Microfluidic nucleic acid detection reagent card box, application method thereof and full-automatic detection equipment

Abstract

The invention discloses a microfluidic nucleic acid detection reagent card box and a use method thereof and full-automatic detection equipment, the microfluidic nucleic acid detection reagent card box comprises a card box body and a rotating switch, the card box body is provided with a plurality of liquid storage tanks which are separated from each other, and a liquid reagent for pre-loading a sample liquid and treating a biological sample; the plurality of independent chambers comprise a reaction chamber for performing nucleic acid extraction and purification on a sample by using a liquid reagent; the amplification cavity is used for performing nucleic acid amplification on the sample; and a waste liquid storage chamber; the plurality of micro-channels are respectively arranged between the rotating switch and each liquid storage tank, the reaction cavity, the waste liquid storage cavity and the amplification cavity, and are distributed around the rotating switch in a divergent manner; the rotary switch is provided with a radial flow guide channel which is used for realizing switching among the micro-channels; according to the invention, liquid adding and pipetting in the whole process of an experiment can be automatically controlled only by arranging a power source, and nucleic acid extraction and amplification can be realized on the closed clamping box by rotating the switch according to a preset program in cooperation with an external instrument for fluorescence detection, so that the result of a sample can be obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular to a microfluidic nucleic acid detection reagent cartridge and a use method thereof, and a fully automatic detection device having the same. Background Art

[0002] Currently, most molecular diagnostic experiments are conducted in professional laboratories, which have negative pressure laminar flow designs and high-efficiency filtration systems for convection with external air to avoid contamination of the laboratory's external environment. Test samples need to be brought to the laboratory through a special channel, and testing can only begin when the sample volume is sufficient. It often takes 6-8 hours, or even longer, from sampling to results, and patients cannot be diagnosed in a timely manner.

[0003] The construction of large biosafety laboratories is suitable for molecular testing of large sample volumes and requires multiple personnel with rich practical experience to operate. This method is costly for emergency or home monitoring of single samples.

[0004] The existing molecular POCT cartridges on the market claim to complete the reaction completely inside the cartridge and will not pollute the external environment. However, the microfluidic cartridges are equipped with separate sample addition switches and drive devices for each liquid storage chamber and reaction chamber, which are costly, bulky, and complex to operate, making it difficult to meet the requirements of outpatient clinics, intensive care units, and home testing. In addition, the reaction system is not completely closed, and many of them are equipped with filter membranes. Air pressure is generated in the process of pushing the liquid, and excess gas is discharged into the air through the filter membrane, which still poses a risk of cross-infection. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a microfluidic nucleic acid detection reagent cartridge and a method of use thereof, and a fully automatic detection device having the same. The cartridge realizes the connection and closing of each liquid storage tank and reaction chamber, reaction chamber and amplification chamber, and waste liquid chamber through a rotatable switch and a plurality of microchannels divergently arranged around the rotating switch. Only a power source for driving the rotating switch is required to automatically control the liquid addition and pipetting of the entire experimental process. In conjunction with an external instrument for fluorescence detection, the switch can be rotated according to a preset program to realize nucleic acid extraction and amplification on the closed cartridge, and sample-in and result-out can be achieved, which has the characteristics of low cost, integration and automation.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] Microfluidic nucleic acid detection reagent cartridge, including:

[0008] A card box body and a rotary switch, wherein the card box body has

[0009] A plurality of separate liquid storage tanks for pre-loading sample liquid and liquid reagents for processing biological samples;

[0010] A plurality of independent chambers, including a reaction chamber: for extracting and purifying nucleic acid from a sample using the liquid reagent; an amplification chamber: for amplifying nucleic acid from a sample; and a waste liquid storage chamber;

[0011] A plurality of microchannels, which are arranged between the rotary switch and each of the liquid storage tank, reaction chamber, waste liquid storage chamber and amplification chamber, and are distributed in a divergent manner around the rotary switch, wherein a microchannel port connected to the reaction chamber is located at the axis of the rotary switch;

[0012] The rotary switch is rotatably disposed on one side surface of the cartridge body, and includes a flow guide area and a flow stop area. The flow guide area is provided with a radial flow guide channel. When the rotary switch is rotated, the flow guide channel connects the microchannels in the flow guide area and places the microchannels in the flow stop area in a closed state.

[0013] Some technical solutions also include a drive module and an airway.

[0014] The driving module has a piston and a piston chamber, and the piston chamber is divided into a front piston chamber and a rear piston chamber in the piston movement direction.

[0015] The air channel includes a first air channel and a second air channel, wherein the first air channel sequentially connects each liquid storage tank, the front piston chamber and the waste liquid storage chamber, and the second air channel connects the rear piston chamber and the reaction chamber.

[0016] The driving module forms a pressure difference between the first air channel and the second air channel by the movement of the piston in the piston chamber to drive the corresponding liquid.

[0017] In some technical solutions, the piston is selected from any one of a piston rod, a hydraulic piston and a piston ball, and

[0018] A sealing member is arranged on the piston to isolate the front piston cavity from the rear piston cavity and to isolate the interior of the cartridge body from the external environment.

[0019] In some technical solutions, the piston is a piston rod, the front end of the piston rod is sleeved with an elastic member, and the rear end of the piston rod is provided with a limit member to achieve reciprocating motion of the piston rod in the piston cavity; and / or,

[0020] The piston is a piston rod, and sealing components are installed on the piston rod at the front and rear end gaps and on the outer peripheral wall separating the front piston cavity and the rear piston cavity.

