CN107012086B

CN107012086B - Real-time fluorescence PCR thermal cycle device and PCR appearance

Info

Publication number: CN107012086B
Application number: CN201710225002.4A
Authority: CN
Inventors: 张晗; 李�杰; 李轻如; 韦晨
Original assignee: Chengdu Hanchen Guangyi Science And Technology Co Ltd
Current assignee: Chengdu Hanchen Guangyi Science And Technology Co Ltd
Priority date: 2017-04-07
Filing date: 2017-04-07
Publication date: 2020-02-18
Anticipated expiration: 2037-04-07
Also published as: CN107012086A

Abstract

The invention discloses a real-time fluorescent PCR thermal cycling device and a PCR instrument, and relates to the field of biological experimental instruments. The real-time fluorescent PCR thermal cycling device comprises a clapboard layer, a sample layer and a temperature control layer which are arranged in sequence; the clapboard layer, the sample layer and the temperature control layer form a closed-loop control system; the clapboard layer and the temperature control layer are respectively connected to the sample layer; the temperature control layer comprises a uniform heating layer and a heat supply layer which are sequentially arranged, the heat supply layer is connected to the uniform heating layer, and the uniform heating layer is connected to the sample layer; a vacuum cavity is formed in the heat equalizing layer, and heat conducting liquid is arranged in the vacuum cavity. The real-time fluorescent PCR thermal cycling device and the PCR instrument realize heat transfer by the reciprocating cycle of absorbing and gasifying the heat conducting liquid in the vacuum cavity of the soaking layer or liquefying the gas-phase heat conducting liquid when meeting cold, thereby improving the uniformity of the temperature of the whole heating surface, improving the quality of PCR reaction and providing more reliable experimental results.

Description

Real-time fluorescence PCR thermal cycle device and PCR appearance

Technical Field

The invention relates to the field of biological experimental instruments, in particular to a real-time fluorescence PCR thermal cycling device and a PCR instrument.

Background

PCR (polymerase chain reaction) is a biological technique that can amplify a specific DNA fragment in vitro. At present, the PCR technology is widely applied to the fields of molecular diagnosis, precise medical treatment, modern agriculture, food safety and the like. In most PCR reactions, periodic temperature increases and decreases are required to amplify a specific DNA fragment.

PCR reactions are typically run in batches of 96-well or 384-well plates in containers and thermal cycling is achieved using a water bath or a conventional metal bath. The water bath circulation realizes the sample and the temperature rise and reduction by repeatedly soaking the sample in the high-temperature constant-temperature pool and the low-temperature constant-temperature pool, and the process is complicated. In order to improve the efficiency, a thermal cycling device is introduced into the PCR instrument to realize periodic temperature rise and temperature fall. The heat cycle device in the prior art has a simple structure and low heat cycle efficiency, and the area heat distribution is uneven, so that the reflection hole is unevenly heated, and the accuracy of the realization result is influenced.

Disclosure of Invention

In view of the above problems, the present invention is directed to: the utility model provides a real-time fluorescence PCR thermal cycle device, the heat on its control by temperature change layer is high-efficient evenly transmit to the sample layer, when realizing fast rising temperature or cooling, the sample is heated evenly, guarantees sample reaction temperature's uniformity to provide more accurate result.

The second objective of the present invention is to provide a real-time fluorescence PCR instrument, which comprises the real-time fluorescence PCR thermal cycling device.

The technical scheme adopted by the invention is as follows:

a real-time fluorescent PCR thermal cycle device comprises a clapboard layer, a sample layer and a temperature control layer which are arranged in sequence; the clapboard layer, the sample layer and the temperature control layer form a closed-loop control system; the clapboard layer and the temperature control layer are respectively connected to the sample layer; the baffle plate layer is transparent so as to transmit light; the temperature control layer comprises a uniform heating layer and a heat supply layer which are sequentially arranged, the heat supply layer is connected to the uniform heating layer, and the uniform heating layer is connected with the sample layer; a vacuum cavity is formed in the heat equalizing layer, and heat conducting liquid is arranged in the vacuum cavity.

Preferably, a heat conduction channel is arranged in the vacuum cavity along the vertical direction, and the heat conduction channel is connected to the side wall of the inner part of the heat equalizing layer.

