;

as shown in fig. 2a, the controller 4 is electrically connected to the power assembly 3, the first temperature regulator 13 and the second temperature regulator 21, and is used for controlling the first temperature regulator 13, the second temperature regulator 21 and the power assembly 3. The controller 4 is electrically connected with the control part of the full-automatic pipetting workstation.

Closed means that the disposable aluminum film is heat-sealed on the PCR plate by the extrusion and heating of the upper cover before the PCR reaction process of the present invention. The pollution of the experiment can be avoided, and the traditional PCR instrument needs to be manually sealed with a layer of film before the experiment. Most of other automatic workstations are equipped with reusable sealing covers, which have poor sealing performance and need to be cleaned after experiments. The invention adopts the aluminum film to reduce the cost while ensuring the complete sealing performance.

The heat-conducting plate can be a fixed sheet metal part, adopts stainless steel materials, is subjected to anodic oxidation treatment on the surface, is not adhered to aluminum at high temperature, and is corrosion-resistant, heat-resistant, uniform in heat transfer and high in heat transfer rate.

Horizontal migration formula seals membrane subassembly, can closely laminate disposable aluminium apron and PCR reaction zone, avoids experimental sample to pollute.

Further, as shown in fig. 2a and 2b, the first temperature regulator 13 and/or the second temperature regulator 21 is provided with a temperature regulation sheet 5. The flake shape can avoid overlarge volume and save space.

Optionally, the temperature adjusting sheet 5 is a peltier patch. Accurate temperature control, rapid temperature rise and temperature reduction. The current direction of the Peltier patch can be controlled by the controller, and positive/negative current is supplied to the Peltier patch, so that the functions of temperature rise and temperature reduction are realized.

Further, when the film sealing assembly 2 is located in the film sealing assembly placing area 121, a gap is left between the second temperature regulator 21 and the film sealing assembly placing area 121. Used for placing the disposable aluminum cover plate.

In one embodiment, as shown in fig. 1, the sealing membrane assembly further comprises a thermal cover plate 22, which can be covered with the second temperature regulator 21. The hot cover plate can be made of engineering plastics, and has the advantages of excellent comprehensive performance, high rigidity, small creep, high mechanical strength and good heat resistance.

Optionally, the main frame of the second temperature regulator 21 is made of aluminum alloy, so that the strength is good, the corrosion resistance is good, and the processing is easy. A temperature regulation sheet may be placed inside the main body frame.

The first temperature regulator 13 further comprises a heat-conducting fin 131, and the heat-conducting fin 131 is arranged between the temperature regulating fin 5 and the PCR reaction region 11. The heat conducting fins 131 can be fixed sheet metal parts, are made of stainless steel, and are corrosion-resistant and heat-resistant.

Further, as shown in fig. 1, the power assembly 3 includes a motor 31, a rotating shaft 32 and a transmission wheel 33, which are connected in sequence, wherein the transmission wheel 33 is driven by the motor 31 to rotate through the transmission shaft 32; the transmission wheel 33 is connected with the film sealing component 2. Optionally, a transmission shaft slot 34 is further disposed between the transmission wheel 33 and the rotation shaft.

Optionally, a transmission rod 35 is connected to the transmission wheel 33, and the transmission wheel is connected to the film sealing assembly through the transmission rod 35.

The transmission rod 35 is fixedly connected with the transmission wheel 33.

Further, a guide rod 36 is rotatably connected to the transmission rod 35, and the guide rod 36 is slidably connected to the bottom of the heat conducting plate 12. The end of the guide rod 36 can be provided with a pulley, and the bottom of the heat conducting plate 12 is provided with a slide rail matched with the heat conducting plate. The two ends of the sliding rail are provided with baffles to prevent the pulley from derailing.

As shown in fig. 6, when the film is not sealed, the film sealing assembly is located in the film sealing assembly placing area 121, when the film sealing is started, the motor is started to drive the driving wheel 33 to rotate counterclockwise, at this time, the driving rod 35 moves along with the driving wheel (to the lower left in fig. 6), as shown in fig. 5, the film sealing assembly is pulled to move towards the PCR reaction area exposing window 122, and the guiding rod 36 is driven to slide at the bottom of the heat conducting plate 12 to form a proper angle so that the film sealing assembly is located above the PCR reaction area exposing window 122. At which time the motor stops moving. When the film sealing is finished, the motor is started to drive the transmission wheel 33 to rotate clockwise, at the moment, the transmission rod 35 generates upward lifting force along with the motion of the transmission wheel, the guide rod is pushed to abut against the bottom of the heat conduction plate to slide back, the transmission is carried out, and under the guide of the guide rod, the transmission rod 35 drives the film sealing component to move in parallel to the film sealing component placing area 121.

The transmission wheel can be a copper gear, the operation noise is low, the transmission is stable, and the speed ratio variation range is wide.

The number of the transmission wheels 33 is at least two, and the transmission wheels are respectively connected with two sides of the film sealing assembly 2.

The motor 31 is a stepping motor.

Optionally, the main structure of the power assembly 3 is located below the PCR assembly. The main structure means that most of the structures of the motor, the transmission shaft, the driving wheel and the like of the power assembly are positioned below the PCR assembly. This arrangement saves space.

The PCR reaction region 11 is provided with a 96-well plate adapter 111. For placing 96-well plates. The 96-pore plate adapter can be made of stainless steel and is corrosion-resistant and heat-resistant.

In one embodiment, the PCR reaction region further comprises a fixed upper cover 112, and the fixed upper cover 112 is disposed above the 96-well plate adaptor. The aluminum alloy can be adopted, the strength is good, the corrosion resistance is good, and the processing is easy.

