OA19784A - System for detecting convective PCR amplification and method for detecting convective PCR application. - Google Patents

Abstract

The application provides a convective PCR amplification detection system and method. The system comprises a microfluidic chip, including a storage structure, a convective PCR tube, an FTA membrane and a waste liquid reeeiving structure, wherein the fïrst end of the convective PCR tube is connected with a storage cavity of the storage structure, the second end of the convective PCR tube is connected with a waste liquid cavity of the waste liquid reeeiving structure, the FTA membrane is arranged in the convective PCR tube to filter a solution flowing from the first end of the convective PCR tube to the second end and adsorb nucleic acids in the solution on the surface of the FTA membrane; a flow control module for enabling the solution in the storage cavity to enter the convective PCR tube and enabling the solution to enter the waste liquid cavity after being filtered by the FTA membrane; a heating module for heating reactants in the convective PCR tube; and an optical detection module for performing fluorescence detection on the reactants in the convective PCR tube. The application can improve the efficiency of nucleic acid analysis.

Description

System For Detecting Convective PCR Amplification And Method For Detecting Convective PCR Amplification

Related Application

The application is based on the Chinese Patent Application No. 201710054798.1, filed on January 24, 2017, with the title of “Convective PCR Amplification Détection System and Convective PCR Amplification Détection Method” and daims its priority, and the entire disclosure of this Chinese patent application is herein incorporated by reference.

Technical Field

The application relates to the field of life medicine détection and diagnosis, and in particular to a convective PCR amplification détection System and a convective PCR amplification détection method.

Background Art

In the prior art, the nucleic acid analysis method often includes two steps, wherein the first step is to lyse a détection sample and then capture and purify nucleic acid templates; and the second step is to implement polymerase chain reaction (PCR) with nucleic acid templates or other isothermal amplification and détection. The PCR is a molecular biology technology used for amplifying spécifie DNA fragments. The PCR generally requires repeated thermal cycling steps at two or three températures for a reaction mixture to perform amplification. As a novel PCR amplification technology, convective polymerase chain reaction (CPCR, hereinafter referred to as convective PCR) relies on one or two constant reaction températures to establish stable température gradient at two ends of a reaction tube (convective PCR tube). Based on the principle of thermohydrodynamics, a periodic motion flow field is produced in the reaction tube, so that amplification samples can perform reciprocating motion between the two ends with different températures of the test tube, thereby obtaining the température 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 step and the nucleic acid amplification step are 1 separated from each other. Therefore, the traditional nucleic acid analysis System and method are inefficient.

Summary of the Invention

The application aims at providing a convective PCR amplification détection System and a convective PCR amplification détection method and aims at solving the problem of relatively low efficiency of the traditional nucleic acid analysis System and method.

In order to achieve the above object, the first aspect of the application provides a convective PCR amplification détection System, comprising: a microfluidic chip, which comprises a storage structure, a convective PCR tube, an FTA membrane and a waste liquid receiving structure, wherein the storage structure has a storage cavity, the waste liquid receiving structure has a waste liquid cavity, the first end of the convective PCR tube is connected with the storage cavity, the second end of the convective PCR tube is connected with the waste liquid cavity, the FTA membrane is arranged in the convective PCR tube to filter a solution flowing from the first end of the convective PCR tube to the second end of the convective PCR tube and adsorb nucleic acids in the solution on the surface of the FTA membrane; a flow control module for enabling the solution in the storage cavity to enter the convective PCR tube and enabling the solution through the FTA membrane to enter the waste liquid cavity; a heating module for heating reactants in the convective PCR tube; and an optical détection module for performing fluorescence détection to the reactants in the convective PCR tube.

Further, the flow control module comprises a rotating body, the rotating body is configured to drive the convective PCR tube to rotate around a central axis, and the storage structure, the first end of the convective PCR tube, the second end of the convective PCR tube and the waste liquid receiving structure are sequentially arranged from a position near the central axis to the position away from another central axis.

Further, the microfluidic chip comprises one layer or at least two layers of supporting membranes with a plurality of micropores, the supporting membranes are arranged between the FTA membrane and the waste liquid cavity, and the supporting membranes are contacted with the FTA membrane.

Further, the microfluidic chip comprises a heat conduction groove structure for introducing 2 heat of the heating module into the convective PCR tube, the heat conduction groove structure comprises a heat conduction groove, and at least the part of the convective PCR tube which is provided with the FTA membrane is positioned in the heat conduction groove.

Further, the microfluidic chip comprises a microporous connecting member, the microporous connecting member is arranged between the heat conduction groove structure and the waste liquid receiving structure, and the microporous connecting member comprises micropores which are connected with the second end of the convective PCR tube and the waste liquid cavity.

Further, the storage structure comprises a storage cavity inlet, the microfluidic chip comprises a soft plug, and the soft plug matches with the storage cavity inlet to seal the storage cavity inlet.

Further, the flow control module comprises a centrifugal module, the centrifugal module comprises a rotating body which is rotatably arranged around the central axis, the microfluidic chip is connected with the rotating body, the rotating body is configured to drive the microfluidic chip to rotate to enable the solution in the storage structure to enter the convective PCR tube and enable the solution to enter the waste liquid cavity after being filtered by the FTA membrane under centrifugal force.

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

Further, the rotating body comprises a rotating dise and a chip fixing member, the rotating dise is rotatably arranged around the central axis, the chip fixing member is fixedly connected with the rotating dise, and the microfluidic chip is fixedly arranged on the chip fixing member.

Further, the fixed connection position of the rotating dise and the chip fixing member is changeable.

Further, the centrifugal module comprises a positioning control element, and the positioning control element is configured to render the convective PCR tube to be in a vertical State.

Further, the centrifugal module comprises a rotation driving mechanism, and the rotation driving mechanism is drivingly connected with the rotating body to drive the rotating body to rotate.

