WO2018137513A1 - System for detecting convective pcr amplification and method for detecting convective pcr amplification - Google Patents

Abstract

Disclosed are a system and a method for detecting convective PCR amplification. The system comprises: a microfluidic chip, comprising a storage structure, a convective PCR tube, an FTA film and a waste liquid receiving structure, wherein a first end of the convective PCR tube is in communication with a storage cavity of the storage structure, a second end of the convective PCR tube is in communication with a waste liquid cavity of the waste liquid receiving structure, and the FTA film is arranged in the convective PCR tube so as to filter a solution flowing from the first end of the convective PCR tube to the second end of same, and is capable of adsorbing nucleic acids in the solution onto the surface of the FTA film; a flow control module, used for making a solution in the storage cavity flow into the convective PCR tube and for making same be filtered via the FTA film, and then enter the waste liquid cavity; a heating module, used for heating materials in the convective PCR tube; and an optical detection module, used for the fluorescence detection of materials in the convective PCR tube.

Description

Convective PCR amplification detection system and convection PCR amplification detection method

Related application

The application is based on the Chinese patent application with the application number of 201710054798.1 and the application date of January 24, 2017, and the invention name is “convection PCR amplification detection system and convective PCR amplification detection method”, and claims its priority. The disclosure of the Chinese patent application is hereby incorporated herein in its entirety.

Technical field

The present application relates to the field of life medicine detection and diagnosis, and in particular to a convection PCR amplification detection system and a convection PCR amplification detection method.

Background technique

In the prior art, the nucleic acid analysis method often includes two steps, wherein the first step cleaves the detection sample, and then captures and purifies the nucleic acid template; the second step performs a polymerase chain reaction on the nucleic acid template (Polymerase Chain Reaction) , PCR) or other isothermal amplification and detection. Polymerase chain reaction is a molecular biology technique used to amplify specific DNA fragments. Polymerase chain reaction generally requires repeated thermal cycling steps between two or three temperatures of the reaction mixture to effect amplification. As a new type of PCR amplification technology, Convective Polymerase Chain Reaction (CPCR, hereinafter referred to as convection PCR) relies on one or two constant reaction temperatures to establish stability at both ends of the reaction tube (convection PCR tube). The temperature gradient, based on the principle of thermohydrodynamics, generates a periodic motion flow field in the reaction tube, so that the amplified sample reciprocates between the ends of the tube at different temperatures, thereby obtaining the temperature conditions required for PCR amplification.

In the prior art, nucleic acid extraction is often performed by manual or semi-automatic instruments, and the nucleic acid extraction process and the nucleic acid amplification process are separated from each other. Therefore, the conventional nucleic acid analysis system and method are inefficient.

Summary of the invention

The purpose of the present application is to provide a convection PCR amplification detection system and a convection PCR amplification detection method, aiming at solving the problem of low efficiency of the conventional nucleic acid analysis system and method.

To achieve the above object, a first aspect of the present application provides a convection PCR amplification detection system, including: a microfluidic chip, the microfluidic chip comprising a storage structure, a convection PCR tube, an FTA membrane, and a waste liquid receiving structure, The storage structure has a storage cavity, the waste liquid receiving structure has a waste liquid cavity, the first end of the convection PCR tube is in communication with the storage cavity, and the second end of the convection PCR tube is connected to the waste liquid cavity The FTA membrane is disposed inside the convection PCR tube to filter a solution flowing from the first end to the second end of the convection PCR tube and is capable of adsorbing nucleic acid in the solution on the surface of the FTA membrane; a flow control module, configured to enter a solution in the storage chamber into the convection PCR tube and filter through the FTA membrane to enter a waste liquid chamber; a heating module for heating the substance in the convection PCR tube; optical detection a module for performing fluorescence detection on a substance in the convection PCR tube.

Further, the flow control module includes a rotating body, the rotating body drives the convection PCR tube to rotate about a central axis, the storage structure, the first end of the convection PCR tube, and the second of the convection PCR tube The end and the waste liquid receiving structure 27 are sequentially arranged from a position close to the center axis to a position away from the center axis.

Further, the microfluidic chip further includes one or more layers of a support film having a plurality of micropores, the support film being disposed between the FTA film and the waste liquid chamber, and the support film Bonded to the FTA film.

Further, the microfluidic chip further includes a heat conduction groove structure for introducing heat of the heating module into the convection PCR tube, the heat conduction groove structure includes a heat conduction groove, and at least the convection PCR tube is configured A portion of the FTA film is located within the thermally conductive slot.

Further, the microfluidic chip further includes a microvia connector disposed between the heat conducting slot structure and the waste liquid receiving structure, the microhole connector comprising respectively communicating the The second end of the convection PCR tube and the micropores of the waste liquid chamber.

Further, the storage structure includes a storage cavity inlet, and the microfluidic chip further includes a soft plug for mating with the storage cavity inlet to close the storage cavity inlet.

Further, the flow control module includes a centrifugal module including a rotating body rotatably disposed about a central axis, the microfluidic chip being coupled to the rotating body, the rotating body for driving the The microfluidic chip is rotated to allow the solution of the storage structure to enter the convection PCR tube under the action of centrifugal force and filtered through the FTA membrane into the waste liquid chamber.

Further, the distance between the microfluidic chip and the central axis is adjustably set.

Further, the rotating body includes a turntable and a chip fixing member, the turntable is rotatably disposed around the central axis, the chip fixing member is fixedly connected to the turntable, and the microfluidic chip is fixedly disposed on the On the chip holder.

Further, the fixed connection position of the turntable and the chip fixing member is variably disposed.

Further, the centrifugation module includes a positioning control element for controlling the convection PCR tube to be in a vertical state.