By adopting the technical scheme, when heat is transferred to the soaking layer from the heat supply layer, the heat-conducting liquid in the vacuum cavity starts to generate the gasification phenomenon of the heat-conducting liquid after being heated in the environment with low vacuum degree, at the moment, the heat-conducting liquid rapidly expands due to the absorption of heat energy, the whole vacuum cavity is rapidly filled with the heat-conducting liquid in a gas phase, and the phenomenon of condensation can be generated when the heat-conducting liquid in the gas phase contacts a relatively cold area. The heat accumulated during evaporation is released by means of the condensation phenomenon, the condensed heat conducting liquid can return to the evaporation heat source by means of the heat conducting channel, the process is carried out in the cavity body repeatedly, the uniformity of the temperature of the whole heating surface is improved, the consistency of the temperature in each reaction hole is ensured, and the quality of PCR reaction is improved.

Preferably, a heat conduction layer is arranged between the sample layer and the soaking layer, and the thickness of the heat conduction layer is smaller than 1 mm.

The heat conducting layer has the function of ensuring that a good contact surface is formed between the temperature control layer and the reaction layer so as to improve the heat transfer efficiency. Therefore, the thickness of the heat conduction layer is controlled to be less than 1mm, otherwise, the heat conduction layer is too thick, and the heat conduction efficiency is reduced.

Preferably, the sample layer includes a transparent pressure-sensitive film and a PCR reaction plate provided with reaction holes, and the transparent pressure-sensitive film is connected to the barrier layer to seal the reaction holes.

Under the action of pressure, the pressure-sensitive membrane and the contact layer of the PCR reaction plate can form tight connection, thereby achieving good sealing effect and preventing different samples from being polluted mutually during PCR reaction. The pressure-sensitive film is set to be transparent, the influence on the light transmittance is small, and the light transmittance of the excitation light source and the fluorescence signal is ensured.

Preferably, the separator layer is coated with a transparent conductive film on which the first thermal resistor is disposed.

Preferably, the transparent conductive film is disposed on a surface of the separator layer on a side connected to the sample layer.

Because the liquid sample evaporates, produce the water smoke on the pressure-sensitive membrane above the reaction hole, this water smoke can reduce the luminousness of pressure-sensitive membrane, reduces the collection of optical detection to fluorescence signal to influence detection effect. Due to the adoption of the technical scheme, the current can be applied to the conductive film, and the temperature can be detected through the first thermal resistor so as to control the temperature between the clapboard layer and the sample layer. When water mist is generated, current can be applied to the conductive film, the temperature of the pressure-sensitive film is increased, evaporated liquid is prevented from being liquefied on the pressure-sensitive film to form water mist, and therefore the collection of fluorescence signals by optical detection is prevented from being reduced.

Preferably, the heat supply layer is formed of one or more thermoelectric modules.

Preferably, the real-time fluorescent PCR thermal cycling device further includes a second thermal resistor disposed on the thermal equalization layer, and the second thermal resistor cooperates with the thermal module to control the temperature of the temperature control layer.

Compared with the electrical module and the thermal module, the thermoelectric module has the characteristics of rapid temperature rise and temperature drop. Through the temperature feedback of the second thermal resistor and the temperature output of the thermoelectric module, the temperature change of the temperature control layer can be accurately and rapidly controlled, the PCR reaction efficiency is improved, and the reaction time is shortened.

Preferably, the temperature control layer further comprises a heat dissipation layer connected to the heat supply layer; the heat dissipation layer comprises a heat radiator and a fan; the radiator is connected to the heat supply layer, and the fan is connected to the lower side of the radiator.

The effect of heat dissipation layer is when the temperature control layer needs the cooling, and the quick heat that will give off is sent out.

A real-time fluorescence PCR instrument comprises an excitation light source, a detection system and the real-time fluorescence PCR thermal cycling device, wherein the excitation light source is arranged above a partition plate layer, and the detection system is connected to a sample layer to receive signals emitted by a sample.

In conclusion, due to the adoption of the technical scheme, the invention has the beneficial effects that:

the heat transfer is realized by the reciprocating circulation of the heat conducting liquid absorbing and gasifying or the gas-phase heat conducting liquid meeting cold liquefaction in the vacuum cavity of the soaking layer, the temperature uniformity of the whole heating surface is improved, the temperature consistency of different areas of the reaction layer is improved, the reaction temperature consistency of each reaction sample is ensured, the quality of PCR reaction is improved, and a more reliable experimental result is provided.