[0021] In some technical solutions, the elastic member is specifically a spring. When the piston rod is driven to move forward, the spring is compressed, and the air pressure in the first air channel connected to the front piston chamber is higher than the air pressure in the second air channel connected to the rear piston chamber, thereby enabling the reagents in each liquid storage chamber to flow to the reaction chamber; when the driving force weakens or withdraws, the piston is reset under the action of the spring restoring force, and the air pressure in the first air channel connected to the front piston chamber is lower than the air pressure in the second air channel connected to the rear piston chamber, thereby enabling the liquid in the reaction chamber to be discharged to the waste liquid storage chamber.

[0022] In some technical solutions, the cartridge body is an integrally formed structure, and an air pressure balance hole is provided on the cartridge body corresponding to the piston cavity.

[0023] Some technical solutions also include a sealing layer, which is made of transparent material;

[0024] The card box body is an integrally formed structure, and a plurality of microfluidic grooves and a plurality of chambers are provided on one side surface thereof. The sealing layer covers and adheres to the surface of the card box body, so that the microfluidic grooves on the card box body form the microfluidic channels, and seals a plurality of chambers to form the respective independent reaction chambers, amplification chambers and waste liquid storage chambers.

[0025] In some technical solutions, the sealing layer is made of a polymer transparent film and is combined with the card box body by thermal bonding, adhesive bonding, solvent bonding, laser welding, microwave or ultrasonic bonding.

[0026] In some technical solutions, the liquid storage tank includes a lysis liquid storage tank, a binding liquid storage tank, a plurality of cleaning liquid storage tanks, a rinse liquid storage tank, a sample storage tank and an eluent storage tank, wherein:

[0027] A magnetic bead placement slot is provided on the microchannel communicating between the sample storage slot and the reaction chamber;

[0028] The amplification chambers are multiple and spaced apart, and a freeze-dried reagent placement slot is provided on the microchannel connecting the elution chamber and each amplification chamber.

[0029] In some technical solutions, the reaction chamber includes a lysis chamber and an elution chamber, the reaction chamber is in an arc-shaped structure as a whole, and the lysis chamber and the elution chamber are respectively located at two ends of the arc;

[0030] There are more than one flow guide channel, which includes a first flow guide channel connecting the lysis chamber with the lysis liquid storage tank, the combination liquid storage tank, multiple cleaning liquid storage tanks, the rinse liquid storage tank, the sample storage tank, and the waste liquid storage chamber, and a second flow guide channel connecting the elution chamber with the elution liquid storage tank and multiple amplification chambers.

[0031] In some technical solutions, the layout area of ​​the amplification chamber adopts a flat design.

[0032] In some technical solutions, the surface of the microfluidic channel is treated with hydrophilicity and hydrophobicity.

[0033] In some technical solutions, the liquid storage tank is arranged on the top of the cartridge body, the reaction chamber, the waste liquid storage chamber and the amplification chamber are arranged from top to bottom on the front of the cartridge body, the microchannel is arranged on one side of the chamber, and the first air channel and the second air channel are arranged on the back of the cartridge body; and / or,

[0034] The driving module is arranged on the side of the cartridge body; the rotating switch is arranged on the back of the cartridge body, located in the gathering area of ​​each micro-channel.

[0035] The method for using the microfluidic nucleic acid detection reagent cartridge comprises the following steps performed in sequence:

[0036] Sample loading, lysis, binding, multi-stage washing and rinsing, elution and amplification; among them,

[0037] The multi-stage cleaning and rinsing steps include: adjusting the rotary switch to connect the first cleaning liquid storage tank and the reaction chamber, driving the piston to push the first cleaning liquid into the reaction chamber, mixing the magnetic beads with the first cleaning liquid, and after the external magnetic rod adsorbs the magnetic beads, adjusting the rotary switch to connect the reaction chamber and the waste liquid storage chamber, loosening the piston, discharging the waste liquid, and completing the first-level cleaning; adjusting the rotary switch to connect the second cleaning liquid storage tank and the reaction chamber, repeating the above liquid addition and liquid discharge operations of the reaction chamber, and performing the second-level cleaning; adjusting the rotary switch to connect the third cleaning liquid storage tank and the reaction chamber, repeating the above liquid addition and liquid discharge operations of the reaction chamber, and performing the third-level cleaning; adjusting the rotary switch to connect the rinsing liquid storage tank and the reaction chamber, repeating the above liquid addition and liquid discharge operations of the reaction chamber, and performing the rinsing.

[0038] In some technical schemes, the elution step includes: using a magnetic rod to move the magnetic beads bound to DNA or RNA to the elution chamber, adjusting the rotary switch to connect the elution liquid storage tank and the elution chamber, driving the piston to move in the piston chamber to allow the elution liquid to enter the elution chamber, withdrawing the magnetic rod to mix the magnetic beads with the elution liquid, and adsorbing the magnetic beads through the external magnetic rod.

[0039] In some technical solutions, the sample adding step includes: adjusting a rotary switch to connect the sample storage tank and the reaction chamber, driving the piston to move in the piston chamber so that the magnetic beads on the sample mixing path enter the reaction chamber, and resetting the rotary switch and the piston; and / or,

[0040] The lysis step includes: adjusting a rotary switch to connect the lysis solution storage tank and the lysis chamber, driving a piston to move in the piston chamber so that the lysis solution enters the lysis chamber to lyse the DNA or RNA; and / or,

[0041] The combining step includes: adjusting a rotary switch to connect the combining liquid storage tank and the reaction chamber, driving a piston to move in the piston chamber to allow the combining liquid to enter the reaction chamber, combining the DNA or RNA with the magnetic beads, adsorbing the magnetic beads by an external magnetic rod, adjusting a rotary switch to connect the reaction chamber and the waste liquid storage chamber, releasing the piston, and discharging the waste liquid; and / or,

[0042] The amplification step includes: adjusting the rotary switch to connect the amplification chambers in sequence, partially loosening the piston, and delivering the eluent containing DNA or RNA to each amplification chamber in sequence. At the same time, the freeze-dried reagent on the path is dissolved and brought into the amplification chamber, and the amplification chamber is heated and cooled to achieve nucleic acid amplification.