Further, the centrifugal module includes a rotary drive mechanism that is drivingly coupled to the rotating body to drive the rotating body to rotate.

Further, the rotational speed of the rotary drive mechanism is adjustably set.

Further, the centrifugal module includes a positioning control element for controlling the convection PCR tube to be in a vertical state, wherein the positioning control element includes a photoelectric switch, the photoelectric switch and the rotary drive The mechanism is coupled and controls the convection PCR tube to be in a vertical state by controlling a rotational angle of the rotary drive mechanism.

Further, the heating module includes a heating element and a temperature measuring element for measuring the temperature of the heating element.

Further, the heating module includes a heating element movably disposed relative to the microfluidic chip to switch between a heated position and a non-heated position, wherein the heating element is adjacent to the heating element The microfluidic chip heats it, and in the non-heating position, the heating element is remote from the microfluidic chip relative to the heating position.

Further, the heating module further includes a linear drive mechanism that is drivingly coupled to the heating element to drive the heating element to switch between the heated position and the non-heated position.

Further, the optical detection module includes an excitation light source movably disposed relative to the microfluidic chip to switch between an excitation position and a non-excitation position, wherein the excitation light source is in the excitation position Concentric with the convective PCR tube, the centerline of the excitation source is spaced from the centerline of the convection PCR tube at the non-excited position.

Further, the optical detection module includes an excitation light source movably disposed relative to the microfluidic chip to switch between an excitation position and a non-excitation position, wherein the excitation light source is in the excitation position Concentric with the convective PCR tube, the centerline of the excitation source is spaced from the centerline of the convection PCR tube in the non-excited position, wherein the excitation source is disposed relatively fixedly to the heating element.

Further, the flow control module includes a suction device in communication with the waste liquid chamber, the suction device for forming a vacuum in the waste liquid chamber to cause a solution of the storage structure to be in a differential pressure The convection PCR tube is passed down and filtered through the FTA membrane to enter the waste liquid chamber.

The second aspect of the present application provides a convective PCR amplification detection method for performing amplification detection using the convective PCR amplification detection system according to any one of the first aspects of the present application, the method comprising: an extraction step, including a filtering step Adding a sample solution containing the nucleic acid to the storage chamber of the storage structure in the filtering step, flowing the sample solution in the storage chamber into the convection PCR tube, and filtering through the FTA membrane, Nucleic acid is adsorbed on the surface of the FTA membrane, the remaining material in the sample solution flows into the waste liquid chamber; an amplification step, after the extracting step, using the convection PCR tube and the heating module pair Amplifying the nucleic acid adsorbed on the surface of the FTA membrane for amplification; and detecting a step of performing fluorescence detection on the amplification product in the convection PCR tube by using the optical detection module.

Further, in the detecting step, the optical detection module performs fluorescence detection on the amplification product in the convection PCR tube while the amplification step.

Further, the amplifying step comprises: rotating the convection PCR tube to a vertical state; injecting an amplification reagent into the convection PCR tube; and heating the substance in the convection PCR tube by the heating module.

Further, the extracting step further includes a purifying step, after the filtering step, adding the purified liquid to the storage chamber, and flowing the purified liquid in the storage chamber into the convection PCR tube, and purifying and adsorbing The purified liquid after the nucleic acid on the surface of the FTA film flows into the waste liquid chamber through the FTA film.

Further, the extracting step further includes a washing step, after the purifying step, adding a washing liquid to the storage chamber, causing the washing liquid in the storage chamber to flow into the convection PCR tube, and washing and adsorbing The washing liquid after the nucleic acid on the surface of the FTA film flows into the waste liquid chamber through the FTA film.

According to the convective PCR amplification detection system and method provided by the present application, when nucleic acid extraction is performed on a sample solution containing nucleic acid, the FTA film 23 is subjected to the sample solution by a flow control device (for example, by rotating the convection PCR tube 22 by a rotating body). By filtration, the nucleic acid is adsorbed on the surface of the FTA film 23, and other substances in the sample solution flow through the FTA film 23 into the waste liquid chamber, thereby realizing the nucleic acid extraction function. When the amplification solution in the convection PCR tube 22 is amplified, the amplification solution in the convection PCR tube 22 and the FTA membrane are heated by the heating module to bring the amplification solution and the FTA membrane to a desired temperature. At the time of amplification, the amplification product in the convection PCR tube 22 is subjected to fluorescence detection using an optical detection module. The convective PCR amplification detection system of the present application realizes the integration of automatic nucleic acid extraction and nucleic acid amplification detection, and constructs an integrated and automated nucleic acid analysis microfluidic chip. The microfluidic chip is an automatic nucleic acid extraction chip, which realizes the automation of the nucleic acid extraction process and improves the nucleic acid extraction efficiency; the microfluidic chip is also a nucleic acid amplification and detection chip. The microfluidic network structure integrated by microfluidic chip realizes the automation and integration of nucleic acid analysis, which provides a reasonable technical platform for improving the efficiency of nucleic acid analysis and improves the overall level of nucleic acid analysis. In addition, the convection PCR amplification detection system can significantly shorten the nucleic acid amplification and detection time, can also reduce the manual operation steps, and improve the accuracy and reliability of nucleic acid analysis.

DRAWINGS

FIG. 1 is a schematic structural diagram of a convection PCR amplification detection system according to an embodiment of the present application.

2 is a schematic structural view of a microfluidic chip in the convective PCR amplification detection system shown in FIG. 1.

3 is a schematic view showing a connection structure of a convection PCR tube, an FTA film, and a support film of a microfluidic chip in the convection PCR amplification detection system shown in FIG. 1.