Drawings

The invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 is a schematic view of the construction of a real-time fluorescent PCR thermal cycler provided in example 1;

FIG. 2 is a schematic cross-sectional view taken along A-A of FIG. 1;

FIG. 3 is a schematic view of the real-time fluorescent PCR thermal cycler provided in example 2.

The labels in the figure are: 100-real-time fluorescent PCR thermal cycling device; 110-a separator layer; 111-transparent conductive film; 113-a first thermal resistance; 130-sample layer; 131-a transparent pressure sensitive film; 133-PCR reaction plate; 135-reaction well; 150-temperature control layer; 151-heat equalizing layer; 153-vacuum chamber; 155-heat conducting channel; 157-heat supply layer; 158-thermally conductive layer; 159-a heat sink layer; 200-real-time fluorescent PCR thermal cycling device; 220-heat supply layer; 240-second thermal resistance; 260-soaking layer.

Detailed Description

All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.

Any feature disclosed in this specification (including any accompanying claims, abstract) may be replaced by alternative features serving equivalent or similar purposes, unless expressly stated otherwise. That is, unless expressly stated otherwise, each feature is only an example of a generic series of equivalent or similar features.

Example 1

As shown in FIG. 1, the present embodiment provides a real-time fluorescent PCR thermal cycling apparatus 100, which comprises a separator layer 110, a sample layer 130 and a temperature control layer 150, which are sequentially arranged from top to bottom. The separator layer 110, sample layer 130, and temperature control layer 150 form a closed loop control system. The spacer layer 110 and the temperature control layer 150 are connected to the sample layer 130, respectively.

The barrier layer 110 is provided transparent to transmit light. The body of the spacer layer 110 is formed by coating a transparent conductive film 111 on a glass or plexiglass coated surface. Preferably, the transparent conductive film 111 is coated on the lower surface of the glass, i.e., the surface of the side of the spacer layer 110 connected to the sample layer 130. A first thermal resistor 113 is disposed on the separator layer 110. The first thermal resistor 113 is disposed on the transparent conductive film 111. The transparent conductive film 111 cooperates with the first heat resistor 113 to control the temperature between the spacer layer 110 and the sample layer 130. The separator layer 110 has a light transmittance of more than 85% for light having a wavelength of 420nm to 680 nm.

The sample layer 130 is where the sample reacts. The sample layer 130 includes a transparent pressure-sensitive film 131 and a PCR reaction plate 133. The transparent pressure-sensitive film 131 has a light transmittance of more than 90% in the visible light wavelength range. The transparent pressure sensitive film 131 is connected to the separator layer 110, and is in contact with the PCR reaction plate 133 under pressure to seal the PCR reaction plate 133, thereby preventing mutual contamination between different samples during PCR reaction. The PCR reaction plate 133 is provided with a reaction well 135 as a container for a reaction sample, and the reaction well 135 is sealed by a transparent pressure-sensitive film 131 during the reaction. The thickness of the PCR reaction plate 133 is preferably 0.5mm to 3 mm.

The temperature control layer 150 includes a soaking layer 151 and a heat supply layer 157. The soaking layer 151 is connected to the sample layer 130, and the heat supply layer 157 is disposed under the soaking layer 151 and connected to the soaking layer 151.

The heat equalizing layer 151 is provided with a vacuum cavity 153 therein, and heat conducting liquid (not shown) is arranged in the vacuum cavity 153, and the heat conducting liquid is gasified by absorbing heat, and the gas phase heat conducting liquid is liquefied by cooling to transfer heat. In order to allow the vaporized heat transfer liquid to transfer heat more efficiently and uniformly, heat transfer channels 155 for providing paths for the heat transfer liquid are provided inside the soaking layer 151, the heat transfer channels 155 are provided in a vertical direction, and the heat transfer channels 155 are formed of dense and gapless capillaries (as shown in fig. 2).

The heat supply layer 157 is composed of one or more thermal modules. In order to improve the heat conduction efficiency, a heat conductive silicone grease is coated between the heat supply layer 157 and the soaking layer 151.

In order to improve the heat conduction efficiency, a heat conduction layer 158 is further disposed between the soaking layer 151 and the sample layer 130. The heat conducting layer 158 is generally formed by a silicone pad with high heat conducting performance and good ductility, and provides a good contact surface between the temperature control layer 150 and the sample layer 130, thereby improving heat transfer efficiency. The thickness of the thermally conductive layer 158 is less than or equal to 1mm, and an excessively thick thermally conductive layer 158 adversely reduces heat transfer efficiency.