Further, the rotational speed of the rotation driving mechanism is adjustable.

Further, the centrifugal module comprises a positioning control element, and the positioning control element is configured to render the convective PCR tube to be in a vertical state, wherein the positioning control element comprises a photoelectric switch sensor, the photoelectric switch sensor is coupled with the rotation driving mechanism and used render the convective PCR tube to be in the vertical State by changing the rotation angle of the rotation driving mechanism.

Further, the heating module comprises a heater and a température measuring sensor for measuring the température of the heater.

Further, the heating module comprises a heater, the heater is movable relative to the microfluidic chip to switch between a heating position and a non-heating position, and in the heating position, the heater contacts with the microfluidic chip to heat the microfluidic chip, and in the non-heating position, the heater is away from the microfluidic chip relative to the heating position.

Further, the heating module comprises a linear driving mechanism, and the linear driving mechanism is drivingly connected with the heater to drive the heater to switch between the heating position and the non-heating position.

Further, the optical détection module comprises an excitation light source, the excitation light source is movable relative to the microfluidic chip to switch between an excitation position and a non-excitation position, and in the excitation position, the excitation light source is concentric with the convective PCR tube, and in the non-excitation position, an interval exists between the central line of the excitation light source and the central line of the convective PCR tube.

Further, the optical détection module comprises an excitation light source, the excitation light source is movable relative to 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 convective PCR tube, and in the non-excitation position, an interval exists between the central line of the excitation light source and the central line of the convective PCR tube, wherein the excitation light source and the heater are fixedly arranged relative to each other.

Further, the flow control module comprises a suction device connected with the waste liquid cavity, the suction device is configured to form a vacuum in the waste liquid cavity to enable the solution in the storage cavity to enter the convective PCR tube and enable the solution to enter the waste liquid cavity after being filtered by the FTA membrane under a pressure différence.

The second aspect of the application provides a convective PCR amplification détection method utilizing the convective PCR amplification détection System of any one of the first aspect of the application, and the method comprises: an extraction step including a filtration step, in the filtration step, a sample solution containing nucleic acids is added into the storage cavity of the storage structure to enable the sample solution in the storage cavity to flow into the convective PCR tube, and after being filtered by the FTA membrane, the nucleic acids are adsorbed on the surface of the FTA membrane, and other substances in the sample solution flow into the waste liquid cavity; an amplification step, after the extraction step, the convective PCR tube and the heating module are utilized to amplify the nucleic acids adsorbed on the surface of the FTA membrane; and a détection step, the optical détection module is utilized to perform fluorescence détection to amplification products in the convective PCR tube.

Further, in the détection step, the optical détection module is utilized to perform fluorescence détection to the amplification products in the convective PCR tube while the amplification step is running.

Further, the amplification step comprises: rotating the convective PCR tube into a vertical State; injecting amplification reagent into the convective PCR tube; and utilizing the heating module to heat reactants in the convective PCR tube.

Further, the extraction step comprises a purification step, after the filtration step, a purification solution is added into the storage cavity to enable the purification solution in the storage cavity to flow into the convective PCR tube, and the purification solution flows into the waste liquid cavity through the FTA membrane after purifying the nucleic acids adsorbed on the surface of the FTA membrane.

Further, the extraction step comprises a washing step, after the purification step, a washing solution is added into the storage cavity to enable the washing solution in the storage cavity to flow into the convective PCR tube, and the washing solution flows into the waste liquid cavity through the FTA membrane after washing the nucleic acids adsorbed on the surface of the FTA membrane.

According to the PCR amplification détection System and method provided by the application, when nucleic acid extraction is performed with the sample solution containing the extracted nucleic acids, the sample solution is filtered by the FTA membrane 23 through the flow control device (for example, by using the rotating body to drive the convective PCR tube 22 to rotate), then the nucleic acids are adsorbed on the surface of the FTA membrane 23, and other reactants in the sample solution enter the waste liquid cavity through the FTA membrane 23, so as to achieve nucleic acid extraction. When the amplification solution in the convective PCR tube 22 is amplified, the amplification solution in the convective PCR tube 22 and the FTA membrane are heated by the heating module to enable the amplification solution and the FTA membrane to achieve a required température. In amplification, the optical détection module is utilized to perform fluorescence détection to the amplification products in the convective PCR tube. The convective PCR amplification détection System of the application achieves the intégration of nucleic acid automatic extraction and nucleic acid amplification détection and provides an integrated and automated nucleic acid analysis microfluidic chip. The microfluidic chip is a nucleic acid automatic extraction chip which achieves the automation of nucleic acid extraction process and improves the nucleic acid extraction efficiency; and the microfluidic chip is also a nucleic acid amplification and détection chip. By virtue of the integrated microfluidic network structure of the microfluidic chip, automation and intégration of nucleic acid analysis are achieved, a reasonable technical platform is provided to improve the nucleic acid analysis efficiency and the overall level of nucleic acid analysis is improved. In addition, the convective PCR amplification détection System can not only significantly shorten the nucleic acid amplification and détection time, but also reduce the manual operation steps and improve the accuracy and reliability of nucleic acid analysis.

Brief Description of the Drawings

Fig. 1 is a structural schematic diagram of a convective PCR amplification détection System of an embodiment of the application.

Fig. 2 is a structural schematic diagram of a microfluidic chip in the convective PCR amplification détection System as shown in Fig. 1.

Fig. 3 is a schematic diagram of a connection structure of a convective PCR tube, an FTA membrane and a supporting membrane of the microfluidic chip in the convective PCR amplification détection System as shown in Fig. 1.

Fig. 4 is a structural schematic diagram of a centrifugal module in the convective PCR 6 amplification détection System as shown in Fig. 1.

Fig. 5 is a schematic diagram of a connection structure of the centrifugal module and the microfluidic chip in the convective PCR amplification détection System as shown in Fig. 1.

