Microfluidic device for mycoplasma pneumoniae detection and use method thereof

Abstract

The invention discloses a centrifugal micro-fluidic device for mycoplasma pneumoniae detection, comprising: a micro-fluidic chip comprising a chip body and at least one detection unit; the chip body is provided with a rotation center; the detection unit comprises a washing liquid chamber; the device comprises a washing liquid chamber, a blasting valve, a sample and magnetic bead liquid chamber, a siphon valve, an eluent chamber and a pneumatic chamber, one end of the blasting valve is communicated with the washing liquid chamber, and the eluent chamber is respectively communicated with one end of the pneumatic chamber and one end of the siphon valve; a magnetic attraction piece is arranged in the mixing chamber, and the other end of the explosion valve, the sample and magnetic bead liquid chamber and the other end of the siphon valve are all communicated with the mixing chamber; the flow dividing valve is communicated with the mixing chamber; the first waste chamber is communicated with the diverter valve; the reaction unit comprises at least one metering chamber, and the at least one reaction chamber and the at least one metering chamber are communicated with the diverter valve. The invention also discloses a use method of the centrifugal microfluidic device. According to the invention, nucleic acid extraction, amplification and detection are integrated, the detection efficiency is high, and the detection cost is low.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a microfluidic device for Mycoplasma pneumoniae detection and a method for using the same. Background Art

[0002] Since the 21st century, a series of infectious disease crises have spurred technological advancements in the health sector and driven structural upgrades in the healthcare industry. The development of portable early diagnostic equipment for public health emergencies has become a major national strategic need. Mycoplasma pneumonia is a contagious respiratory disease that commonly affects children and adolescents. Reportedly, the incidence of mycoplasma pneumonia has been increasing annually, with outpatient visits exceeding 60% in many locations in 2023. The initial clinical manifestations of mycoplasma pneumonia are similar to those of a cold or flu, making it easy to overlook, leading to disease progression and multiple organ damage. Therefore, timely diagnosis is crucial for subsequent treatment and alleviating patient suffering.

[0003] Currently, the main methods for detecting Mycoplasma pneumoniae include culture, serological testing, and pathogen detection. However, these methods each have limitations: culture is time-consuming, and serological testing lacks sensitivity and specificity. Compared with the above two methods, pathogen detection has the advantages of high sensitivity, specificity, and rapidity. Among them, methods such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase isothermal amplification (RPA) further shorten the detection time while retaining high sensitivity and specificity. They are currently widely used detection methods in medicine. However, before the amplification reaction, they require tedious processes such as sample pretreatment and nucleic acid extraction, which require more manual participation and consume more reagents. This not only increases the cost of detection but also increases the burden on medical staff. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a microfluidic device for Mycoplasma pneumoniae detection and a method for using the same.

[0005] In order to achieve the above-mentioned purpose, the technical solution provided by one embodiment of the present invention is as follows:

[0006] A centrifugal microfluidic device for Mycoplasma pneumoniae detection, comprising:

[0007] A microfluidic chip comprising a chip body and at least one detection unit disposed on the chip body, wherein the chip body has a rotation center, and the detection unit comprises:

[0008] a washing liquid chamber, adjacent to the rotation center;

[0009] A bursting valve, a sample and magnetic bead liquid chamber, a siphon valve, an eluent chamber, and a pneumatic chamber are sequentially arranged in a circumferential direction, one end of the bursting valve is connected to a side of the washing liquid chamber away from the rotation center, and the eluent chamber is respectively connected to one end of the siphon valve and the pneumatic chamber;

[0010] A mixing chamber is provided in which a magnetic attraction member is provided, and the other end of the burst valve, the sample and magnetic bead liquid chamber, and the other end of the siphon valve are all connected to a side of the mixing chamber close to the rotation center;

[0011] a diverter valve connected to the other side of the mixing chamber away from the rotation center;

[0012] a first waste chamber connected to the diverter valve;

[0013] The reaction unit includes at least one metering chamber and at least one reaction chamber connected to the at least one metering chamber, and the at least one metering chamber is connected to the diverter valve.