4 is a schematic structural view of a centrifugal module in the convective PCR amplification detection system shown in FIG. 1.

FIG. 5 is a schematic diagram showing the connection structure of a centrifugal module and a microfluidic chip in the convective PCR amplification detection system shown in FIG. 1. FIG.

6 is a schematic diagram showing a connection structure of an excitation module of an heating module and an optical detection module in the convection PCR amplification detection system shown in FIG. 1.

7 is a schematic structural view of a heating module in the convection PCR amplification detection system shown in FIG. 1.

FIG. 8 is a schematic structural diagram of a receiving module of an optical detecting module in the convective PCR amplification detecting system shown in FIG. 1. FIG.

detailed description

The technical solutions in the embodiments of the present application are clearly and completely described in the following with reference to the accompanying drawings, and the embodiments described are only a part of the embodiments of the present application, but not all embodiments. The following description of one exemplary embodiment is merely illustrative, and is in no way intended to limit the invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present application without departing from the inventive scope are the scope of the present application.

The relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments are not intended to limit the scope of the application, unless otherwise specified. In the meantime, it should be understood that the dimensions of the various parts shown in the drawings are not drawn in the actual scale relationship for the convenience of the description. Techniques, methods and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the techniques, methods and apparatus should be considered as part of the authorization specification. In all of the examples shown and discussed herein, any specific values are to be construed as illustrative only and not as a limitation. Accordingly, other examples of the exemplary embodiments may have different values. It should be noted that similar reference numerals and letters indicate similar items in the following figures, and therefore, once an item is defined in one figure, it is not required to be further discussed in the subsequent figures.

In the description of the present application, it should be understood that the use of the terms "first", "second" and the like to define a component is merely for the purpose of facilitating the distinction between the corresponding components, and if not otherwise stated, the above words are not special. The meaning is therefore not to be construed as limiting the scope of the application.

In the description of the present application, it is to be understood that orientations such as "front, back, up, down, left, right", "horizontal, vertical, vertical, horizontal" and "top, bottom" and the like are indicated. Or the positional relationship is generally based on the orientation or positional relationship shown in the drawings, and is merely for the convenience of the description of the present application and the simplified description, which are not intended to indicate or imply the indicated device or component. It must be constructed and operated in a specific orientation or in a specific orientation, and thus is not to be construed as limiting the scope of the application; the orientations "inside and outside" refer to the inside and outside of the contour of the components themselves.

1 to 8 show a convective PCR amplification detection system of an embodiment of the present application.

The convection PCR amplification detection system mainly comprises a microfluidic chip, a flow control module, a heating module and an optical detection module. The microfluidic chip mainly includes a storage structure 21, a convection PCR tube 22, an FTA film 23, and a waste liquid receiving structure 27. The storage structure 21 has a storage cavity. The waste liquid receiving structure 27 has a waste liquid chamber. The first end of the convection PCR tube 22 is in communication with the storage chamber, and the second end of the convection PCR tube 22 is in communication with the waste liquid chamber. The FTA film 23 is disposed inside the convection PCR tube 22 to filter the sample solution flowing from the first end to the second end of the convection PCR tube 22 and is capable of adsorbing nucleic acid in the solution on the surface of the FTA film 23. The flow control module is used to allow the solution in the storage chamber to enter the convection PCR tube 22 and be filtered through the FTA membrane 23 to enter the waste chamber. The heating module is used to heat the contents of the convection PCR tube 22. The optical detection module is used to perform fluorescence detection on the substance in the convection PCR tube 22.

In the embodiment of the present application, when extracting the sample solution containing the nucleic acid, the FTA film 23 filters the sample solution by the flow control device (for example, rotating the convection PCR tube 22 by the rotating body), and the nucleic acid is adsorbed to the FTA film 23 The surface of the sample solution flows through the FTA membrane 23 into the waste chamber to effect nucleic acid extraction. When the amplification solution in the convection PCR tube 22 is amplified, the amplification solution in the convection PCR tube 22 and the FTA membrane are heated by the heating module to bring the amplification solution and the FTA membrane to a desired temperature. At the time of amplification, the amplification product in the convection PCR tube 22 is subjected to fluorescence detection using an optical detection module.

According to the above description, the convective PCR amplification detection system of the embodiment of the present invention realizes the integration of automatic nucleic acid extraction and nucleic acid amplification detection, and constructs an integrated and automated nucleic acid analysis microfluidic chip. The microfluidic chip is an automatic nucleic acid extraction chip, which realizes the automation of the nucleic acid extraction process and improves the nucleic acid extraction efficiency; the microfluidic chip is also a nucleic acid amplification and detection chip, and realizes the microfluidic network structure integrated by the microfluidic chip. The automation and integration of nucleic acid analysis provides a reasonable technical platform for improving the efficiency of nucleic acid analysis, and improves the overall level of nucleic acid analysis. In addition, the convection PCR amplification detection system can significantly shorten the nucleic acid amplification and detection time, can also reduce the manual operation steps, and improve the accuracy and reliability of nucleic acid analysis.

Therefore, the embodiments of the present application combine the nucleic acid extraction method based on the solid phase carrier and the convection PCR amplification detection technology to fully utilize the characteristics and advantages of the two, thereby simplifying the complexity of the integrated nucleic acid extraction and amplification detection system. It can also reduce the time of nucleic acid analysis, improve the efficiency of nucleic acid analysis, and lay a solid foundation for disease diagnosis in a fast environment.