Fig. 6 is a schematic diagram of a connection structure of a heating module and an excitation module of an optical détection module in the convective PCR amplification détection System as shown in Fig. 1.

Fig. 7 is a structural schematic diagram of the heating module in the convective PCR amplification détection System as shown in Fig. 1.

Fig. 8 is a structural schematic diagram of a receiving module of the optical détection module in the convective PCR amplification détection System as shown in Fig. 1.

Detailed Description of the Invention

The technical schemes in the embodiments of the application are clearly and completely described below in conjunction with varions accompanying drawings of the application. Obviously, the described embodiments are only one part of the embodiments of the application rather than ail of the embodiments. The following description of one exemplary embodiment is merely illustrative, and is in no way intended to limit the application and the use thereof. Based on the embodiments in the application, ail the other embodiments obtained by those of ordinary skill in the art without créative labor are still within the scope of the claimed application.

The relative arrangements of parts 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, for the convenience of description, it should be understood that the dimensions of the varions parts shown in the drawings are not drawn in the actual scale relation. Technologies, methods and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods and equipment should be considered as part of the authorized spécification. In ail of examples shown and discussed herein, any spécifie values are to be construed as illustrative only and not as a limitation. Accordingly, other examples of the exemplary embodiments may hâve different values. It should be noted that similar reference numerals and letters indicate similar items in the following drawings, and therefore, once an item is defined in one drawing, it is not required to be further discussed in the subséquent drawings.

In the description of the application, it should be understood that, the use of the terms such as “first”, “second” and the like to define the parts is merely for the purpose of facilitating the distinction of the corresponding parts. The above words hâve no spécial meanings, unless otherwise indicated. Therefore, the words are not to be construed as limiting the protection scope of the application.

In the description of the application, it is to be understood that directional or positional relations indicated by orientation words such as “front, back, up, down, left, right”, “transverse, longitudinal, vertical, horizontal” and “top, bottom” and the like are generally the directional or positional relations shown on the basis of the drawings. The orientation words are merely for the convenience of description of the application and the simplification of the description. Unless indicated to the contrary, the orientation words are not intended to indicate and imply the indicated device or the element must hâve the spécifie orientation or must be constructed and operated in the spécifie orientation. Thus, the orientation words are not to be construed as limiting the protection scope of the application; and the orientation words “inside and outside” refer to the inside and outside relative to the profile of the respective part.

Figs. 1-8 show a convective PCR amplification détection System of an embodiment of the application.

The convective PCR amplification détection System mainly comprises a microfluidic chip, a flow control module, a heating module and an optical détection module. The microfluidic chip mainly comprises a storage structure 21, a convective PCR tube 22, an FTA membrane 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 cavity. The first end of the convective PCR tube 22 is connected with the storage cavity, and the second end of the convective PCR tube 22 is connected with the waste liquid cavity. The FTA membrane 23 is arranged in the convective PCR tube 22 to filter a sample solution flowing from the first end of the convective PCR tube 22 to the second end of the convective PCR tube 22 and enable nucleic acids in the solution to be adsorbed on the surface of the FTA membrane 23. The flow control module is configured to enable the solution in the storage cavity to enter the convective PCR tube 22 and enable the solution to enter the waste liquid cavity after being filtered by the FTA membrane 23. The 8 heating module is configured to heat reactants in the convective PCR tube 22. The optical détection module is configured to perform fluorescence détection to the reactants in the convective PCR tube 22.

In the embodiment of the application, when the sample solution containing the nucleic acids is extracted, the sample solution is filtered by the FTA membrane 23 through the flow control device (for example, by using the rotating body to drive the convective PCR tube 22 to rotate), then the nucleic acids are adsorbed on the surface of the FTA membrane 23, and other substances in the sample solution enter the waste liquid cavity through the FTA membrane 23, so as to achieve the nucleic acid extraction. When the amplification solution in the convective PCR tube 22 is amplified, the amplification solution in the convective PCR tube 22 and the FTA membrane are heated by the heating module to enable the amplification solution and the FTA membrane to achieve a required température. In amplification, the optical détection module is utilized to perform fluorescence détection to amplification products in the convective PCR tube 22.

It can be known from the above description that, the convective PCR amplification détection System of the embodiment of the application performs the intégration of nucleic acid automatic extraction and nucleic acid amplification détection and constructs an integrated and automated nucleic acid analysis microfluidic chip. The microfluidic chip is a nucleic acid automatic extraction chip which performs the automation of nucleic acid extraction process and improves the nucleic acid extraction efficiency; and the microfluidic chip is also a nucleic acid amplification and détection chip. By virtue of the integrated microfluidic network structure of the microfluidic chip, automation and intégration of nucleic acid analysis are realized, a reasonable technical platform is provided for improving the nucleic acid analysis efficiency and the overall level of nucleic acid analysis is improved. In addition, the convective PCR amplification détection System can not only significantly shorten the nucleic acid amplification and détection time, but also reduce the manual operation steps and improve the accuracy and reliability of nucleic acid analysis.

Thus, in the embodiment of the application, the nucleic acid extraction method based on a solid-phase carrier and the convective PCR amplification détection technology are mutually combined to fully realize the characteristics and advantages of the two, thereby not only simplifying the complexity of the integrated nucleic acid extraction and amplification détection

System, but also reducing the nucleic acid analysis time, improving the nucleic acid analysis efïiciency and laying a solid foundation for disease diagnosis in an on-site fast environment.

In the embodiment, the flow control module is a centrifugal module, and the centrifugal module comprises a rotating body which is rotatably arranged around a central axis. The microfluidic chip is connected with the rotating body, and the rotating body is configured to drive the microfluidic chip to rotate, so as to enable the solution in the storage structure 21 to enter the convective PCR tube 22 and enable the solution to enter the waste liquid cavity after being filtered by the FTA membrane 23 under centrifugal force.