[0014] As a further improvement of the present invention, a first microchannel is connected between the washing liquid chamber and one end of the bursting valve, a second microchannel is connected between the sample and magnetic bead liquid chamber and the mixing chamber, a third microchannel is connected between the pneumatic chamber and the eluent chamber, and a fourth microchannel is connected between the eluent chamber and the siphon valve. The third microchannel and the fourth microchannel converge and are connected to the eluent chamber.

[0015] As a further improvement of the present invention, the width of the first microchannel is 0.2 mm and the depth is 0.2 mm.

[0016] As a further improvement of the present invention, the width of the second microchannel is 0.55 mm, the depth is 2 mm, and the length is 2 mm.

[0017] As a further improvement of the present invention, the width of the third microchannel and the fourth microchannel are both 0.3 mm, and the depth is both 0.3 mm. The width of the siphon valve is 0.3 mm, the depth is 0.3 mm, and the length is 5 mm.

[0018] As a further improvement of the present invention, the difference between the minimum radial distance from the rotation center to the siphon valve and the minimum radial distance from the rotation center to the eluent chamber is 0-4 mm.

[0019] As a further improvement of the present invention, a delayed release structure is connected between the mixing chamber and the diverter valve.

[0020] As a further improvement of the present invention, the delayed-release structure is formed by folding a microchannel, wherein the microchannel has a width of 0.2 mm, a depth of 0.2 mm, and a total length of 120 mm.

[0021] As a further improvement of the present invention, the magnetic attraction member includes a magnet sheet, and the magnet sheet has a length of 6 mm, a width of 4 mm, and a thickness of 0.5 mm.

[0022] A method for using a microfluidic device for Mycoplasma pneumoniae detection, using the centrifugal microfluidic device, comprises the following steps:

[0023] (1) adding the sample to the sample and magnetic bead liquid chamber, and waiting for the magnetic beads to specifically bind to the Mycoplasma pneumoniae DNA;

[0024] (2) Rotate counterclockwise to allow the sample and magnetic bead mixture to enter the mixing chamber, wait until the magnetic beads are attracted by the magnetic element, and then increase the rotation speed counterclockwise to allow the waste liquid to enter the first waste chamber under the action of the diverter valve;

[0025] (3) Continue to rotate counterclockwise and increase the speed, so that the washing liquid in the washing liquid chamber breaks through the capillary pressure barrier of the burst valve and enters the mixing chamber, so that the washing liquid flushes the magnetic beads adsorbed by the magnetic element in the mixing chamber, and the waste liquid after flushing enters the first waste chamber through the diverter valve;

[0026] (4) Then rotate clockwise to force the eluent in the eluent chamber into the pneumatic chamber;

[0027] (5) Rotate clockwise again and reduce the speed to expand the air in the pneumatic chamber, and the eluent breaks through the siphon valve and enters the mixing chamber;

[0028] (6) The eluent carries away the DNA on the magnetic beads in the mixing chamber, and under the action of the diverter valve, the eluent carries the DNA into the metering chamber;

[0029] (7) Then, the clockwise rotation continues and the speed increases, and the DNA enters the reaction chamber and reacts with the reagents in the reaction chamber.

[0030] The beneficial effects of the present invention are:

[0031] (1) The washing solution, magnetic bead solution, eluent and reagents of the present invention are all pre-loaded in the microfluidic chip, thereby simplifying the operation process. In addition, the present invention prevents the liquid and reagents from contacting the external environment, avoiding contamination and evaporation.

[0032] (2) The present invention cooperates with the burst valve, siphon valve, pneumatic chamber, eluent chamber and diverter valve, and does not require additional driving equipment. The valve can be opened by simply adjusting the rotation speed of the microfluidic chip, which is beneficial to the integration and miniaturization of the microfluidic chip.

[0033] (3) The present invention forms a nucleic acid extraction structure by cooperating the sample with the magnetic bead liquid chamber, the mixing chamber and the elution liquid chamber, thereby achieving the adsorption of nucleic acids by magnetic beads, the adsorption of magnetic beads with nucleic acids by magnetic elements and the flushing of nucleic acids on magnetic beads by elution liquid. There is no need for separate nucleic acid purification treatment, thus saving manpower and material costs.