In order to achieve rapid cooling of the temperature control layer 150, the temperature control layer 150 is further provided with a heat dissipation layer 159. The heat dissipation layer 159 is located below the heat supply layer 157, and the heat dissipation layer 159 is connected to the heat supply layer 157. The heat dissipation layer 159 includes a heat sink (not shown) connected to the heat supply layer 157 for directly cooling the heat supply layer 157, and a fan (not shown) located below the heat sink for assisting in cooling the heat supply layer 157. In order to improve heat conduction efficiency, a heat conductive silicone grease is coated between the heat supply layer 157 and the heat dissipation layer 159.

The present embodiment further provides a real-time fluorescence PCR instrument, which includes an excitation light source, a detection system and the real-time fluorescence PCR thermal cycling apparatus 100. An excitation light source is positioned above the spacer layer 110 and a detection system is coupled to the sample layer 130 to receive signals from the sample.

Example 2

As shown in fig. 3, the present embodiment provides a real-time fluorescent PCR thermal cycling apparatus 200, which is substantially the same as that provided in embodiment 1, except that the heat supply layer 220 in the present embodiment is composed of one or more thermoelectric modules. When a forward voltage is applied to the heat supply layer 220, the upper surface of the heat supply layer is heated, and the lower surface is cooled; when a negative voltage is applied thereto, the upper surface of the heat supply layer 220 is cooled and the lower surface is heated. Compared with the electrical module and the thermal module, the thermoelectric module has the characteristics of rapid temperature rise and temperature drop.

In addition, the real-time fluorescent PCR thermal cycling apparatus 200 provided by the present embodiment further includes a second thermal resistor 240, and preferably, the second thermal resistor 240 is disposed on the soaking layer 260. Through the temperature feedback of the second thermal resistor 240 and the temperature output of the thermoelectric module, the temperature change of the temperature control layer can be accurately and rapidly controlled, the PCR reaction efficiency is improved, and the reaction time is shortened.

The invention is not limited to the foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification and any novel method or process steps or any novel combination of features disclosed.

Claims

1. A real-time fluorescence PCR circulating device comprises a clapboard layer, a sample layer and a temperature control layer which are arranged from top to bottom in sequence; the separation plate layer, the sample layer and the temperature control layer form a closed-loop control system, the separation plate layer and the temperature control layer are respectively connected to the sample layer, the separation plate layer is coated with a transparent conductive film, a first thermal resistor is arranged on the transparent conductive film, and the transparent conductive film is arranged on the surface of one side, connected with the sample layer, of the separation plate layer; the temperature control layer comprises a uniform heating layer and a heat supply layer which are sequentially arranged, the heat supply layer is connected to the uniform heating layer, the uniform heating layer is connected to the sample layer, a heat conduction layer is arranged between the sample layer and the uniform heating layer, and the thickness of the heat conduction layer is smaller than or equal to 1 mm; the vacuum heat-insulating layer is characterized in that a vacuum cavity is formed in the soaking layer, heat-conducting liquid is arranged in the vacuum cavity, a heat-conducting channel which provides a path for the heat-conducting liquid and is arranged in the vertical direction is arranged in the vacuum cavity, and the heat-conducting channel is connected to the side wall of the inside of the soaking layer.

2. The real-time fluorescent PCR thermal cycler of claim 1, wherein the sample layer comprises a transparent pressure sensitive film and a PCR reaction plate provided with reaction wells, the transparent pressure sensitive film being connected to the separator layer to seal the reaction wells.

3. The real-time fluorescent PCR thermal cycler of claim 1, wherein the heat supply layer is comprised of one or more thermoelectric modules.

4. The real-time fluorescent PCR thermal cycler of claim 3, further comprising a second thermal resistor disposed with the thermal spreader layer, the second thermal resistor cooperating with the thermal module to control the temperature of the temperature control layer.

5. The real-time fluorescent PCR thermal cycler of claim 1, wherein the temperature control layer further comprises a heat dissipation layer connected to the heat supply layer; the heat dissipation layer comprises a heat radiator and a fan; the heat sink is connected to the heat supply layer, and the fan is connected to a lower side of the heat sink.

6. A real-time fluorescent PCR instrument comprising an excitation light source, a detection system, and the real-time fluorescent PCR thermal cycling apparatus according to any one of claims 1 to 5; the excitation light source is arranged above the clapboard layer, and the detection system is connected to the sample layer to receive signals emitted by the sample.