[0043] The fully automatic detection device includes the microfluidic nucleic acid detection reagent cartridge as described above, and

[0044] A housing, wherein the microfluidic nucleic acid detection reagent cartridge is detachably placed in the housing;

[0045] A first control unit, used to control the rotary switch to rotate according to a preset program;

[0046] A second control unit, used to control the driving device to move according to a preset program;

[0047] A temperature control module, used to increase or decrease the temperature of the amplification chamber;

[0048] The detection module is used to detect the optical signal of the amplification cavity.

[0049] The present invention adopts the above technical solution to have at least the following beneficial effects:

[0050] 1. Microfluidic technology is used to integrate nucleic acid extraction and amplification into a miniaturized and convenient integrated reagent cartridge. The sealed cartridge effectively eliminates aerosol contamination, solving the problem that nucleic acid testing requires a professional testing environment;

[0051] 2. A transparent film layer is used to cover the surface of the cartridge body, and the chamber is sealed to form a microchannel. All liquids and gases circulate inside the cartridge. There is no filter membrane, and the internal gas will not be exchanged with the outside, and the external environment will not be polluted. Preferably, multiple seals are provided on the piston in the driving module to isolate the front and rear cavities of the piston and the cartridge body from the external environment.

[0052] 3. The layout area of ​​the amplification chamber of the present invention adopts a flat design, which can increase the heat conduction area, achieve rapid temperature rise and fall, and minimize the experimental time under the same reagents and operating conditions;

[0053] 4. A freeze-dried reagent placement tank is provided in the eluent delivery path of the present invention, and the freeze-dried reagent will be dissolved during the eluent delivery process and brought into the amplification chamber, making it possible to store the reagent cartridge for a long time;

[0054] 5. The reagent cartridge of the present invention adopts a rotatable flow channel switch, which has better stability than a linear reciprocating switch. The overall structure is a flat plate, which is light and small in size, ingeniously designed, compact in structure, and can be adapted to a variety of external nucleic acid detection equipment. It is easy to operate and has efficient detection, and truly realizes sample-in and result-out, making it more suitable for outpatient clinics, intensive care units, and home testing scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings and their marks required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0056] Figure 1 This is a schematic elevation view of a microfluidic nucleic acid detection reagent cartridge according to an embodiment of the present invention;

[0057] Figure 2 It is a front view of the microfluidic nucleic acid detection reagent cartridge according to an embodiment of the present invention;

[0058] Figure 3 A top view of the microfluidic nucleic acid detection reagent cartridge according to an embodiment of the present invention;

[0059] Figure 4 It is a rear view of the microfluidic nucleic acid detection reagent cartridge according to an embodiment of the present invention;

[0060] Figure 5 It is a right view of the microfluidic nucleic acid detection reagent cartridge according to an embodiment of the present invention;

[0061] Figure 6 This is a working state diagram of the piston when it is reset according to an embodiment of the present invention;

[0062] Figure 7 This is a working state diagram of the piston driving according to an embodiment of the present invention;

[0063] Figure 8 It is a structural schematic diagram of a rotary switch according to an embodiment of the present invention;

[0064] Fig. 9 A schematic diagram of a microfluidic channel in a default state of a reagent cartridge according to an embodiment of the present invention;

[0065] Fig.10A schematic diagram of a microfluidic channel in a state where a reagent cartridge is transporting a sample according to an embodiment of the present invention;

[0066] Fig.11 A schematic diagram of a microfluidic channel in a state where a lysate is transported by a reagent cartridge according to an embodiment of the present invention;

[0067] Fig.12 A schematic diagram of a microfluidic channel in a state where a reagent cartridge according to an embodiment of the present invention is transporting a binding liquid;

[0068] Fig.13 A schematic diagram of a microfluidic channel in a state where the reagent cartridge according to an embodiment of the present invention discharges waste liquid;

[0069] Fig.14 A schematic diagram of a microfluidic channel in a state where a primary cleaning solution is transported by a reagent cartridge according to an embodiment of the present invention;

[0070] Fig.15 A schematic diagram of a microfluidic channel in a state where a secondary cleaning solution is transported by a reagent cartridge according to an embodiment of the present invention;

[0071] Fig.16 A schematic diagram of a microfluidic channel in a state where a three-stage cleaning solution is transported by a reagent cartridge according to an embodiment of the present invention;

[0072] Fig.17 A schematic diagram of a microfluidic channel in a state where a reagent cartridge according to an embodiment of the present invention is transporting a rinse solution;

[0073] Fig.18 Schematic diagram of the microfluidic channel in the state of transporting eluent of the reagent cartridge according to an embodiment of the present invention.

[0074] The meanings of the symbols in the figure are as follows:

[0075] 11 - lysis solution storage tank, 12 - binding solution storage tank, 13 - first cleaning solution storage tank, 14 - second cleaning solution storage tank, 15 - third cleaning solution storage tank, 16 - rinse solution storage tank, 17 - sample storage tank, 18 - eluent storage tank;

[0076] 21 - lysis chamber, 22 - elution chamber;

[0077] 30-waste liquid storage chamber; 40-amplification chamber;

[0078] 50 - driving module, 51 - piston, 52 - sealing element, 53 - limiting piece;

[0079] 61 – first airway, 62 – second airway;

[0080] 70 - rotary switch, 71 - first flow guide, 72 - second flow guide;

[0081] 80 - Air pressure balance hole. DETAILED DESCRIPTION

[0082] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings and other implementation methods can be obtained based on these drawings without creative work.