[0034] (4) The microfluidic device of the present invention can precisely control fluids in a tiny space, achieving efficient mixing and reaction of samples and reagents, thereby significantly reducing the amount of samples and reagents used. At the same time, the microfluidic device can integrate multiple functional units such as nucleic acid extraction, amplification, and detection into one, realizing integrated rapid detection. It has the characteristics of high throughput, rapidity, and high sensitivity, high detection efficiency, and low detection cost, which significantly alleviates the work pressure of medical workers and provides strong technical support for clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 Schematic diagram of the structure of a microfluidic chip according to a preferred embodiment of the present invention;

[0037] Figure 2 Schematic diagram of the structure of a single detection unit in a preferred embodiment of the present invention;

[0038] Figure 3 A schematic diagram of the release principle of a centrifugal pneumatic valve according to a preferred embodiment of the present invention;

[0039] Figure 4 A flow chart of liquid release according to a preferred embodiment of the present invention;

[0040] Figure 5 A detection flow chart of a preferred embodiment of the present invention;

[0041] In the figure: 1. Microfluidic chip, 2. Chip body, 21. Rotation center, 3. Detection unit, 30. Washing liquid chamber, 31. Burst valve, 32. Sample and magnetic bead liquid chamber, 33. Siphon valve, 34. Eluent chamber, 35. Pneumatic chamber, 36. Mixing chamber, 37. Diverter valve, 38. First waste chamber, 39. Reaction unit, 391. Metering chamber, 392. Reaction chamber, 41. First microchannel, 42. Second microchannel, 43. Third microchannel, 44. Fourth microchannel, 5. Delayed release structure, 51. Microchannel. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0043] See also Figure 1 、 Figure 2 The embodiment of the present application discloses a centrifugal microfluidic device for detecting Mycoplasma pneumoniae, comprising a microfluidic chip 1. The microfluidic chip 1 comprises a chip body 2, at least one detection unit 3 arranged on the chip body 2, and the chip body 2 has a rotation center 21. The detection unit 3 comprises a washing liquid chamber 30, a bursting valve 31, a sample and magnetic bead liquid chamber 32, a siphon valve 33, an eluent chamber 34, a pneumatic chamber 35, a mixing chamber 36, a diverter valve 37, a first waste chamber 38 and a reaction unit 39. The washing liquid chamber 34 is close to the rotation center 21, and the washing liquid chamber 34 is pre-encapsulated with washing liquid for washing impurities on the magnetic beads. The bursting valve 31, the sample and magnetic bead liquid chamber 32, the siphon valve 33, the eluent chamber 34 and the pneumatic chamber 35 are arranged in sequence along the circumferential direction. One end of the bursting valve 31 is connected to the side of the washing liquid chamber 34 away from the rotation center 21, and the eluent chamber 34 is respectively connected to one end of the siphon valve 33 and the pneumatic chamber 35. A magnetic element (not shown) is provided in the mixing chamber 36. The magnetic element attracts the DNA-bound magnetic beads released from the sample and magnetic bead liquid chamber 32, while the remaining liquid can pass through unimpeded. The other end of the burst valve 31, the sample and magnetic bead liquid chamber 32, and the other end of the siphon valve 33 are all connected to the side of the mixing chamber 36 close to the rotation center 21. The diverter valve 37 is connected to the other side of the mixing chamber 36 away from the rotation center 21. The first waste chamber 38 is connected to the diverter valve 37. The reaction unit 39 includes at least one metering chamber 391 and at least one reaction chamber 392 connected to the at least one metering chamber 391. The at least one metering chamber 391 is connected to the diverter valve 37.

[0044] The chip body 2 is preferably made of PMMA, a transparent material with a diameter of 130 mm and a thickness of 3 mm. The chip body 2 includes a bottom plate and a top plate. The wash liquid chamber 30, burst valve 31, sample and magnetic bead liquid chamber 32, siphon valve 33, eluent chamber 34, pneumatic chamber 35, mixing chamber 36, diverter valve 37, first waste chamber 38, and reaction unit 39 are all disposed on the bottom plate. Wash liquid is pre-installed in the wash liquid chamber 30, magnetic bead liquid is pre-installed in the sample and magnetic bead liquid chamber 32, eluent is pre-installed in the eluent chamber 34, and reagents are pre-installed in the reaction chamber 392 of the reaction unit 39. Finally, the top plate and bottom plate are bonded together.