In this embodiment, the flow control module is a centrifugal module, and the centrifugal module includes a rotating body rotatably disposed about a central axis. The microfluidic chip is connected to the rotating body, and the rotating body is used to drive the microfluidic chip to rotate, so that the solution of the storage structure 21 enters the convection PCR tube 22 under the action of centrifugal force and flows through the FTA membrane 23 to enter the waste liquid chamber.

In an embodiment not shown, the flow control module can include a suction device in communication with the waste chamber for reducing the pressure within the waste chamber to cause the solution of the storage structure to enter under the pressure differential The convection PCR tube was filtered through the FTA membrane and then entered into the waste chamber. The suction device can be, for example, a vacuum pump, a syringe or the like.

In another embodiment not shown, the flow control module may also include a boosting device in communication with the storage chamber for increasing the pressure within the storage chamber to cause the solution within the storage chamber to be under pressure differential After entering the convection PCR tube 22 and filtering through the FTA membrane 23, it enters the waste liquid chamber. The supercharging device can be, for example, a booster pump or the like.

As shown in FIG. 2, in the embodiment, the microfluidic chip specifically includes a soft plug 20, the foregoing storage structure 21, the convection PCR tube 22, the FTA film 23, the microvia connector 24, the heat conducting slot structure 25, and the chip fitting. 26. The waste liquid receiving structure 27 and the support film 28 described above.

As shown in FIG. 5, the storage structure 21, the first end of the convection PCR tube 22, the second end of the convection PCR tube 22, and the waste liquid receiving structure 27 are from a position near the central axis of the rotating body to a central axis away from the rotating body. The positions are arranged in sequence, which facilitates the orderly flow of the sample solution in the shortest flow when the rotating body of the centrifugal module drives the microfluidic chip to rotate.

As shown in FIG. 2, the storage structure 21 is located on top of the convection PCR tube 22. The storage structure 21 includes a storage chamber inlet and a storage chamber outlet. Both the storage chamber inlet and the storage chamber outlet are in communication with the storage chamber. The storage chamber inlet is for injecting a solution or a liquid such as a sample solution, a purification liquid, a washing liquid into the storage chamber. The storage chamber outlet is coupled to the first end of the convection PCR tube 22 to provide the convective PCR tube 22 with the reagents required for each step.

The soft plug 20 is adapted to cooperate with the inlet of the storage chamber to close the inlet of the storage chamber. As shown in FIG. 1, FIG. 2 and FIG. 5, in the present embodiment, the inlet of the storage chamber is located at the top of the storage structure 21, and the soft plug 20 is fixed at the top of the storage structure 21. The soft plug 20 can prevent the splash of the reaction reagent during the centrifugation operation.

The FTA membrane 23 is integrated at the bottom of the convection PCR tube 22, i.e., near the second end of the convection PCR tube 22. The lysed or unlysed sample solution is filtered through a microchannel of convection PCR tube 22 under centrifugal force and filtered through FTA membrane 23, where the nucleic acid is captured by FTA membrane 23. FTA membrane 23 can not only achieve sample lysis (or select externally pre-completed sample lysis), nucleic acid adsorption, but also directly participate in convective PCR amplification reaction due to its solid-state storage lysis reagent, providing amplification for convective PCR amplification reaction. Template to achieve integration of nucleic acid extraction and nucleic acid amplification.

A support film 28 having a plurality of micropores is disposed between the FTA film 23 and the waste liquid chamber, and the support film 28 is bonded to the FTA film 23. As shown in FIG. 3, the FTA film 23 and the support film 28 are tightly fixed to the bottom of the convection PCR tube 22. Wherein, the micropores of the support film 28 are required to pass through a substance other than the nucleic acid in the sample solution (the nucleic acid has been adsorbed and captured by the FTA film 23) and the purification liquid and the washing liquid, so that the work of the convection PCR amplification detection system is not adversely affected. influences.

The support film 28 is bonded to the FTA film 23 to support the FTA film 23. In addition, after the support film 28 is disposed at the bottom of the FTA film 23, the support film 28 can cooperate with the FTA film 23 to prevent the amplification reagent from entering the waste liquid chamber. In this embodiment, the waste liquid chamber is not in communication with the atmosphere, and the inner diameter of the upper convection PCR tube 22 of the FTA membrane 23 is small. Therefore, the surface tension of the convection PCR tube 22, the back pressure in the waste liquid chamber, and the FTA film and the support film. The combination of the resistance of 28 prevents the amplification reagent from entering the waste chamber during the convection PCR amplification reaction.

As an example of the support film, the support film may be a porous film having a micropore diameter satisfying the above requirements. The porous membrane is a separation membrane containing from 10 to 100 million pores per square centimeter, a porosity of 70% to 80% of the total volume, a uniform pore diameter, and a pore diameter ranging from 0.02 to 20 μm. As another example of the support film, the support film may also be made of a fiber material.

The heat transfer slot structure 25 is used to introduce heat from the heating module into the convection PCR tube 22. A portion of the convection PCR tube 22 in which at least the FTA film 23 is disposed is located within the heat transfer groove structure 25. The microvia connector 24 is disposed between the thermally conductive slot structure 25 and the waste receiving structure 27. The microvia connector 24 includes microwells that communicate with the second end of the convection PCR tube 22 and the waste chamber, respectively.

As shown in FIG. 1, FIG. 2 and FIG. 5, the heat conducting groove structure 25 includes a heat conducting groove penetrating vertically, and the convection PCR tube 22 is tightly fitted over the inside of the heat conducting groove. The microvia connector 24 includes a cover plate disposed at the top of the waste liquid receiving structure 27 and a communication tube disposed on the cover plate. The communication tube is tightly fitted under the inner side of the heat conduction groove, and the waste liquid receiving structure 27 is located below the cover plate of the micro hole connection member 24, and is closely connected with the micro hole connection member 24, and the upper end of the communication tube and the convection PCR tube 22 are Two end docking. The waste chamber collects the waste liquid produced by the nucleic acid extraction process. The cover plate is located below the heat transfer groove structure 25 and the lower end of the communication pipe communicates with the waste liquid chamber. The thermally conductive slot structure 25 and the microvia connector 24 are secured together by a die fitting 26.