In an embodiment which is not shown herein, the flow control module may comprise a suction device connected with the waste liquid cavity, the suction device is configured to reduce the pressure in the waste liquid cavity to enable the solution in the storage structure to enter the convective PCR tube and enable the solution to enter the waste liquid cavity after being filtered by the FTA membrane under a pressure différence. For example, the suction device may be a vacuum pump, a syringe and the like.

In another embodiment which is not shown herein, the flow control module may also comprise a boosting device connected with the storage cavity, the boosting device is configured to increase the pressure in the storage cavity to enable the solution in the storage cavity to enter the convective PCR tube 22 and enable the solution to enter the waste liquid cavity after being filtered by the FTA membrane 23 under a pressure différence. For example, the boosting device may be a boosting pump and the like.

As shown in Fig. 2, in the embodiment, the microfluidic chip specifically comprises a soft plug 20, the storage structure 21, the convective PCR tube 22, the FTA membrane 23, a microporous connecting member 24, a heat conduction groove structure 25, a chip matching member 26, the waste liquid receiving structure 27 and a supporting membrane 28.

As shown in Fig. 5, the storage structure 21, the first end of the convective PCR tube 22, the second end of the convective PCR tube 22 and the waste liquid receiving structure 27 are sequentially arranged from the position near the central axis of the rotating body to the position away from the central axis, and the arrangement is conductive to enabling the sample solution to flow orderly in a shortest flow path when the rotating body of the centrifugal module drives the microfluidic chip to rotate.

As shown in Fig. 2, the storage structure 21 is positioned at the top of the convective PCR tube 22. The storage structure 21 comprises a storage cavity inlet and a storage cavity outlet. Both the storage cavity inlet and the storage cavity outlet are connected with the storage cavity. The storage chamber inlet is configured to inject the sample solution, a purification solution, a washing solution and other solutions or liquid into the storage cavity. The storage cavity outlet is connected with the first end of the convective PCR tube 22 to provide reaction reagents required by various steps to the convective PCR tube 22.

The soft plug 20 is configured to match with the storage cavity inlet to seal the storage cavity inlet. As shown in Figs. 1, 2 and 5, the storage cavity inlet in the embodiment is positioned 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 reagents during centrifugal operation.

The FTA membrane 23 is integrated at the bottom of the convective PCR tube 22, i.e., near the second end of the convective PCR tube 22. The lysed or unlysed sample solution flows through a microchannel of the convective PCR tube 22 under centrifugal force and is filtered by the FTA membrane 23, wherein the nucleic acids are captured by the FTA membrane 23. The FTA membrane 23 with lysis reagents preserved in solid state can not only perform sample lysis (or select to pre-complete the sample lysis extemally) and nucleic acid adsorption, but also directly participate in the convective PCR amplification reaction to provide amplification templates for the convective PCR amplification reaction, so as to perform the intégration of nucleic acid extraction and nucleic acid amplification.

The supporting membrane 28 with a plurality of micropores is arranged between the FTA membrane 23 and the waste liquid cavity, and the supporting membrane 28 is contacted with the FTA membrane 23. As shown in Fig. 3, the FTA membrane 23 and the supporting membrane 28 are tightly fixed at the bottom of the convective PCR tube 22. Wherein, the micropores of the supporting membrane 28 are required to enable reactants other than the nucleic acids in the sample solution (the nucleic acids hâve been adsorbed by the FTA membrane 23) as well as the purification solution and the washing solution to pass through without adverse effects on the work of the convective PCR amplification détection System.

The supporting membrane 28 is contacted with the FTA membrane 23, so as to be capable of supporting the FTA membrane 23. In addition, after the supporting membrane 28 is arranged at the bottom of the FTA membrane 23, the supporting membrane 28 can cooperate with the FTA membrane 23 to prevent the amplification reagent from entering the waste liquid cavity. In this embodiment, the waste liquid cavity is not connected with the atmosphère, and the inner diameter of the convective PCR tube 22 at the upper part of the FTA membrane 23 is smaller. Therefore, under the combined action of the surface tension of the convective PCR tube 22, the back pressure in the waste liquid cavity and the résistance of the FTA membrane and the supporting membrane 28, the amplification reagent can be prevented from entering the waste liquid cavity during the convective PCR amplification reaction.

As an example of the supporting membrane, the supporting membrane can be a porous membrane with the pore diameter of the micropores meeting the above requirements. Wherein, the porous membrane is a séparation membrane containing 10 to 100 million pores per square centimeter, wherein the porosity is 70%-80% of the total volume, the pore diameter is uniform, and the pore diameter range is 0.02-20pm. As another example of the supporting membrane, the supporting membrane may also be made of a fiber material.

The heat conduction groove structure 25 is configured to introduce heat of the heating module into the convective PCR tube 22. At least the part of the convective PCR tube 22 which is provided with the FTA membrane 23 is positioned in the heat conduction groove structure 25. The microporous connecting member 24 is arranged between the heat conduction groove structure 25 and the waste liquid receiving structure 27. The microporous connecting member 24 comprises the micropores which are connected with the second end of the convective PCR tube 22 and the waste liquid cavity.

As shown in Figs 1, 2 and 5, the heat conduction groove structure 25 comprises an up and down through heat conduction groove, and the convective PCR tube 22 is tightly matched above the interior of the heat conduction groove. The microporous connecting member 24 comprises a cover plate arranged at the top part of the waste liquid receiving structure 27 and a connecting tube arranged on the cover plate. The connecting tube is tightly matched below the interior of the heat conduction groove, the waste liquid receiving structure 27 is positioned below the cover plate of the microporous connecting member 24 and tightly connected with the microporous connecting member 24 together, and the upper end of the connecting tube is in butt joint with the 12 second end of the convective PCR tube 22. The waste liquid cavity collects waste liquid produced in the nucleic acid extraction process. The cover plate is positioned below the beat conduction groove structure 25, and the lower end of the connecting tube is connected with the waste liquid cavity. The heat conduction groove structure 25 and the microporous connecting member 24 are locked together by the chip matching member.