[0045] To facilitate the rotation of the microfluidic chip 1, the chip body 2 can be connected to a rotation drive mechanism, which drives the chip body 2 to rotate about the rotation center 21. The rotation drive mechanism can be a motor, or a combination of a motor and a turntable. It is understood that the rotation drive mechanism is not limited to the above structure, and other structures that can achieve rotation of the chip body 2 are also acceptable and are not limited here.

[0046] A first microchannel 41 is connected between the washing liquid chamber 30 and one end of the burst valve 31, a second microchannel 42 is connected between the sample and magnetic bead liquid chamber 32 and the mixing chamber 36, a third microchannel 43 is connected between the pneumatic chamber 35 and the eluent chamber 34, and a fourth microchannel 44 is connected between the eluent chamber 34 and the siphon valve 33. The third microchannel 43 and the fourth microchannel 44 converge and are connected to the eluent chamber 34.

[0047] The first microchannel 41 has a width of 0.2 mm and a depth of 0.2 mm. The washing liquid chamber 30 is pre-filled with 250 microliters of anhydrous ethanol.

[0048] The second microchannel 42 has a width of 0.55 mm, a depth of 2 mm, and a length of 2 mm. The sample and magnetic bead liquid chamber 32 is pre-packaged with a magnetic bead liquid. During testing, the sample is injected into the sample and magnetic bead liquid chamber 32 through a sample injection hole (not shown). The sample and magnetic bead liquid mix to achieve specific binding between the DNA and the magnetic beads. The magnetic bead liquid can be set to 190 microliters. The sample injection hole can be opened on the top plate to correspond to the sample and magnetic bead liquid chamber 32.

[0049] The width of the third microchannel 43 and the depth of the fourth microchannel 44 are both 0.3 mm, and the siphon valve 33 has a width of 0.3 mm, a depth of 0.3 mm, and a length of 5 mm.

[0050] The eluent chamber 34 is pre-packed with eluent. Siphon valve 33 works in conjunction with pneumatic chamber 35 to control the release of eluent from the eluent chamber 34 by varying the air pressure within the pneumatic chamber 35, thereby eluting the DNA bound to the magnetic beads. Specifically, 70 μL of DNA eluent is pre-packed in the eluent chamber 34.

[0051] See also Figure 3As shown, the release of eluent occurs in two stages: the first is a high-speed rotation stage, where a large centrifugal force presses the liquid into the pneumatic chamber 35, compressing the air in the pneumatic chamber 35. The second is a low-speed rotation stage, where the centrifugal force weakens, causing the gas in the pneumatic chamber 35 to expand, pushing the liquid into the siphon valve 33, triggering the siphon effect and completing the release of the liquid. The position of the siphon valve 33 plays a crucial role in the release of the eluent. If the siphon valve 33 is too far from the rotation center 21, during high-speed operation, the liquid can easily reach the siphon apex and break through the siphon apex, triggering the siphon effect and completing the release prematurely. If the siphon valve 33 is too close to the rotation center 21, during the deceleration stage, the gas in the pneumatic chamber 35 expands, and the force generated may not be sufficient to push the liquid past the siphon apex, thus failing to trigger the siphon effect. The present invention preferably has a minimum radial distance d1 between the rotation center 21 and the siphon valve 33 and a minimum radial distance d2 between the rotation center 21 and the eluent chamber 34 of 0-4 mm, thereby achieving stable release of the eluent. It is further preferred that the difference between the minimum radial distance d1 from the rotation center 21 to the siphon valve 33 and the minimum radial distance d2 from the rotation center 21 to the eluent chamber 34 is 1 mm.

[0052] In the present invention, a delayed-release structure 5 is preferably connected between the mixing chamber 36 and the diverter valve 37. When liquid flows into the delayed-release structure 5, its release rate is significantly slowed, thereby ensuring that the liquid in the mixing chamber 36 is slowly released and that there is sufficient reaction time for the reaction to proceed fully. Specifically, the delayed-release structure 5 is formed by folding a microchannel 51. The microchannel 51 has a width of 0.2 mm, a depth of 0.2 mm, and a total length of 120 mm. The head end of the microchannel 51 is connected to the mixing chamber 36, and the tail end of the microchannel 51 is connected to the diverter valve 37.