[0083] In order to simplify the drawings, only the parts related to the invention are schematically shown in each figure, and they do not represent the actual structure of the product. In addition, in order to simplify the drawings and facilitate understanding, in some figures, only one of the parts with the same structure or function is schematically drawn or marked. In this article, "one" not only means "only one", but also means "more than one".

[0084] It should be further understood that the term “and / or” used in the specification and appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0085] In this document, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0086] In one embodiment of the present invention, in conjunction with reference Figures 1 to 5 , shows a microfluidic nucleic acid detection reagent cartridge, comprising an integrally assembled cartridge body and a rotary switch 70.

[0087] The cartridge body has a plurality of separate liquid storage tanks for pre-loading sample liquid and liquid reagents for processing biological samples; a plurality of independent chambers, including a reaction chamber for extracting and purifying nucleic acid from samples using the liquid reagents; an amplification chamber 40 for amplifying nucleic acid from samples; and a waste liquid storage chamber 30; a plurality of microfluidics, which are arranged between the rotary switch and each liquid storage tank, the reaction chamber, the waste liquid storage chamber 30 and the amplification chamber 40, and are distributed in a divergent manner around the rotary switch, wherein a microfluidic port connected to the reaction chamber is located at the axis of the rotary switch.

[0088] The rotary switch is rotatably arranged on one side surface of the cartridge body, and comprises a flow guide area and a flow stop area. The flow guide area is provided with a radial flow guide channel. When the rotary switch is rotated, the flow guide channel connects the microchannel in the flow guide area and makes the microchannel in the flow stop area in a closed state.

[0089] In this embodiment, a rotary switch 70 is adopted to connect the corresponding microchannels distributed divergently around it, so as to realize liquid addition and pipetting in the whole process of the experiment. It is easy to operate and has a compact overall layout, which is suitable for manufacturing a more portable and miniaturized molecular POCT cartridge. In addition, the use of a rotatable channel switch has better stability than a linear reciprocating switch, which can ensure effective switching between experimental steps and continuous liquid addition and pipetting processes, thereby obtaining reliable detection results.

[0090] In the above embodiment, a driving module 50 and an air channel are also included. The driving module 50 has a piston 51 and a piston chamber. The piston chamber is divided into a front piston chamber and a rear piston chamber in the direction of movement of the piston 51. The air channel includes a first air channel 61 and a second air channel 62. The first air channel 61 sequentially connects each liquid storage tank, the front piston chamber and the waste liquid storage chamber 30, and the second air channel 62 connects the rear piston chamber and the reaction chamber. The driving module 50 forms a pressure difference between the first air channel 61 and the second air channel 62 through the movement of the piston 51 in the piston chamber to drive the corresponding liquid.

[0091] In a specific embodiment, the piston 51 is a piston rod. Of course, it can also be a hydraulic piston or a piston ball. Figure 6 and Figure 7 The piston rod in the present application is driven by an external motor, which has a spring sleeved on the front end of the piston rod and a limit plate 53 installed on the rear end of the piston rod. When the external motor drives the piston rod to move in the piston chamber, the space of the front piston chamber is compressed, the space of the rear piston chamber is expanded, and the air pressure of the first air channel 61 connected to the front piston chamber is increased, and the air pressure of the second air channel 62 connected to the rear piston chamber is reduced. In this way, the reagents in each liquid storage chamber can flow to the reaction chamber; when the driving force of the external motor is weakened or withdrawn, the piston 51 is driven to withdraw by the restoring force of the spring, the space of the front piston chamber is expanded, and the space of the rear piston chamber is compressed, and the air pressure of the first air channel 61 connected to the front piston chamber is reduced, and the air pressure of the second air channel 62 connected to the rear piston chamber is increased. In this way, the liquid in the reaction chamber can be discharged to the waste liquid storage chamber 30.

[0092] In some optional embodiments, multiple sets of seals 52 are provided on the piston rod at the front and rear end gaps and on the peripheral wall separating the front piston cavity and the rear piston cavity. In this embodiment, multiple seals 52 are provided correspondingly on the piston 51 in the driving module 50 to achieve isolation of the front and rear cavities of the piston and isolation of the interior of the cartridge body from the external environment, effectively preventing aerosol contamination and solving the dilemma of nucleic acid testing for professional testing environments.

[0093] Preferably, a pressure balance hole 80 is provided on the cartridge body corresponding to the piston cavity, for achieving pressure balance inside the cartridge when the piston 51 moves, thereby avoiding liquid backflow due to uneven pressure inside the cartridge.

[0094] In some preferred embodiments, the card box body is an integrally formed structure, and a plurality of microfluidics and a plurality of chambers are opened on one side surface thereof, and a sealing layer covers and adheres to the surface of the card box body, so that the microfluidics on the card box body form a microfluidic channel, and a plurality of chambers are sealed to form independent reaction chambers, amplification chambers 40 and waste liquid storage chambers 30; specifically, the sealing layer adopts a polymer transparent film, and is combined with the card box body by thermal bonding, gluing, solvent bonding, laser welding, microwave or ultrasonic bonding.

[0095] In this embodiment, a transparent film layer is used to cover the surface of the card box body, and the chamber is sealed to form a microchannel. All liquids and gases circulate inside the card box. There is no filter membrane, and the internal gas will not be exchanged with the outside, and the external environment will not be polluted.