In this embodiment, the heat conduction groove structure 25 can not only transfer the heat of the heater 11 to the convective PCR tube 22 to transfer energy for the convective PCR amplification reaction, but also fix the microporous connecting member 24 and the convective PCR tube 22. The microporous connecting member 24 tightly connects the heat conduction groove structure 25 with the waste liquid receiving structure 27, thereby being capable of also effectively reducing the absorption of the heat of the heating module by the waste liquid receiving structure 27.

Preferably, the distance between the microfluidic chip and the central axis of the rotor is adjustable. This setting may adjust the magnitude of the centrifugal force received by each part of the microfluidic chip, thereby controlling the time required for enabling the sample solution or the purification solution or the like to pass through the FTA membrane 23.

The centrifugal module comprises a rotation driving mechanism, and the rotation driving mechanism is drivingly connected with the rotating body to drive the rotating body to rotate. Preferably, the rotational speed of the rotation driving mechanism is adjustable.

In this embodiment, the rotation driving mechanism is a rotating motor 2 specifically. The reaction reagent can pass through the FTA membrane 23 in the microfluidic chip under the centrifugal force produced by the rotating motor 2 driving a rotating dise 3 to rotate, so as to sequentially complété nucleic acid adsorption, purification and washing steps to complété nucleic acid extraction.

The centrifugal module comprises a positioning control element, and the positioning control element is configured to render the convective PCR tube 22 to be in a vertical State. In this embodiment, the positioning control element comprises a photoelectric switch sensor 6.

As shown in Figs. 1 and 4, in this embodiment, the centrifugal module specifically comprises the rotating motor 2, the rotating dise 3, a rotating motor fixing member 4, a chip fixing member 5, the photoelectric switch sensor 6, a photoelectric switch sensor fixing base 7 13 and a rotating motor fixing base 8.

The rotating body comprises the rotating dise 3 and the chip fixing member 5. The rotating dise 3 is rotatably arranged around the central axis. The chip fixing member 5 is fixedly connected with the rotating dise 3. The microfluidic chip is fixedly arranged on the chip fixing member 5.

As shown in Fig. 4, the rotating dise 3 is fixed on a rotating shaft of the rotating motor 2 through the rotating motor fixing member 4. The chip fixing member 5 is fixed on the rotating dise 3. The rotating motor 2 is mounted on a base plate 1 through the rotating motor fixing base 8. When the microfluidic chip is positioned in a vertical position that the first end of the convective PCR tube 22 is at the top and the second end is at the bottom, the bottom of the chip fixing member 5 is positioned in an accommodating groove of the photoelectric switch sensor 6 to sense the position of the microfluidic chip. The photoelectric switch sensor 6 is mounted on the base plate 1 through the photoelectric switch sensor fixing base 7.

The centrifugal force is produced by the rotating motor 2 driving the rotating dise 3 to rotate, so as to enable a lysed or unlysed test sample or the reaction reagent for purification to flow through a microchannel in the convective PCR tube 22 from the storage structure 21 and be filtered by the FTA membrane 23 to perform capture and purification of nucleic acid templates under the centrifugal force.

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

The photoelectric switch sensor 6 is arranged below the rotating dise 3, the photoelectric switch sensor 6 is coupled with the rotating motor 2 and Controls the rotation angle of the rotating motor 2 to realize positioning control of the rotating motor 2, so as to enable the convective PCR tube 22 to be in a vertical state and meet the convective PCR amplification reaction conditions.

The fixed connection position of the rotating dise 3 and the chip fixing member 5 is changeable to adjust the distance between the microfluidic chip and the central axis of the rotating body. Therefore, the adjustment of the centrifugal force on the microfluidic chip is performed.

The heating module comprises a heater 11, the heater 11 is movable relative to the rotation axis of the microfluidic chip to switch between a heating position and a non-heating position. In the heating position, the heater 11 contacts with the microfluidic chip to heat the microfluidic chip, and in the non-heating position, the heater 11 is away from the microfluidic chip relative to the heating position.

The heating module comprises a linear driving mechanism, and the linear driving mechanism is drivingly connected with the heater 11 to drive the heater to switch between the heating position and the non-heating position.

Preferably, the linear driving mechanism is a linear motor 9. The linear motor 9 is driven, the heater 11 contacts to the heat conduction groove structure 25 at the bottom of the microfluidic chip, and the heating module is started, so that heat produced by the heater 11 may be transferred to the convective PCR tube 22 through the heat conduction groove structure 25 for providing energy for the convective PCR amplification reaction.

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

As shown in Figs. 1, 6 and 7, in this embodiment, the heating module specifically comprises the linear motor 9, a blocking piece 10, the heater 11, a first heating fixing member 12, the température measuring element 13, a second heating fixing member 14 and a linear motor fixing base 15.

The heater 11 is tightly matched between the blocking piece 10 and the first heating fixing member 12. The first heating fixing member 12 is made of a métal material having a high heat capacity. The température measuring element 13 is mounted on the first heating fixing member 12 to complété the température measurement. The heater 11, the first heating fixing member 12 and the blocking piece 10 are mounted on the second heating fixing piece 14. The second heating fixing member 14 is made of a non-metal material having a low heat capacity. The second heating fixing member 14 is fixed on the rotating shaft of the linear motor 9.

The linear motor 9 is mounted on the base plate 1 through the linear motor fixing base 15. The motion of the linear motor 9 is controlled to enable the heater 11 to be close to the heat conduction groove structure 25 of the microfluidic chip, thereby realizing contact heating of the microfluidic chip by the heater 11. The température measuring element 13 is deeply buried in the 15 middle of the first heating fixing member 12 to achieve température détection.