[0053] In an embodiment of the present invention, the magnetic attraction member includes a magnet sheet having a length of 6 mm, a width of 4 mm, and a thickness of 0.5 mm.

[0054] Preferably, the washing liquid chamber 30 is in a fan-shaped shape, so that the washing liquid in the washing liquid chamber 30 can be easily released under the action of centrifugal force.

[0055] The diverter valve 37 is preferably a Coriolis valve connected to the tail end of the delayed-release structure 8. It is used to divert waste liquid and purified DNA. The Coriolis valve ensures a relatively stable diversion effect, with waste liquid flowing into the first waste chamber 38 and purified DNA flowing into the metering chamber 391. The metering chamber 391 is used to temporarily store the purified DNA, ensuring that the amplification reaction of the purified DNA after entering the reaction chamber 392 can proceed simultaneously. A fifth microchannel 45 is preferably connected between the metering chamber 391 and the reaction chamber 392. The fifth microchannel 45 has a width of 0.2 mm and a depth of 0.2 mm. The reaction chamber 392 is pre-packaged with a fluorescent reagent for constant-temperature detection of Mycoplasma pneumoniae. The recombinase, single-stranded DNA binding protein, and DNA polymerase in the reaction system use specific primers to perform a rapid nucleic acid amplification reaction. As the amplified product accumulates, the specific molecular probe releases a fluorescent signal under the action of the exonuclease. Using a fluorescence detection device, the amplification process of the target fragment can be monitored in real time. When the purified DNA in the metering chamber 391 flows into the reaction chamber 392, the amplification reaction begins. The metering chamber 391 at the tail end is also connected to a second waste chamber 393, which is used to collect impurities such as excess eluent, nucleic acids and proteins.

[0056] Except for the sample, the rest of the liquids and reagents are pre-loaded in the microfluidic chip 1. Therefore, when using, it is only necessary to add the sample into the sample and magnetic bead liquid chamber 3. Figure 4 As shown, wait for the magnetic beads to specifically bind to the Mycoplasma pneumoniae DNA, then start centrifugation, rotate counterclockwise to allow the sample and magnetic bead solution to enter the mixing chamber 36, wait for 30 seconds, the small magnetic beads are attracted by the micro-magnetic sheet, continue to rotate counterclockwise and increase the speed, so that the waste liquid passes through the delayed release structure 5 and enters the first waste chamber 38 under the action of the Coriolis force valve, then rotate counterclockwise and increase the speed, so that the anhydrous ethanol in the washing liquid chamber 30 breaks through the capillary pressure barrier of the burst valve 31 and enters the mixing chamber 36, so that the anhydrous ethanol flushes the small magnetic beads attracted by the micro-magnetic sheet in the mixing chamber 36, and the flushed waste liquid The liquid passes through the delayed-release mechanism 5 and enters the first waste chamber 38 under the action of the Coriolis force valve. It then rotates clockwise, forcing the elution liquid in the eluent chamber 34 into the pneumatic chamber 35. Further clockwise rotation and reduced speed cause the air in the pneumatic chamber 35 to expand, allowing the eluent to break through the siphon valve 33 and enter the mixing chamber 36, carrying away the DNA on the small magnetic beads in the mixing chamber 36. It then enters the delayed-release mechanism 5. Under the action of the Coriolis force valve, the eluent carries the DNA into the metering chamber 391. Then, clockwise rotation and increased speed allow the DNA to enter the reaction chamber 392 and react with a constant-temperature amplification fluorescent reagent. This microfluidic chip can be used to detect Mycoplasma pneumoniae. Based on these test results, clinicians can use them to assist in diagnosing Mycoplasma pneumoniae by integrating them with other clinical information, such as the patient's medical history, physical signs, and in vivo tests.