[0096] In some specific embodiments, the above-mentioned liquid storage tanks include a lysis liquid storage tank 11, a binding liquid storage tank 12, a first cleaning liquid storage tank 13, a second cleaning liquid storage tank 14, a third cleaning liquid storage tank 15, a rinse liquid storage tank 16, a sample storage tank 17 and an elution liquid storage tank 18; wherein, a magnetic bead placement tank is arranged on the microchannel connected between the sample storage tank 17 and the reaction chamber; the reaction chamber includes a lysis chamber 21 and an elution chamber 22, and the reaction chamber is an arc-shaped structure as a whole, and the lysis chamber 21 and the elution chamber 22 are respectively located at the two ends of the arc. When the card box is placed upright, the lysis chamber 21 and the elution chamber 22 form two chambers that are connected in the middle but are independent of each other, and cooperate with an external magnetic rod to perform rapid solid-liquid separation, thereby improving the purification efficiency of genetic material.

[0097] Amplification chambers 40 are arranged in multiple intervals, and a freeze-dried reagent placement slot is set on the microfluidic channel connecting the elution chamber 22 and each amplification chamber 40. In some specific embodiments, 6-8 amplification chambers can be set. With the instrument of multiple fluorescence channels, 1-30 targets can be detected at a time to achieve super multiplex PCR detection; please refer to Figure 8The rotary switch involves more than one flow channel, including a first flow channel 71 connecting the lysis chamber with the lysis solution storage tank, the binding solution storage tank, multiple cleaning solution storage tanks, the rinse solution storage tank, the sample storage tank, and the waste liquid storage chamber, and a second flow channel 72 connecting the elution chamber with the elution solution storage tank and multiple amplification chambers. It should be pointed out that the number of cleaning solution storage tanks can be designed according to the cleaning requirements of DNA or RNA, and the number of amplification chambers can be designed according to the detection accuracy.

[0098] In a preferred embodiment, multiple liquid storage tanks are arranged at intervals and are evenly distributed to the top of the cartridge body; the cartridge body is square, and on the front of the cartridge body, the reaction chamber, the waste liquid storage chamber 30 and the amplification chamber 40 are arranged from top to bottom, the microchannel is arranged on one side of the chamber, the first air channel 61 and the second air channel 62 are arranged on the back of the cartridge body, and the transparent film layer covers the front, back and top surface of the cartridge body; in addition, the drive module 50 is arranged on the side of the cartridge body; the rotary switch 70 is arranged on the back of the cartridge body, located in the collection area of ​​each microchannel. With such a design, the microchannels on the cartridge body are regularly arranged and concentrated, the chambers and liquid storage tanks are reasonably arranged, the reagent cartridge has a compact structure, and the whole process of nucleic acid extraction, PCR amplification and fluorescence detection is realized in the same cartridge, making the cartridge more miniaturized and convenient. The flat-plate structure design allows each nucleic acid detector to independently operate 2 or more at the same time, which meets the needs of outpatient monitoring and home self-testing.

[0099] In another preferred embodiment, the layout area of ​​the amplification chamber 40 adopts a flat design, which can increase the heat conduction area and achieve rapid temperature rise and fall, thereby minimizing the experimental time under the same reagents and operating conditions.

[0100] In another preferred embodiment, the surface of the microchannel is treated with hydrophilicity and hydrophobicity to prevent fluid from being blocked in the microchannel. Specifically, a microchannel with hydrophilic-hydrophobic separation can be prepared by plasma jet, or the surface can be coated with hydrophilic-hydrophobic materials for modification.

[0101] The POCT reagent cartridge of the present invention can realize nucleic acid extraction, amplification, fluorescence quantification on the cartridge in conjunction with a power source, and finally obtain the result, so that the sample is in and the result is out. The cartridge is completely sealed relative to the outside, and all liquids and gases circulate inside the cartridge. There is no filter membrane, and the internal gas will not be exchanged with the outside, and the external environment will not be polluted.

[0102] In another embodiment of the present invention, a method for using a microfluidic nucleic acid detection reagent cartridge is provided, as follows:

[0103] See also Figure 8In the present microfluidic nucleic acid detection reagent cartridge, in the default state, the rotary valve is located at the original position in the figure. In this position, the microchannel above the rotary valve is not connected to any microchannel, and all chambers are in a closed state.

[0104] See also Fig. 9 , turn the rotary valve to the position of the sample storage tank 17, so that the sample storage tank 17 is connected to the reaction chamber; drive the piston 51, the liquid sample in the sample storage tank 17 flows through the magnetic bead placement slot and enters the reaction chamber, and the magnetic beads are also thawed and brought into the reaction chamber; rotate the rotary valve to the original position, release the piston 51, and the piston 51 is pulled back under the action of the spring force.

[0105] See also Fig.10 , turn the rotary valve to the lysis liquid position, so that the lysis liquid storage tank 11 is connected to the reaction chamber; drive the piston 51, and the liquid in the lysis liquid storage tank 11 enters the reaction chamber; drive the piston 51 to reciprocate, so that the lysis liquid and the sample are fully mixed, and the DNA or RNA in the cells is lysed.

[0106] See also Fig.11 After the lysis is completed, the rotary valve is turned to the binding liquid position, so that the binding liquid storage tank 12 is connected to the reaction chamber; the piston 51 is driven, and the liquid in the binding liquid storage tank 12 enters the reaction chamber, and under the action of the binding liquid, the DNA or RNA is combined with the magnetic beads; the external magnetic rod moves to the reaction chamber and is close to the film outside the reaction chamber, and the magnetic rod adsorbs the magnetic beads.