The température measuring element 13 may be, for example, a thermal resistor or a thermocouple or the like; and the heater 11 may be, for example, a semiconductor refrigerator or a heating resistor film or the like.

As shown in Figs. 1 and 6, the optical détection module includes an excitation module and a receiving module.

The excitation module comprises an excitation light source, the excitation light source is movable relative to the rotation 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 convective PCR tube 22, and in the non-excitation position, an interval exists between the central line of the excitation light source and the central line of the convective PCR tube 22.

As shown in Figs. 1 and 6, the excitation module specifically comprises an LED lamp 16, an excitation filter 17, an LED lamp fixing base 18, and an LED lamp fixing support 19. In this embodiment, the LED lamp 16 serves as an excitation light source.

As shown in Fig. 6, the excitation light source and the heater 11 are fixedly arranged relative to each other. The linear motor 9 can drive the excitation source of the optical détection module to be at the top of the convective PCR tube 22 while driving the heater 11 to be close to the heat conduction groove structure 25, thereby providing excitation light for fluorescence détection in the convective PCR amplification reaction.

Specifically, the LED lamp 16 is fixed on the LED lamp fixing base 18, and the LED lamp fixing base 18 is mounted on the second heating fixing member 14 through the LED lamp fixing support 19. The excitation filter 17 is positioned at the bottom of the LED lamp fixing base 18. The LED lamp 16 is close to the excitation filter 17 and positioned at the upper part thereof. The LED lamp fixing base 18 is mounted on the second heating fixing member 14. The LED lamp 16 of the excitation module can be driven to move to the excitation position at the top of the convective PCR tube 22 through the linear motor 9, so as to control the LED lamp 16 to open and provide the excitation light for the fluorescence détection in the convective PCR amplification reaction.

See Fig. 8. In this embodiment, the receiving module comprises a fluorescence detector 29, a rubber gasket 30 and a receiving filter 31. The receiving filter 31 is fixed by the rubber gasket 30 and close to the front of a caméra of a fluorescence detector 29. The caméra of the fluorescence detector 29 is just facing the middle part of the convective PCR tube 22 to perform real-time optical détection to amplification products of the convective PCR amplification reaction.

The receiving filter 31 is fixed in the optical détection module through the rubber gasket 30 to overcome the influence of extemal light on fluorescent signais. The fluorescence detector 29 may adopt an industrial CCD, a smart mobile phone caméra or other types of caméras, and may also be a photodiode, a photomultiplier and other photoelectric sensors.

This embodiment also provides a convective PCR amplification détection method utilizing the above convective PCR amplification détection System to perform convective PCR amplification détection. The method comprises: an extraction step including a filtration step, in the filtration step, a sample solution containing nucleic acids is added into the storage cavity of the storage structure 21 to enable the sample solution in the storage cavity to flow into the convective PCR tube 22, and after being filtered by the FTA membrane 23, the nucleic acids are adsorbed on the surface of the FTA membrane 23, and other substances in the sample solution flow into the waste liquid cavity; an amplification step, after the extraction step, the convective PCR tube 22 and the heating module are utilized to amplify the nucleic acids adsorbed on the surface of the FTA membrane 23; and a détection step, the optical détection module is utilized to perform fluorescence détection to amplification products in the convective PCR tube 22 while the amplification step is performed.

The convective PCR amplification détection method has the same advantages as the above convective PCR amplification détection System.

Preferably, the amplification step comprises: rotating the convective PCR tube 22 into a vertical State; injecting an amplification reagent into the convective PCR tube 22; and utilizing the heating module to heat reactants in the convective PCR tube 22.

Further, the extraction step comprises a purification step, after the filtration step, a purification solution is added into the storage cavity of the storage structure 21 to enable the purification solution in the storage cavity to flow into the convective PCR tube 22, and the purification solution flows into the waste liquid cavity through the FTA membrane 23 after 17 purifying the nucleic acids adsorbed on the surface of the FTA membrane 23.

Further, the extraction step comprises a washing step, after the purification step, a washing solution is added into the storage cavity of the storage structure 21 to enable the washing solution in the storage cavity to flow into the convective PCR tube 22, and the washing solution flows into the waste liquid cavity through the FTA membrane 23 after washing the nucleic acids adsorbed on the surface of the FTA membrane 23.

In this embodiment, enabling the sample solution in the storage cavity to flow into the convective PCR tube 22, enabling the purification solution in the storage cavity to flow into the convective PCR tube 22 and enabling the washing solution in the storage cavity to flow into the convective PCR tube 22 are performed by rotating the rotating body to drive the microfluidic chip to rotate. For the embodiment in which the flow device comprises the suction device connected with the waste liquid cavity and/or the boosting device connected with the storage cavity, the step of rotating the rotating body to drive the microfluidic chip to rotate can be replaced by the step of reducing the pressure in the waste liquid cavity by the suction device and/or increasing the pressure in the storage cavity by the boosting device.

The convective PCR amplification détection method of this embodiment will be specifically described as below.

Extraction step. The sample solution is added into the storage cavity of the storage structure 21 through the storage cavity inlet by a pipetting gun or an automatic sample adding needle, and then the storage cavity inlet is sealed by the soft plug 20. The rotating motor 2 is started, the high-speed rotation of the microfluidic chip is driven by the rotating dise 3, the sample solution in the storage cavity of the storage structure 21 flows into the convective PCR tube 22 under the centrifugal force, then passes through the FTA membrane 23 and flows into the waste liquid cavity under the centrifugal force, so as to enable the nucleic acids in the sample solution to be adsorbed on the surface of the FTA membrane 23 to complété the filtration step. The purification solution and the washing solution are sequentially injected into the storage structure 21 in the same way to complété the purification step and the washing step; and the fast extraction of the nucleic acids is finally completed.