[0057] like Figure 5 As shown, the embodiment of the present application also discloses a method for using a microfluidic device for Mycoplasma pneumoniae detection, using the above-mentioned centrifugal microfluidic device, comprising the following steps:

[0058] S1: adding the sample to the sample and magnetic bead liquid chamber 32, and waiting for the magnetic beads to specifically bind to the Mycoplasma pneumoniae DNA;

[0059] S2: Rotate counterclockwise to allow the sample and magnetic bead mixture to enter the mixing chamber 36, wait for the magnetic beads to be attracted by the magnetic element, and then increase the speed counterclockwise to allow the waste liquid to enter the first waste chamber 38 under the action of the diverter valve 37;

[0060] S3: Continue to rotate counterclockwise and increase the speed, so that the washing liquid in the washing liquid chamber 30 breaks through the capillary pressure barrier of the burst valve 31 and enters the mixing chamber 36, so that the washing liquid flushes the magnetic beads attracted by the magnetic element in the mixing chamber 36, and the waste liquid after flushing enters the first waste chamber 38 through the diverter valve 37;

[0061] S4: Then, the valve rotates clockwise to allow the eluent in the eluent chamber 34 to flow into the pneumatic chamber 35;

[0062] S5: Rotate clockwise again and reduce the speed to expand the air in the pneumatic chamber 35, and the eluent breaks through the siphon valve 33 and enters the mixing chamber 36;

[0063] S6: The eluent carries away the DNA on the magnetic beads in the mixing chamber 36. Under the action of the diverter valve 37, the eluent carries the DNA into the metering chamber 391.

[0064] S7: Then, the clockwise rotation is continued and the speed is increased, and the DNA enters the reaction chamber 392 and reacts with the reagents in the reaction chamber 392.

[0065] To further illustrate the method for using the microfluidic device for Mycoplasma pneumoniae detection of the present invention, as a preferred embodiment, the method includes the following steps:

[0066] (1) Add the sample to the sample and magnetic bead liquid chamber 32 and wait for 2 minutes to allow the magnetic beads to specifically bind to the Mycoplasma pneumoniae DNA.

[0067] (2) Rotate counterclockwise at 800 rpm to allow the sample and magnetic bead mixture to enter the mixing chamber 36. Wait for 30 seconds until the magnetic beads are attracted by the magnet. Increase the rotation speed counterclockwise to allow the waste liquid to enter the first waste chamber 38 under the action of the Coriolis force valve.

[0068] (3) Continue to rotate counterclockwise and increase the speed to 2000 rpm, so that the anhydrous ethanol in the washing liquid chamber 30 breaks through the capillary pressure barrier of the burst valve 31 and enters the mixing chamber 36, so that the anhydrous ethanol flushes the magnetic beads adsorbed by the magnet sheet in the mixing chamber 36, and the waste liquid after flushing enters the first waste chamber 38 through the Coriolis force valve.

[0069] (4) Then rotate clockwise at a speed of 3000 rpm to allow the eluent in the eluent chamber 34 to flow into the pneumatic chamber 35.

[0070] (5) Rotate clockwise again and reduce the speed to 1000 rpm, so that the air in the pneumatic chamber 35 expands, and the DNA eluate breaks through the siphon valve 33 and enters the mixing chamber 36.

[0071] (6) The DNA elution liquid carries away the DNA on the magnetic beads in the mixing chamber 36 , and under the action of the Coriolis force valve, the DNA elution liquid carries the DNA into the metering chamber 391 .

[0072] (7) Then, the rotation speed is increased clockwise to 3000 rpm, and the DNA enters the reaction chamber 392 and reacts with the constant temperature amplification fluorescent reagent in the reaction chamber 392.

[0073] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0074] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A centrifugal microfluidic device for Mycoplasma pneumoniae detection, characterized in that: include: A microfluidic chip comprising a chip body and at least one detection unit disposed on the chip body, wherein the chip body has a rotation center, and the detection unit comprises: a washing liquid chamber, adjacent to the rotation center; A bursting valve, a sample and magnetic bead liquid chamber, a siphon valve, an eluent chamber, and a pneumatic chamber are sequentially arranged in a circumferential direction, one end of the bursting valve is connected to a side of the washing liquid chamber away from the rotation center, and the eluent chamber is respectively connected to one end of the siphon valve and the pneumatic chamber; A mixing chamber is provided in which a magnetic attraction member is provided, and the other end of the burst valve, the sample and magnetic bead liquid chamber, and the other end of the siphon valve are all connected to a side of the mixing chamber close to the rotation center; a diverter valve connected to the other side of the mixing chamber away from the rotation center; a first waste chamber connected to the diverter valve; The reaction unit includes at least one metering chamber and at least one reaction chamber connected to the at least one metering chamber, and the at least one metering chamber is connected to the diverter valve.