[0107] See also Fig.12 , turn the rotary valve to the waste liquid discharge position, so that the waste liquid storage chamber 30 is connected with the reaction chamber; release the piston 51, the piston 51 is pulled back under the action of the spring force, and the waste liquid is discharged into the waste liquid storage chamber 30, and only the magnetic beads combined with DNA or RNA are left in the reaction chamber.

[0108] See also Fig.13 , perform the first cleaning, turn the rotary valve to the first-level cleaning liquid position, so that the first cleaning liquid storage tank 13 is connected to the reaction chamber; drive the piston 51, and the cleaning liquid in the first cleaning liquid storage tank 13 enters the reaction chamber; withdraw the magnetic rod, so that the magnetic beads are mixed with the cleaning liquid; drive the piston 51 to reciprocate, so that the cleaning liquid and the magnetic beads bound to DNA or RNA are fully mixed; the external magnetic rod moves to the reaction chamber and clings to the film outside the reaction chamber, and the magnetic rod adsorbs the magnetic beads at this time; turn the rotary valve to the waste liquid discharge position, so that the waste liquid storage chamber 30 is connected to the reaction chamber; release the piston 51, the piston 51 is pulled back under the action of the spring force, and the waste liquid is discharged into the waste liquid storage chamber 30, and only the magnetic beads bound to DNA or RNA are left in the reaction chamber.

[0109] See also Fig.14, perform the second cleaning, turn the rotary valve to the secondary cleaning liquid position, so that the second cleaning liquid storage tank 14 is connected to the reaction chamber; drive the piston 51, and the cleaning liquid in the second cleaning liquid storage tank 14 enters the reaction chamber; withdraw the magnetic rod, so that the magnetic beads are mixed with the cleaning liquid; drive the piston 51 to reciprocate, so that the cleaning liquid and the magnetic beads bound to DNA or RNA are fully mixed; the external magnetic rod moves to the reaction chamber and clings to the film outside the reaction chamber, and the magnetic rod adsorbs the magnetic beads at this time; turn the rotary valve to the waste liquid discharge position, so that the waste liquid storage chamber 30 is connected to the reaction chamber; release the piston 51, the piston 51 is pulled back under the action of the spring force, and the waste liquid is discharged into the waste liquid storage chamber 30, and only the magnetic beads bound to DNA or RNA are left in the reaction chamber.

[0110] See also Fig.15 , perform the third cleaning, turn the rotary valve to the third cleaning liquid position, so that the third cleaning liquid storage tank 15 is connected to the reaction chamber; drive the piston 51, and the cleaning liquid in the third cleaning liquid storage tank 15 enters the reaction chamber; withdraw the magnetic rod, so that the magnetic beads are mixed with the cleaning liquid; drive the piston 51 to reciprocate, so that the cleaning liquid and the magnetic beads bound to DNA or RNA are fully mixed; the external magnetic rod moves to the reaction chamber and clings to the film outside the reaction chamber, and the magnetic rod adsorbs the magnetic beads at this time; turn the rotary valve to the waste liquid discharge position, so that the waste liquid storage chamber 30 is connected to the reaction chamber; release the piston 51, the piston 51 is pulled back under the action of the spring force, and the waste liquid is discharged into the waste liquid storage chamber 30, and only the magnetic beads bound to DNA or RNA are left in the reaction chamber.

[0111] See also Fig.16 , and rinse. The purpose of rinsing is to remove the residual ethanol on the DNA or RNA and reduce the effect of ethanol on PCR. Turn the rotary valve to the rinse liquid position so that the rinse liquid storage tank 16 is connected to the reaction chamber; drive the piston 51, and the rinse liquid in the rinse liquid storage tank 16 enters the reaction chamber; withdraw the magnetic rod so that the magnetic beads are mixed with the rinse liquid; push the piston 51 back and forth so that the rinse liquid is fully mixed with the magnetic beads bound to the DNA or RNA; the external magnetic rod moves to the reaction chamber and clings to the film outside the reaction chamber, and the magnetic rod adsorbs the magnetic beads at this time; turn the rotary valve to the waste liquid discharge position so that the waste liquid storage chamber 30 is connected to the reaction chamber; release the piston 51, and the piston 51 is pulled back under the action of the spring force, and the waste liquid is discharged into the waste liquid storage chamber 30, leaving only the magnetic beads bound to the DNA or RNA in the reaction chamber. This embodiment combines the rinse process with the closed cartridge structure design to reduce the inhibitory effect of ethanol on the PCR reaction and solve the technical difficulties in the molecular POCT industry: the contradiction between the cleaning effect and PCR inhibition.

[0112] See also Fig.17, elution is performed. Before elution, the magnetic rod is moved, and the magnetic beads bound to DNA or RNA are moved to the elution chamber 22 by the magnetic rod. The rotary valve is turned to the eluent position, so that the eluent storage tank 18 is connected to the elution chamber 22; the piston 51 is driven, and the eluent in the eluent storage tank 18 enters the elution chamber 22; the magnetic rod is withdrawn, so that the magnetic beads are mixed with the eluent; the piston 51 is driven to reciprocate, so that the eluent and the magnetic beads bound to DNA or RNA are fully mixed, and the DNA or RNA is eluted into the eluent; the external magnetic rod moves to the elution chamber 22 and is close to the film outside the elution chamber 22. At this time, the magnetic rod adsorbs the magnetic beads. This embodiment realizes rapid solid-liquid separation through the arc-shaped reaction chamber design and the magnetic rod moving along the arc-shaped trajectory, thereby improving the purification efficiency of genetic material.