In the present embodiment, the heat conducting groove structure 25 can transfer the heat of the heating element 11 to the convection PCR tube 22, transfer heat for the convection PCR amplification reaction, and fix the microporous connector 24 and the convection PCR tube 22. The microporous connector 24 tightly connects the heat conducting groove structure 25 and the waste liquid receiving structure 27, and can also effectively reduce the heat of the heating module from being absorbed by the waste liquid receiving structure 27.

Preferably, the distance between the microfluidic chip and the central axis of the rotor is adjustably arranged. This arrangement can adjust the magnitude of the centrifugal force received by each portion of the microfluidic chip, thereby controlling the time required for the sample solution or the purification solution or the like to pass through the FTA film 23.

The centrifugal module includes a rotary drive mechanism that is drivingly coupled to the rotating body to drive the rotating body to rotate. Preferably, the rotational speed of the rotary drive mechanism is adjustably set.

In the present embodiment, the rotary drive mechanism is specifically a rotary electric machine 2. The centrifugal force generated by the rotation of the rotary motor 2 to drive the turntable 3 causes the reaction reagent to pass through the FTA film 23 in the microfluidic chip, and sequentially performs nucleic acid adsorption, purification, and washing steps to complete nucleic acid extraction.

The centrifugation module includes a positioning control element for controlling the convective PCR tube 22 to be in a vertical state. In this embodiment, the positioning control element includes a photoelectric switch 6.

As shown in FIG. 1 and FIG. 4, in the embodiment, the centrifugal module specifically includes a rotating electrical machine 2, a turntable 3, a rotating electrical machine fixing member 4, a chip fixing member 5, a photoelectric switch 6, a photoelectric switch fixing seat 7, and a rotating motor fixed. Block 8.

The aforementioned rotating body includes a turntable 3 and a chip fixing member 5. The turntable 3 is rotatably disposed about a central axis. The chip holder 5 is fixedly connected to the turntable 3. The microfluidic chip is fixedly disposed on the chip holder 5.

As shown in FIG. 4, the turntable 3 is fixed to the rotating shaft of the rotary electric machine 2 by a rotary motor fixing member 4. The chip holder 5 is fixed to the turntable 3. The rotary electric machine 2 is mounted on the base plate 1 via a rotary motor mount 8. When the microfluidic chip is at the first end of the convection PCR tube 22 and the second end is in the lower vertical position, the bottom of the chip holder 5 is located in the accommodating groove of the photoelectric switch 6 to sense the microfluidic chip. s position. The photoelectric switch 6 is mounted on the base plate 1 via a photoelectric switch mount 7.

The rotation of the turntable 3 is driven by the rotary electric machine 2 to generate centrifugal force, so that the cracked or uncracked test sample, or the reagent for purification, flows from the storage structure 21 through the microchannel in the middle of the convection PCR tube 22 under the action of centrifugal force. , filtering from the FTA membrane 23 to achieve capture and purification of the nucleic acid template.

The adjustment of the centrifugal force of the microfluidic chip can be achieved by adjusting the rotational speed of the rotating electrical machine 2, thereby controlling the time required for the detection of the sample or purification reagent through the FTA membrane 23.

The photoelectric switch 6 is disposed under the turntable 3, and the photoelectric switch 6 is coupled with the rotary electric machine 2 and controls the rotation angle of the rotary electric machine 2 to realize the positioning control of the rotary electric machine 2, so that the convection PCR tube 22 is in a vertical state, and the convection PCR amplification reaction condition is satisfied. .

The fixed connection position of the turntable 3 to the chip holder 5 is variably provided to adjust the distance between the microfluidic chip and the central axis of the rotating body. Thereby, the adjustment of the centrifugal force of the microfluidic chip is realized.

The heating module includes a heating element 11 movably disposed relative to a rotational axis of the microfluidic chip to switch between a heated position and a non-heated position, wherein in the heated position, the heating element 11 is adjacent to the microfluidic chip to Heating is performed, and in the non-heating position, the heating element 11 is remote from the microfluidic chip relative to the heating position.

The heating module also includes a linear drive mechanism that is drivingly coupled to the heating element 11 to drive the heating element to switch between a heated position and a non-heated position.

Preferably, the linear drive mechanism is a linear motor 9. The linear motor 9 is driven to fasten the heating element 11 to the heat conducting groove structure 25 at the bottom of the microfluidic chip, and the heating module is activated to transfer the heat generated by the heating element 11 to the convection PCR tube 22 via the heat conducting groove structure 25 for convection. The PCR amplification reaction provides heat.

In addition, the heating module may further comprise a temperature measuring element 13 for measuring the temperature of the heating element 11.

As shown in FIG. 1 , FIG. 6 and FIG. 7 , in the embodiment, the heating module specifically includes a linear motor 9 , a blocking piece 10 , a heating element 11 , a first heating fixture 12 , a temperature measuring component 13 , and a second heating fixture. 14. Linear motor mount 15.

The heating element 11 is tightly fitted between the flap 10 and the first heating fixture 12. The first heating fixture 12 is made of a metal material having a high heat capacity. The temperature measuring element 13 is mounted on the first heating fixture 12 to complete the temperature measurement. The heating element 11, the first heating fixture 12, and the flap 10 are mounted on the second heating fixture 14. The second heating fixture 14 is made of a non-metallic material having a low heat capacity. The second heating fixture 14 is fixed to the rotating shaft of the linear motor 9.