2. The centrifugal microfluidic device for Mycoplasma pneumoniae detection according to claim 1, characterized in that: A first microchannel is connected between the washing liquid chamber and one end of the burst valve, a second microchannel is connected between the sample and magnetic bead liquid chamber and the mixing chamber, a third microchannel is connected between the pneumatic chamber and the eluent chamber, and a fourth microchannel is connected between the eluent chamber and the siphon valve. The third microchannel and the fourth microchannel converge and are connected to the eluent chamber.

3. The centrifugal microfluidic device for Mycoplasma pneumoniae detection according to claim 2, characterized in that: The first microchannel has a width of 0.2 mm and a depth of 0.2 mm.

4. The centrifugal microfluidic device for Mycoplasma pneumoniae detection according to claim 2, characterized in that: The second microchannel has a width of 0.55 mm, a depth of 2 mm, and a length of 2 mm.

5. The centrifugal microfluidic device for Mycoplasma pneumoniae detection according to claim 2, characterized in that: The width of the third microchannel and the fourth microchannel are both 0.3 mm and the depth is both 0.3 mm. The width of the siphon valve is 0.3 mm, the depth is 0.3 mm and the length is 5 mm.

6. The centrifugal microfluidic device for Mycoplasma pneumoniae detection according to claim 1, characterized in that: The difference between the minimum radial distance from the rotation center to the siphon valve and the minimum radial distance from the rotation center to the eluent chamber is 0-4 mm.

7. The microfluidic device for Mycoplasma pneumoniae detection according to claim 1, characterized in that: A delayed release structure is communicated between the mixing chamber and the diverter valve.

8. The microfluidic device for Mycoplasma pneumoniae detection according to claim 7, characterized in that: The delayed-release structure is formed by folding a microchannel, wherein the microchannel has a width of 0.2 mm, a depth of 0.2 mm, and a total length of 120 mm.

9. The microfluidic device for Mycoplasma pneumoniae detection according to claim 1, characterized in that: The magnetic attraction member includes a magnet sheet with a length of 6 mm, a width of 4 mm, and a thickness of 0.5 mm.

10. A method for using a microfluidic device for Mycoplasma pneumoniae detection, characterized by using the centrifugal microfluidic device according to any one of claims 1 to 9, comprising the following steps: (1) adding the sample to the sample and magnetic bead liquid chamber, and waiting for the magnetic beads to specifically bind to the Mycoplasma pneumoniae DNA; (2) Rotate counterclockwise to allow the sample and magnetic bead mixture to enter the mixing chamber, wait until the magnetic beads are attracted by the magnetic element, and then increase the rotation speed counterclockwise to allow the waste liquid to enter the first waste chamber under the action of the diverter valve; (3) Continue to rotate counterclockwise and increase the speed, so that the washing liquid in the washing liquid chamber breaks through the capillary pressure barrier of the burst valve and enters the mixing chamber, so that the washing liquid flushes the magnetic beads adsorbed by the magnetic element in the mixing chamber, and the waste liquid after flushing enters the first waste chamber through the diverter valve; (4) Then, clockwise, the eluent in the eluent chamber is forced into the pneumatic chamber; (5) Rotate clockwise again and reduce the speed to expand the air in the pneumatic chamber, and the eluent breaks through the siphon valve and enters the mixing chamber; (6) The eluent carries away the DNA on the magnetic beads in the mixing chamber, and under the action of the diverter valve, the eluent carries the DNA into the metering chamber; (7) Then, the clockwise rotation continues and the speed increases, and the DNA enters the reaction chamber and reacts with the reagents in the reaction chamber.

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