[0113] The rotary valve is turned to the first PCR position so that the elution chamber 22 is connected to the first amplification chamber, the piston 51 is loosened to a certain extent, the piston 51 is pulled back under the action of the spring force, and the eluent containing DNA or RNA is transported to the first amplification chamber; the rotary valve is turned to the second PCR position so that the elution chamber 22 is connected to the second amplification chamber, the piston 51 is further loosened to a certain extent, the piston 51 is pulled back under the action of the spring force, and the eluent containing DNA or RNA is transported to the second amplification chamber; the above operation is continued to transport all the eluent containing DNA or RNA to all amplification chambers. There is a freeze-dried reagent placement slot in the PCR delivery pipeline, and the freeze-dried reagent will be dissolved during the eluent delivery process and brought to the amplification chamber 40.

[0114] After elution is completed, the instrument heats and cools the amplification chamber 40 to achieve nucleic acid amplification. During the heating and cooling process, fluorescence detection is performed on the chamber to finally obtain a concentration value.

[0115] In another embodiment of the present invention, a fully automatic detection device is also provided, including: the above-mentioned microfluidic nucleic acid detection reagent cartridge; a shell, in which the former microfluidic nucleic acid detection reagent cartridge is detachably placed; a first control unit, used to control the rotation of the rotary switch according to a preset program; a second control unit, used to control the movement of the driving device according to a preset program; a temperature control module, used to heat and cool the amplification chamber; and a detection module, used to detect the optical signal of the amplification chamber.

[0116] In the implementation steps of the present invention, a rotary switch is used to sequentially connect the cleaning liquid storage tanks and the reaction chambers at various levels, so that the cleaning liquid and the magnetic beads are fully mixed, and then the rotary switch is adjusted to connect the reaction chamber and the waste liquid storage chamber for drainage, and the above reaction chamber addition and drainage operations are repeated to achieve multiple cleaning of the magnetic beads; further, the present application also connects the rinse liquid storage tank and the reaction chamber by adjusting the rotary switch, and repeats the above reaction chamber addition and drainage steps to achieve the rinsing of the magnetic beads, which can fully remove the residual ethanol on the DNA and RNA, and reduce the inhibitory effect of ethanol on the PCR reaction. The cleaning effect of the present invention is significant, avoiding the inhibitory effect of reagent ethanol on the PCR reaction, and improving the accuracy of the test results.

[0117] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A microfluidic nucleic acid detection reagent cartridge, characterized in that: include: A card box body and a rotary switch, wherein the card box body has A plurality of separate liquid storage tanks for pre-loading sample liquid and liquid reagents for processing biological samples; A plurality of independent chambers, including a reaction chamber: for extracting and purifying nucleic acid from a sample using the liquid reagent; an amplification chamber: for amplifying nucleic acid from a sample; and a waste liquid storage chamber; A plurality of microchannels, which are arranged between the rotary switch and each of the liquid storage tank, reaction chamber, waste liquid storage chamber and amplification chamber, and are distributed in a divergent manner around the rotary switch, wherein a microchannel port connected to the reaction chamber is located at the axis of the rotary switch; The rotary switch is rotatably disposed on one side surface of the cartridge body, and includes a flow guide area and a flow stop area. The flow guide area is provided with a radial flow guide channel. When the rotary switch is rotated, the flow guide channel connects the microchannels in the flow guide area and places the microchannels in the flow stop area in a closed state.

2. The microfluidic nucleic acid detection reagent cartridge according to claim 1, characterized in that: It also includes a drive module and airway, The driving module has a piston and a piston chamber, and the piston chamber is divided into a front piston chamber and a rear piston chamber in the piston movement direction. The air channel includes a first air channel and a second air channel, wherein the first air channel sequentially connects each liquid storage tank, the front piston chamber and the waste liquid storage chamber, and the second air channel connects the rear piston chamber and the reaction chamber. The driving module forms a pressure difference between the first air channel and the second air channel by the movement of the piston in the piston chamber to drive the corresponding liquid.

3. The microfluidic nucleic acid detection reagent cartridge according to claim 2, characterized in that: The piston is selected from any one of a piston rod, a hydraulic piston and a piston ball, and A sealing member is arranged on the piston to isolate the front piston cavity from the rear piston cavity and to isolate the interior of the cartridge body from the external environment.

4. The microfluidic nucleic acid detection reagent cartridge according to claim 2, characterized in that: The piston is a piston rod, the front end of the piston rod is sleeved with an elastic member, and the rear end of the piston rod is provided with a limit member to achieve reciprocating motion of the piston rod in the piston cavity; and / or, The piston is a piston rod, and sealing components are installed on the piston rod at the front and rear end gaps and on the outer peripheral wall separating the front piston cavity and the rear piston cavity.

5. The microfluidic nucleic acid detection reagent cartridge according to claim 4, characterized in that: The elastic member is a spring. When the driving piston rod moves forward, the spring is compressed. When the driving force weakens or withdraws, the piston is reset under the action of the spring restoring force.

6. The microfluidic nucleic acid detection reagent cartridge according to claim 2, characterized in that: The cartridge body is an integrally formed structure, and an air pressure balance hole is provided on the cartridge body corresponding to the piston cavity.

7. The microfluidic nucleic acid detection reagent cartridge according to claim 1, characterized in that: Also included is a sealing layer, which is made of a transparent material; The card box body is an integrally formed structure, and a plurality of microfluidic grooves and a plurality of chambers are provided on one side surface thereof. The sealing layer covers and adheres to the surface of the card box body, so that the microfluidic grooves on the card box body form the microfluidic channels, and seals a plurality of chambers to form the respective independent reaction chambers, amplification chambers and waste liquid storage chambers.

8. The microfluidic nucleic acid detection reagent cartridge according to claim 7, characterized in that: The sealing layer is made of a polymer transparent film and is combined with the card box body by means of thermal bonding, gluing, solvent bonding, laser welding, microwave or ultrasonic bonding.