The linear motor 9 is mounted on the base plate 1 via a linear motor mount 15. By controlling the movement of the linear motor 9, the heating element 11 is brought into close contact with the heat conducting groove structure 25 of the microfluidic chip to achieve contact heating of the microfluidic chip by the heating element 11. The temperature measuring element 13 is deeply buried in the middle of the first heating fixture 12 to achieve temperature detection.

The temperature measuring element 13 may be, for example, a thermal resistor or a thermocouple. The heating element 11 may be, for example, a semiconductor refrigerator or a heating resistor film.

As shown in FIGS. 1 and 6, the optical detection module includes an excitation module and a reception module.

The excitation module includes an excitation light source movably disposed relative to a rotational axis of the microfluidic chip to switch between an excitation position and a non-excitation position. In the excitation position, the excitation light source is concentric with the convection PCR tube 22 in a non-excited position. The centerline of the excitation source is spaced from the centerline of the convection PCR tube 22.

As shown in FIG. 1 and FIG. 6 , the excitation module specifically includes an LED lamp 16 , an excitation filter 17 , an LED lamp holder 18 , and an LED lamp fixing bracket 19 . In this embodiment, the LED lamp 16 serves as an excitation light source.

As shown in FIG. 6, the excitation light source is disposed relatively fixedly to the heating element 11. The linear motor 9 can drive the excitation source of the optical detection module at the top of the convection PCR tube 22 while driving the heating element 11 in close contact with the thermally conductive slot structure 25 to provide excitation light for fluorescence detection in a convective PCR amplification reaction.

Specifically, the LED lamp 16 is fixed on the LED lamp holder 18, and the LED lamp holder 18 is mounted on the second heating fixture 14 through the LED lamp fixing bracket 19. The excitation filter 17 is located at the bottom of the LED lamp mount 18. The LED lamp 16 is in close contact with the excitation filter 17 and is located at the upper portion thereof. The LED lamp mount 18 is mounted on the second heating fixture 14. The LED lamp 16 of the excitation module can be driven by the linear motor 9 to move to the excitation position at the top of the convection PCR tube 22, and the LED lamp 16 is controlled to be turned on to provide excitation light for fluorescence detection in the convection PCR amplification reaction.

See Figure 8. In this embodiment, the receiving module includes a fluorescence detector 29, a rubber gasket 30, and a receiving filter 31. The receiving filter 31 is fixed by a rubber gasket 30 and is in close contact with the front of the camera of the fluorescence detector 29. The camera of the fluorescence detector 29 is facing the middle of the convection PCR tube 22 to perform real-time optical detection of the convective PCR amplification reaction.

The optical filter module 30 fixes the receiving filter 31 through the rubber gasket 30 to overcome the influence of external light on the fluorescent signal. The fluorescence detector 29 can be an industrial CCD, a smart phone camera or other type of camera, or a photoelectric sensor such as a photodiode or a photomultiplier tube.

This embodiment also provides a convective PCR amplification detection method for performing convective PCR amplification detection using the convective PCR amplification detection system described above. The method comprises: an extracting step comprising a filtering step of adding a sample solution containing the nucleic acid into the storage chamber of the storage structure 21 in the filtering step, causing the sample solution in the storage chamber to flow into the convection PCR tube 22, and filtering through the FTA membrane 23 The nucleic acid is adsorbed on the surface of the FTA film 23, and the remaining substance in the sample solution flows into the waste liquid chamber; the amplification step, after the extraction step, the nucleic acid adsorbed on the surface of the FTA film 23 is performed by the convection PCR tube 22 and the heating module. Amplification; detection step, while the amplification step is performed, the amplification product in the convection PCR tube 22 is fluorescently detected by the optical detection module.

The convection PCR amplification detection method has the same advantages as the aforementioned convection PCR amplification detection system.

Preferably, the step of amplifying comprises: rotating the convective PCR tube 22 to a vertical state; injecting an amplification reagent into the convection PCR tube 22; and heating the substance in the convection PCR tube 22 with a heating module.

Further, the extracting step further includes a purifying step of adding the purifying liquid to the storage chamber of the storage structure 21 after the filtering step, and flowing the purified liquid in the storage chamber into the convection PCR tube 22 to be purified and adsorbed on the surface of the FTA film 23. The purified liquid after the nucleic acid flows into the waste liquid chamber through the FTA film 23.

Further, the extracting step further includes a washing step of adding the washing liquid into the storage chamber of the storage structure 21 after the purifying step, causing the washing liquid in the storage chamber to flow into the convection PCR tube 22, and washing the surface adsorbed on the surface of the FTA film 23. The washing liquid after the nucleic acid flows into the waste liquid chamber through the FTA film 23.

In this embodiment, the sample solution in the storage chamber is caused to flow into the convection PCR tube 22, and the purified liquid in the storage chamber is flowed into the convection PCR tube 22, so that the washing liquid in the storage chamber flows to the convection PCR tube 22. This is achieved by rotating the rotating body to drive the microfluidic chip to rotate. For the embodiment in which the flow device includes a suction device in communication with the waste liquid chamber and/or a pressurization device in communication with the storage chamber, the step of rotating the rotary body to drive the microfluidic chip to rotate can be performed by the suction device. The pressure within the chamber is reduced and/or the step of increasing the pressure within the storage chamber by the boosting device is replaced.

The convection PCR amplification detection method of the present embodiment will be specifically described below.