Amplification step. By means of the photoelectric switch sensor 6 below the rotating dise 3 the microfluidic chip can be rotated into the vertical State. Then, the amplification reagent is 18 injected into the convective PCR tube 22. The linear motor 9 is driven to enable the heater 11 of the heating module to be close to the heat conduction groove structure 25, the heating module is started, a heating température is set at 95 °C, and the LED excitation module is simultaneously positioned over the convective PCR tube 22 to start the convective PCR amplification.

Détection step. In the meantime of the amplification step, the excitation module in the excitation position is opened to light the convective PCR tube 22, and the real-time fluorescence détection is performed on the amplification product of the convective PCR amplification reaction through the receiving module.

Through the above operation, the above convective PCR amplification détection System can be utilized to perform integrated operation of nucleic acid fast extraction, nucleic acid amplification and détection.

Ail or part of the steps in the embodiment may be completed by hardware or be completed by instructing the relevant hardware through a program. The relevant program may be stored in a computer readable storage medium. The storage medium may be a read only memory, a magnetic dise or an optical dise or the like.

It can be known from the above description that, the embodiments of the application provide a convective PCR amplification détection System and a convective PCR amplification détection method integrating the nucleic acid extraction function. Compared with the prior art, the above embodiments of the application hâve the following bénéficiai effects:

The FTA membrane is utilized as a solid phase to complété the nucleic acid fast extraction, provide the templates for the subséquent convective PCR amplification reaction and perform the integrated operation of nucleic acid extraction and nucleic acid amplification.

The nucleic acid fast extraction is performed by utilizing the centrifugal force, thereby improving the nucleic acid extraction efficiency and quality and providing a foundation for the subséquent convective PCR amplification reaction.

The application has the advantages of small volume, simple structure and operation, high level of automation, intégration and the like and reduces the complexity of the device and the research cost.

The invention is not limited to the embodiment/s illustrated in the drawings. Accordingly it should be understood that where features mentioned in the appended daims are followed by reference signs, such signs are included solely for the purpose of enhancing the intelligibility of the daims and are in no way limiting on the scope of the daims.

Finally, it should be noted that, the above embodiments are merely used for explaining the technical schemes of the application rather*than limiting the application. Although the application is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the spécifie embodiments of the application can still be modified or part of the technical features can be replaced equivalently without departing from the spirit of the technical schemes of the application, and such modifications and substitutions should fall within the protection scope of the technical schemes of the application.

Claims (10)

1. Claims

   1. A convective PCR amplification détection System, comprising:

   a microfluidic chip, comprising a storage structure (21), a convective PCR tube (22), an FTA membrane (23) and a waste liquid receiving structure (27), wherein the storage structure (21) has a storage cavity, the waste liquid receiving 5 structure (27) has a waste liquid cavity, a first end of the convective PCR tube (22) is connected with the storage cavity, a second end of the convective PCR tube (22) is connected with the waste liquid cavity, the FTA membrane (23) is arranged in the convective PCR tube (22) to filter a solution flowing from the first end of the convective PCR tube (22) to the second end of the convective 10 PCR tube (22) and adsorb nucleic acids in the solution on a surface of the FTA membrane (23);

   a flow control module for enabling the solution in the storage cavity to enter the convective PCR tube (22) and enabling the solution through the FTA membrane (23) to enter the waste liquid cavity;

   a heating module for heating reactants in the convective PCR tube (22); and an optical détection module for performing fluorescence détection to the reactants in the convective PCR tube (22).
2. 2. The convective PCR amplification détection System according to claim 1, wherein the flow control module comprises a rotating body, the rotating body is ₂q configured to drive the convective PCR tube (22) to rotate around a central axis, and the storage structure (21), the first end of the convective PCR tube (22), the second end of the convective PCR tube (22) and the waste liquid receiving structure (27) are sequentially arranged from a position near the central axis to another position away from the central axis.

   _2>
3. 3. The convective PCR amplification détection System according to claim

   1, wherein the microfluidic chip comprises one layer or at least two layers of supporting membranes (28) with a plurality of micropores, the supporting membranes (28) are arranged between the FTA membrane (23) and the waste liquid cavity, and the supporting membranes (28) are contacted with the FTA 5 membrane (23).
4. 4 . The convective PCR amplification détection System according to claim 1, wherein the microfluidic chip comprises a heat conduction groove structure (25) for introducing heat of the heating module into the convective PCR tube (22), the heat conduction groove structure (25) comprises a heat conduction 1$ groove, and at least the part of the convective PCR tube (22) which is provided with the FTA membrane (23) is positioned in the heat conduction groove.
5. 5 . The convective PCR amplification détection System according to claim 4, wherein the microfluidic chip comprises a microporous connecting member (24), the microporous connecting member (24) is arranged between the heat conduction groove structure (25) and the waste liquid receiving structure (27), and the microporous connecting member (24) comprises micropores which are connected with the second end of the convective PCR tube (22) and the waste liquid cavity.
6. 6 . The convective PCR amplification détection system according to claim 1, wherein the storage structure (21) comprises a storage cavity inlet, the microfluidic chip comprises a soft plug (20), and the soft plug (20) matches with the storage cavity inlet to seal the storage cavity inlet.
7. 7 . The convective PCR amplification détection system according to claim 1, wherein the flow control module comprises a centrifugal module, the centrifugal module comprises a rotating body which is rotatably arranged around 22 the central axis, the microfluidic chip is connected with the rotating body, the rotating body is configured to drive the microfluidic chip to rotate to enable the solution in the storage structure (21) to enter the convective PCR tube (22) and enable the solution to enter the waste liquid cavity after being filtered by the FTA 5 membrane (23) under centrifugal force.
8. 8 . The convective PCR amplification détection System according to claim 7, wherein the distance between the microfluidic chip and the central axis is adjustable.
9. 9 . The convective PCR amplification détection System according to claim 1G 7, wherein the rotating body comprises a rotating dise (3) and a chip fixing member (5), the rotating dise (3) is rotatably arranged around the central axis, the chip fixing member (5) is fixedly connected with the rotating dise (3), and the microfluidic chip is fixedly arranged on the chip fixing member (5).