9. The microfluidic nucleic acid detection reagent cartridge according to claim 1, characterized in that: The liquid storage tanks include a lysate storage tank, a binding liquid storage tank, a plurality of cleaning liquid storage tanks, a rinse liquid storage tank, a sample storage tank and an eluent storage tank, wherein: A magnetic bead placement slot is provided on the microchannel communicating between the sample storage slot and the reaction chamber; The amplification chambers are multiple and spaced apart, and a freeze-dried reagent placement slot is provided on the microchannel connecting the elution chamber and each amplification chamber.

10. The microfluidic nucleic acid detection reagent cartridge according to claim 9, characterized in that: The reaction chamber comprises a lysis chamber and an elution chamber, the reaction chamber is in an arc-shaped structure as a whole, and the lysis chamber and the elution chamber are respectively located at two ends of the arc; There are more than one flow guide channel, which includes a first flow guide channel connecting the lysis chamber with the lysis liquid storage tank, the combination liquid storage tank, multiple cleaning liquid storage tanks, the rinse liquid storage tank, the sample storage tank, and the waste liquid storage chamber, and a second flow guide channel connecting the elution chamber with the elution liquid storage tank and multiple amplification chambers.

11. The microfluidic nucleic acid detection reagent cartridge according to claim 1, characterized in that: The layout area of ​​the amplification chamber adopts a flat design.

12. The microfluidic nucleic acid detection reagent cartridge according to claim 1, characterized in that: The surface of the microfluidic channel is treated with hydrophilicity and hydrophobicity.

13. The microfluidic nucleic acid detection reagent cartridge according to claim 2, characterized in that: The liquid storage tank is arranged on the top of the cartridge body, the reaction chamber, the waste liquid storage chamber and the amplification chamber are arranged from top to bottom on the front of the cartridge body, the microchannel is arranged on one side of the chamber, and the first air channel and the second air channel are arranged on the back of the cartridge body; and / or, The driving module is arranged on the side of the cartridge body; the rotating switch is arranged on the back of the cartridge body, located in the gathering area of ​​each micro-channel.

14. A method for using the microfluidic nucleic acid detection reagent cartridge according to any one of claims 2 to 13, characterized in that: It includes the following steps, performed in order: Sample loading, lysis, binding, multi-stage washing and rinsing, elution and amplification; among them, The multi-stage cleaning and rinsing steps include: adjusting the rotary switch to connect the first cleaning liquid storage tank and the reaction chamber, driving the piston to push the first cleaning liquid into the reaction chamber, mixing the magnetic beads with the first cleaning liquid, and after the external magnetic rod adsorbs the magnetic beads, adjusting the rotary switch to connect the reaction chamber and the waste liquid storage chamber, loosening the piston, discharging the waste liquid, and completing the first-level cleaning; adjusting the rotary switch to connect the second cleaning liquid storage tank and the reaction chamber, repeating the above liquid addition and liquid discharge operations of the reaction chamber, and performing the second-level cleaning; adjusting the rotary switch to connect the third cleaning liquid storage tank and the reaction chamber, repeating the above liquid addition and liquid discharge operations of the reaction chamber, and performing the third-level cleaning; adjusting the rotary switch to connect the rinsing liquid storage tank and the reaction chamber, repeating the above liquid addition and liquid discharge operations of the reaction chamber, and performing rinsing.

15. The method of use according to claim 14, characterized in that: The elution step includes: using a magnetic rod to move the magnetic beads bound to DNA or RNA to the elution chamber, adjusting the rotary switch to connect the elution liquid storage tank and the elution chamber, driving the piston to move in the piston chamber to allow the elution liquid to enter the elution chamber, withdrawing the magnetic rod, mixing the magnetic beads with the elution liquid, and adsorbing the magnetic beads through the external magnetic rod.

16. The method of use according to claim 14, characterized in that: The sample adding step includes: adjusting the rotary switch to connect the sample storage tank and the reaction chamber, driving the piston to move in the piston chamber so that the magnetic beads on the sample mixing path enter the reaction chamber, and resetting the rotary switch and the piston; and / or, The lysis step includes: adjusting a rotary switch to connect the lysis solution storage tank and the lysis chamber, driving a piston to move in the piston chamber so that the lysis solution enters the lysis chamber to lyse the DNA or RNA; and / or, The combining step includes: adjusting a rotary switch to connect the combining liquid storage tank and the reaction chamber, driving a piston to move in the piston chamber to allow the combining liquid to enter the reaction chamber, combining the DNA or RNA with the magnetic beads, adsorbing the magnetic beads by an external magnetic rod, adjusting a rotary switch to connect the reaction chamber and the waste liquid storage chamber, releasing the piston, and discharging the waste liquid; and / or, The amplification step includes: adjusting the rotary switch to connect the amplification chambers in sequence, partially loosening the piston, and delivering the eluent containing DNA or RNA to each amplification chamber in sequence. At the same time, the freeze-dried reagent on the path is dissolved and brought into the amplification chamber, and the amplification chamber is heated and cooled to achieve nucleic acid amplification.

17. Fully automatic testing equipment, characterized in that, include The microfluidic nucleic acid detection reagent cartridge according to any one of claims 1 to 13, and A housing, wherein the microfluidic nucleic acid detection reagent cartridge is detachably placed in the housing; A first control unit, used to control the rotary switch to rotate according to a preset program; A second control unit, used to control the driving device to move according to a preset program; A temperature control module, used to increase or decrease the temperature of the amplification chamber; The detection module is used to detect the optical signal of the amplification cavity.

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