Extraction step. The sample solution is introduced into the storage chamber of the storage structure 21 through the inlet of the storage chamber by a pipette or an automatic needle, and the inlet of the storage chamber is closed by the soft plug 20. The rotary electric machine 2 is started, and the microfluidic chip is driven to rotate at a high speed by the turntable 3. Under the action of the centrifugal force, the sample solution in the storage cavity of the storage structure 21 flows into the convection PCR tube 22, and then flows through the FTA film 23 to the waste. In the liquid chamber, the nucleic acid in the sample solution is adsorbed on the surface of the FTA film 23, and the filtration step is completed. The purification liquid and the washing liquid are sequentially injected into the storage structure 21 in the same manner to complete the purification step and the washing step; finally, the rapid extraction of the nucleic acid is completed.

Amplification step. The microfluidic chip is rotated to the vertical state by the photoelectric switch 6 under the turntable 3. The amplification reagent is then injected into the convection PCR tube 22. The linear motor 9 is driven so that the heating element 11 of the heating module is in close contact with the heat conducting groove structure 25, the heating module is activated, the heating temperature is set to 95 ° C, and the LED excitation module is located directly above the convection PCR tube 22 to start convection PCR amplification.

Detection step. At the same time as the amplification step, the excitation module that has been in the excitation position is turned on, the convection PCR tube 22 is lighted, and the fluorination PCR amplification reaction product is detected by real-time fluorescence detection through the receiving module.

Through the above operation, the above-mentioned convection PCR amplification detection system can be utilized to realize the integrated operation of rapid nucleic acid extraction and nucleic acid amplification and detection.

All or part of the steps of implementing the above embodiments may be performed by hardware, or may be instructed by a program to perform related hardware. The related programs can be stored in a computer readable storage medium. The storage medium may be a read only memory, a magnetic disk or an optical disk or the like.

According to the above description, the embodiment of the present application provides a convection PCR amplification detection system and a convection PCR amplification detection method for integrating nucleic acid extraction functions. Compared with the prior art, the above embodiments of the present application have the following beneficial effects:

The FTA membrane is used as a solid phase to complete the rapid extraction of nucleic acid, and provides a template for subsequent convection PCR amplification reaction, thereby realizing the integrated operation of nucleic acid extraction and nucleic acid amplification.

Using centrifugal force to achieve rapid extraction of nucleic acids, improve nucleic acid extraction efficiency and quality, and provide a basis for subsequent convective PCR amplification reactions.

The utility model has the advantages of small volume, structure, simple operation, high degree of automation, integration, etc., which reduces the complexity and research cost of the device.

It should be noted that the above embodiments are only used to explain the technical solutions of the present application and are not limited thereto; although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that The specific embodiments of the application are modified or equivalently replaced with some technical features; without departing from the spirit of the technical solution of the present application, it should be included in the scope of the technical solutions claimed in the present application.

Claims (25)

1. A convective PCR amplification detection system, comprising:

   The microfluidic chip comprises a storage structure (21), a convection PCR tube (22), an FTA film (23) and a waste receiving structure (27), the storage structure (21) having a storage cavity, and the waste liquid receiving structure (27) having a waste chamber, the first end of the convection PCR tube (22) being in communication with the storage chamber, the second end of the convection PCR tube (22) being in communication with the waste chamber, the FTA a membrane (23) disposed inside the convection PCR tube (22) to filter a solution flowing from the first end to the second end of the convection PCR tube (22) and capable of adsorbing nucleic acids in the solution at the The surface of the FTA film (23);

   a flow control module, configured to enter a solution in the storage chamber into the convection PCR tube (22) and filter through the FTA membrane (23) into the waste liquid chamber;

   a heating module for heating the substance in the convection PCR tube (22);