   10 .The convective PCR amplification détection System according to claim 15 9, wherein the fixed connection position of the rotating dise (3) and the chip fixing member (5) is changeable.
10. 11 . The convective PCR amplification détection System according to claim 7, wherein the centrifugal module comprises a positioning control element, and the positioning control element is configured to render the convective PCR tube 20 (22) to be in a vertical state.

    12 .The convective PCR amplification détection System according to claim 7, wherein the centrifugal module comprises a rotation driving mechanism, and the rotation driving mechanism is drivingly connected with the rotating body to drive the rotating body to rotate.

    13 .The convective PCR amplification détection System according to claim 12, wherein the rotational speed of the ro^tion driving mechanism is adjustable.

    14 .The convective PCR amplification détection System according to claim 12, wherein the centrifugal module comprises a positioning control element, and 5 the positioning control element is configured to render the convective PCR tube (22) to be in a vertical state, wherein the positioning control element comprises a photoelectric switch sensor (6), the photoelectric switch sensor (6) is coupled with the rotation driving mechanism and configured to render the convective PCR tube (22) to be in the vertical state by changing the rotation angle of the 10 rotation driving mechanism.

    15 .The convective PCR amplification détection System according to claim 1, wherein the heating module comprises a heater (11) and a température measuring element for measuring the température of the heater (11).

    16 .The convective PCR amplification détection System according to claim 15 1, wherein the heating module comprises a heater (11), the heater (11) is movable relative to the microfluidic chip to switch between a heating position and a non-heating position, and in the heating position, the heater (11) contacts with the microfluidic chip to beat the microfluidic chip, and in the non-heating position, the heater (11) is away from the microfluidic chip relative to the heating 2o position.

    17 .The convective PCR amplification détection System according to claim 16, wherein the heating module comprises a linear driving mechanism, and the linear driving mechanism is drivingly connected with the heater (11) to drive the heater to switch between the heating position and the non-heating position.

    18 .The convective PCR amplification détection System according to claim 1, wherein the optical détection module comprises an excitation light source, the excitation light source is movable relative to the microfluidic chip to switch between an excitation position and a non-excitation position, and in the 5 excitation position, the excitation light source is concentric with the convective PCR tube (22), and in the non-excitation position, an interval exists between the central line of the excitation light source and the central line of the convective PCR tube (22).

    19 .The convective PCR amplification détection System according to claim 10 16, wherein the optical détection module comprises an excitation light source, the excitation light source is movable relative to the microfluidic chip to switch between an excitation position and a non-excitation position, and in the excitation position, the excitation light source is concentric with the convective PCR tube (22), and in the non-excitation position, an interval exists between the 15 central line of the excitation light source and the central line of the convective PCR tube (22), wherein the excitation light source and the heater (11) are fixedly arranged relative to each other.

    20 .The convective PCR amplification détection System according to claim 1, wherein the flow control module comprises a suction device connected with ²⁰ the waste liquid cavity, the suction device is configured to reduce the pressure in the waste liquid cavity to enable the solution in the storage cavity to enter the convective PCR tube (22) and enable the solution to enter the waste liquid cavity after being filtered by the FTA membrane (23) under a pressure différence; and/or the flow control module comprises a boosting device connected with the ²5 storage cavity, the boosting device is configured to increase the pressure in the storage cavity to enable the solution in the storage cavity to enter the convective

    PCR tube (22) and enable the solution to enter the waste liquid cavity after being filtered by the FTA membrane (23) under a pressure différence.

    21 . A convective PCR amplification détection method utilizing the convective PCR amplification détection System according to claim 1, comprising:

    5 an extraction step comprising a filtration step, in the filtration step, a sample solution containing nucleic acids is added into the storage cavity of the storage structure (21) to enable the sample solution in the storage cavity to flow into the convective PCR tube (22), and after being filtered by the FTA membrane (23), the nucleic acids are adsorbed on the surface, of the FTA membrane (23), and 1Q other substances in the sample solution flow into the waste liquid cavity;

    an amplification step, after the extraction step, utilizing the convective PCR tube (22) and the heating module to amplify by taking the nucleic acids adsorbed on the surface of the FTA membrane (23) as templates; and a détection step, utilizing the optical détection module to perform fluorescence détection to amplification products in the convective PCR tube (22).

    22.The convective PCR amplification détection method according to claim 21, wherein in the détection step, utilizing the optical détection module to perform fluorescence détection to the amplification products in the convective PCR tube (22) while the amplification step is running.

    20 23.The convective PCR amplification détection method according to claim

    21, wherein the amplification step comprises:

    rotating the convective PCR tube (22) into a vertical State;

    injecting an amplification reagent into the convective PCR tube (22); and utilizing the heating module to heat reactants in the convective PCR tube (22).

    24 .The convective PCR amplification détection method according to claim 21, wherein the extraction step comprises a purification step, after the filtration step, a purification solution is added into the storage cavity to enable the purification solution in the storage cavity to flow into the convective PCR tube 5 (22), and the purification solution flows into the waste liquid cavity through the

    FTA membrane (23) after purifying the nucleic acids adsorbed on the surface of the FTA membrane (23).

    25 . The convective PCR amplification détection method according to claim 24, wherein the extraction step comprises a washing step, after the purification step, a 10 washing solution is added into the storage cavity to enable the washing solution in the storage cavity to flow into the convective PCR tube (22), and the washing solution flows into the waste liquid cavity through the FTA membrane (23) after washing the nucleic acids adsorbed on the surface of the FTA membrane (23).