The invention provides a hardware architecture of a controller. The controller of the invention can be provided with control programs with different settings according to requirements.

The controller can be a single chip microcomputer which can be an 8-bit minimum system. The controller may also be a different brand and model, or a higher number of controllers or processors. The temperature controller may be used to install an associated control program. After the relevant control programs are installed, the temperature controller can receive signals of the temperature sensor and adjust the heating capacity of the heating plate according to needs, so that the temperature in the incubator is controlled. The temperature controller in the prior art is adopted.

Optionally, the controller 4 includes a control circuit board, and the control circuit board may be disposed below the temperature adjustment sheet 5, so as to save space.

Optionally, the control circuit board can be electrically connected or in signal connection with an external full-automatic pipetting workstation, and then the PCR reaction instrument can be controlled by the full-automatic pipetting workstation, so that automation is realized.

In one embodiment, the PCR reaction region further comprises a fixing base 113 located at the lowermost part of the PCR reaction region for placing the components of the entire reaction region. Stainless steel can be adopted, and the material is corrosion-resistant and heat-resistant.

In one embodiment, the enclosed PCR reactor of the present invention has the length, width, height dimensions: 360 +/-35 mm, 230 +/-35 mm and 320 +/-20 mm.

The closed PCR reaction instrument which can be embedded into the full-automatic liquid transfer workstation is applied to the full-automatic liquid transfer workstation.

When in use, the utility model is used,

a. the products to be reacted and reagents are placed in a 96-well PCR plate. The 96-well PCR plate was placed in the PCR reaction region 11 in FIG. 1. The disposable aluminum cover plate is placed in the gap between the film sealing component 2 and the heat conducting plate 12.

b. Through an external communication command, the motor 31 is operated to rotate, so that the driving wheel 33 is driven to rotate, and the film sealing assembly 2 is driven to move horizontally. So that the second temperature regulator 21 completely covers the PCR reaction region 11, and the second temperature regulator 21 presses the disposable aluminum cover plate onto the 96-well PCR plate. So that the disposable aluminum cover plate is seamlessly jointed with the 96-hole PCR plate.

c. Through an external communication command, the control circuit board in fig. 2a is operated to supply forward/reverse current to the peltier patch 5, so that the functions of temperature rise and temperature reduction are realized.

d. Through an external communication command, the control circuit board in fig. 2a is operated to supply forward/reverse current to the peltier patches in the membrane sealing assembly 2 in fig. 1, so that the functions of temperature rise and temperature reduction are realized.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. An enclosed PCR reaction apparatus that can be inserted into a fully automatic pipetting station, said PCR reaction apparatus comprising at least:

a PCR assembly (1) comprising a PCR reaction zone (11) and a heat-conducting plate (12); the heat conducting plate (12) is arranged above the PCR reaction area (11); the heat conducting plate (12) is divided into a membrane sealing component placing area (121) and a PCR reaction area exposure window (122); a first temperature regulator (13) is arranged below the PCR reaction zone (11);

the membrane sealing component (2) is arranged above the heat conducting plate (12), and the membrane sealing component (2) can horizontally move from the membrane sealing component placing area (121) to a PCR reaction area exposure window (122); the membrane sealing assembly (2) comprises a second temperature regulator (21), and the second temperature regulator (21) can cover the PCR reaction zone (11);

the power assembly (3) is connected with the film sealing assembly (2) and used for providing power for the movement of the film sealing assembly (2), the power assembly (3) comprises a motor (31), a rotating shaft (32) and a driving wheel (33) which are sequentially connected, and the driving wheel (33) is driven by the motor (31) to rotate through the transmission shaft (32); a transmission rod (35) is connected to the transmission wheel (33), the transmission wheel (33) is connected with the film sealing assembly (2) through the transmission rod (35), a guide rod (36) is rotatably connected to the transmission rod (35), and the guide rod (36) is in sliding connection with the bottom of the heat conducting plate (12);

and the controller (4) is electrically connected with the power assembly (3), the first temperature regulator (13) and the second temperature regulator (21) and is used for controlling the first temperature regulator (13), the second temperature regulator (21) and the power assembly (3).

2. The enclosed PCR reactor according to claim 1, characterized in that the first temperature regulator (13) and/or the second temperature regulator (21) is provided with a temperature regulating blade (5).

3. The enclosed PCR reactor according to claim 2, wherein the temperature adjusting plate (5) is a peltier patch.

4. The enclosed type PCR reaction instrument as set forth in claim 2, wherein the first temperature regulator (13) further comprises a heat conductive sheet (131), the heat conductive sheet (131) being disposed between the temperature regulating sheet (5) and the PCR reaction region (11).

5. The enclosed type PCR reactor as set forth in claim 1, wherein a gap is left between the second temperature regulator (21) and the membrane sealing member placing region (121) when the membrane sealing member (2) is in the membrane sealing member placing region (121).

6. The enclosed type PCR reaction apparatus according to claim 1, wherein the number of the driving wheels (33) is at least two, and the driving wheels are respectively connected with both sides of the membrane sealing assembly (2).

7. The enclosed PCR reactor according to claim 1, wherein the motor (31) is a stepper motor.

8. The enclosed PCR reactor according to claim 1, characterized in that the main structure of the power module (3) is located below the PCR module.

9. The enclosed PCR reactor according to claim 1, wherein the PCR reaction zone (11) is provided with a 96-well plate adaptor (111).

10. Use of the closed PCR reactor according to any one of claims 1 to 9 to be inserted into a fully automated pipetting station.