   An optical detection module for performing fluorescence detection on a substance in the convection PCR tube (22).
2. The convection PCR amplification detection system according to claim 1, wherein the flow control module comprises a rotating body, and the rotating body drives the convection PCR tube (22) to rotate about a central axis, the storage structure ( 21) a first end of the convection PCR tube (22), a second end of the convection PCR tube (22), and the waste liquid receiving structure (27) from a position near the central axis to away from the The positions of the central axes are arranged in sequence.
3. The convective PCR amplification detection system according to claim 1, wherein the microfluidic chip further comprises one or more layers of a support film (28) having a plurality of micropores, the support film (28) Provided between the FTA film (23) and the waste liquid chamber and the support film (28) is bonded to the FTA film (23).
4. The convection PCR amplification detection system according to claim 1, wherein the microfluidic chip further comprises a heat conduction groove structure for introducing heat of the heating module into the convection PCR tube (22) ( 25) The heat conducting groove structure (25) comprises a heat conducting groove, and at least a portion of the convection PCR tube (22) in which the FTA film (23) is disposed is located in the heat conducting groove.
5. The convection PCR amplification detection system according to claim 4, wherein the microfluidic chip further comprises a microvia connector (24), and the microvia connector (24) is disposed on the heat conduction slot structure Between (25) and the waste receiving structure (27), the microporous connector (24) includes micropores that respectively communicate the second end of the convection PCR tube (22) with the waste liquid chamber.
6. The convective PCR amplification detection system according to claim 1, wherein said storage structure (21) comprises a storage cavity inlet, said microfluidic chip further comprising a soft plug (20), said soft plug (20) ) for mating with the storage chamber inlet to enclose the storage chamber inlet.
7. The convective PCR amplification detection system according to claim 1, wherein said flow control module comprises a centrifugal module, said centrifugal module comprising a rotating body rotatably disposed about a central axis, said microfluidic chip and The rotating body is connected, the rotating body is configured to drive the microfluidic chip to rotate, so that the solution of the storage structure (21) enters the convection PCR tube (22) under the action of centrifugal force and passes through the FTA The membrane (23) is filtered and then introduced into the waste chamber.
8. The convective PCR amplification detection system according to claim 7, wherein the distance between the microfluidic chip and the central axis is adjustably set.
9. The convective PCR amplification detecting system according to claim 7, wherein said rotating body comprises a turntable (3) and a chip fixing member (5), said turntable (3) being rotatably disposed around said central axis The chip fixing member (5) is fixedly connected to the turntable (3), and the microfluidic chip is fixedly disposed on the chip fixing member (5).
10. The convective PCR amplification detection system according to claim 9, characterized in that the fixed connection position of the turntable (3) and the chip holder (5) is changeably arranged.
11. The convective PCR amplification detection system according to claim 7, wherein the centrifugation module comprises a positioning control element for controlling the convection PCR tube (22) to be in a vertical state.
12. The convective PCR amplification detecting system according to claim 7, wherein said centrifugal module includes a rotational driving mechanism, said rotational driving mechanism being drivingly coupled to said rotating body to drive said rotating body to rotate.
13. The convection PCR amplification detection system according to claim 12, wherein the rotational speed of the rotational drive mechanism is adjustably set.
14. The convection PCR amplification detection system according to claim 12, wherein the centrifugation module comprises a positioning control element, and the positioning control element is configured to control the convection PCR tube (22) to be in a vertical state, wherein The positioning control element includes a photoelectric switch (6) coupled to the rotary drive mechanism and controlling the convection PCR tube (22) to be in a vertical state by controlling a rotation angle of the rotary drive mechanism .
15. The convective PCR amplification detection system according to claim 1, characterized in that the heating module comprises a heating element (11) and a temperature measuring element (13) for measuring the temperature of the heating element (11).
16. The convective PCR amplification detection system according to claim 1, wherein said heating module comprises a heating element (11), said heating element (11) being movably disposed relative to said microfluidic chip to Switching between a heated position in which the heating element (11) is adjacent to the microfluidic chip to heat it, and a non-heating position in which the heating element (11) The microfluidic chip is remote from the heating position.
17. The convection PCR amplification detection system according to claim 16, wherein the heating module further comprises a linear drive mechanism, the linear drive mechanism being drivingly coupled to the heating element (11) to drive the heating element Switching between the heated position and the non-heated position.
18. The convective PCR amplification detection system according to claim 1, wherein said optical detection module comprises an excitation light source movably disposed relative to said microfluidic chip to be in an excited position and non-excited Switching between positions at which the excitation source is concentric with the convection PCR tube (22), at the non-excited position, the centerline of the excitation source and the convection PCR tube (22) The center line has a gap.
19. The convective PCR amplification detection system according to claim 16, wherein said optical detection module comprises an excitation light source movably disposed relative to said microfluidic chip to be in an excited position and non-excited Switching between positions at which the excitation source is concentric with the convection PCR tube (22), at the non-excited position, the centerline of the excitation source and the convection PCR tube (22) The center line has a spacing, wherein the excitation source is arranged relatively fixedly to the heating element (11).
20. The convective PCR amplification detection system according to claim 1, wherein said flow control module includes a suction device in communication with said waste liquid chamber, said suction device for reducing said waste liquid chamber The pressure is such that the solution in the storage chamber enters the convection PCR tube (22) under the pressure difference and is filtered through the FTA membrane (23) into the waste liquid chamber; and/or The flow control module includes a boosting device in communication with the storage chamber, the boosting device for increasing a pressure within the storage chamber to cause a solution in the storage chamber to enter the convection under a pressure differential The PCR tube (22) is filtered through the FTA membrane (23) and enters the waste chamber.
21. A convective PCR amplification detection method for performing amplification detection using the convective PCR amplification detection system of claim 1, wherein the method comprises:

    An extraction step comprising a filtration step in which a sample solution containing nucleic acid is added to a storage chamber of the storage structure (21), and a sample solution in the storage chamber is flowed to the convection PCR tube (22) Is filtered through the FTA membrane (23), the nucleic acid is adsorbed on the surface of the FTA membrane (23), and the remaining substance in the sample solution flows into the waste liquid chamber;

    An amplification step, after the extracting step, using the convection PCR tube (22) and the heating module to amplify the nucleic acid adsorbed on the surface of the FTA membrane (23) as a template;

    And a detecting step of performing fluorescence detection on the amplification product in the convection PCR tube (22) by using the optical detection module.
22. The convection PCR amplification detection method according to claim 21, wherein in the detecting step, the optical detection module is used to expand the convection PCR tube (22) while the amplification step The product is subjected to fluorescence detection.
23. The convection PCR amplification detection method according to claim 21, wherein the amplifying step comprises:

    Rotating the convection PCR tube (22) to a vertical state;

    Injecting an amplification reagent into the convection PCR tube (22);

    The material in the convection PCR tube (22) is heated by the heating module.
24. The convection PCR amplification detection method according to claim 21, wherein the extracting step further comprises a purification step, after the filtering step, adding a purification liquid to the storage chamber to make the storage chamber The purified liquid flows into the convection PCR tube (22), and the purified liquid after the nucleic acid adsorbed on the surface of the FTA membrane (23) is purified and flows into the waste liquid chamber through the FTA membrane (23). .
25. The convection PCR amplification detection method according to claim 24, wherein the extracting step further comprises a washing step, after the purifying step, adding a washing liquid to the storage chamber to make the storage chamber The washing liquid flows into the convection PCR tube (22), and the washing liquid after washing the nucleic acid adsorbed on the surface of the FTA membrane (23) flows into the waste liquid chamber through the FTA membrane (23). .

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