Bacteria DNA sampling chip and sampling method

Abstract

The invention discloses a sampling chip and a sampling method for DNA of bacteria. The chip is composed of a bottom plate, a middle plate and an upper plate which are integrated through thermocompression bonding, a reaction cavity, a liquid storage cavity, a waste liquid cavity and microfluidic channels which are communicated with one another are sequentially arranged on the bottom plate, paraffin valves capable of being controlled by heating are arranged in the channels, and heating pieces are arranged at the bottom of the reaction cavity. According to the chip, a gas source is introduced through a gas inlet, and the steps of sample loading, cell lysis, supernate removal, ethanol cleaning, pneumatic drying, DNA elution and the like are realized in a closed flow path. The method comprises the following steps: loading and sealing a bacterium-containing sample, heating, splitting and combining, discharging supernatant under the driving of a gas source, introducing a cleaning solution, heating and volatilizing residual liquid, injecting an eluent, desorbing DNA at a constant temperature, and finally collecting the eluent. According to the scheme, extraction of bacterial DNA is completed in a single chip, dependence on external equipment such as centrifugation and pipetting is reduced, and a DNA sample which can be directly used for downstream amplification or sequencing analysis is obtained.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of physical / chemical pretreatment and sample preparation of biological samples, and relates to a bacterial DNA sampling chip and a sampling method. BACKGROUND

[0002] At present, the bacterial DNA extraction method commonly used in laboratories is mainly based on the silica gel adsorption principle. Generally, the bacterial sample is mixed with silica particles or solution, DNA is combined with silica gel through incubation, and then the supernatant is removed and washed through multiple centrifugation or pipetting, and finally the purified DNA sample is obtained by eluting the DNA from the silica gel with an eluent. The process generally includes steps such as water bath heating, supernatant removal, ethanol washing, blow-drying and elution, and requires the use of multiple laboratory equipment such as centrifuge, water bath or thermostat, and the transfer between steps and time control are completed manually by the operator.

[0003] This traditional method has the problems of complicated operation process, long experimental period and high labor cost, and multiple pipetting and manual operation can easily introduce cross contamination. In addition, in order to ensure the extraction efficiency and reproducibility, the experience operation of professional technicians and strict environmental control are also needed, which makes it difficult to carry out DNA extraction work in resource-limited or field conditions.

[0004] In recent years, some DNA extraction systems based on automatic devices have been introduced, but most of them still adopt modular or semi-automatic design, usually requiring external liquid pumps, valve control systems or special interfaces, and the device is large in size and high in cost, which is not conducive to portable application or large-scale popularization and use.

[0005] Therefore, there is an urgent need for a fully automatic DNA sampling scheme with higher integration degree, more convenient operation and without complex peripherals, to realize rapid and reliable bacterial genomic DNA extraction, and to provide efficient and convenient sample preparation means for various application scenarios such as clinical diagnosis, environmental monitoring and on-site detection. SUMMARY

[0006] The purpose of the present application is to solve the problems of complicated process, reliance on professional equipment such as centrifuge and pipette, and easy introduction of cross contamination in the existing bacterial DNA sampling method. The present application provides a bacterial DNA sampling chip and a sampling method.

[0007] The technical solution adopted by the present application to solve the technical problems is: a bacterial DNA sampling chip, comprising a bottom plate, a middle plate and an upper plate which are integrally bonded by hot pressing, comprising: The reaction group comprises a reaction cavity, a first micro-fluidic channel and a second micro-fluidic channel, the first micro-fluidic channel and the second micro-fluidic channel are connected at two sides of the reaction cavity respectively, a first paraffin valve is arranged in the first micro-fluidic channel, and a second paraffin valve is arranged in the second micro-fluidic channel; The air inlet group comprises a first air inlet and an air inlet channel arranged on the bottom plate, and the air inlet channel is connected between the first air inlet and the first micro-fluidic channel. The ethanol cleaning group comprises a first liquid storage cavity, a second air inlet and a third micro-fluidic channel arranged on the bottom plate, the second air inlet and the third micro-fluidic channel are connected at two sides of the first liquid storage cavity respectively, the third micro-fluidic channel is connected with the first micro-fluidic channel, and a third paraffin valve is arranged in the third micro-fluidic channel. The elution group comprises a second liquid storage cavity, a third air inlet and a fourth micro-fluidic channel arranged on the bottom plate, the third air inlet and the fourth micro-fluidic channel are connected at two sides of the second liquid storage cavity respectively, the fourth micro-fluidic channel is connected with the first micro-fluidic channel, and a fourth paraffin valve is arranged in the fourth micro-fluidic channel. The first paraffin valve, the second paraffin valve, the third paraffin valve, the fourth paraffin valve and the bottom of the reaction cavity are provided with heating pieces.

[0008] Preferably, the chip further comprises: The waste liquid group comprises a waste liquid cavity and a waste liquid discharge channel arranged on the bottom plate, the waste liquid discharge channel is connected between the second micro-fluidic channel and the waste liquid cavity, a fifth paraffin valve is arranged in a branch at one side of the waste liquid discharge channel, and the bottom of the fifth paraffin valve is provided with a heating piece.

[0009] Preferably, the bottom plate is further provided with a liquid outlet channel and a liquid outlet, the liquid outlet channel is connected between the second micro-fluidic channel and the liquid outlet, a sixth paraffin valve is arranged in the liquid outlet channel, and the bottom of the sixth paraffin valve is provided with a heating piece.

[0010] Preferably, the upper plate is provided with a sample adding port above the reaction cavity.

[0011] Preferably, the reaction cavity is provided with a silicon dioxide adsorption film.

[0012] The application further provides a sampling method based on the bacterial DNA sampling chip, comprising the following steps: S1, sample loading and sealing: injecting the bacterial suspension into the sample adding port, mixing with the silicon dioxide adsorption film pre-placed in the reaction cavity, and then sealing the sample adding port by using a sealing film; S2, cell lysis and DNA binding: the control program starts, the heating plate at the bottom of the reaction chamber is heated to 40 DEG C and kept for 10 min, so that the bacterial cells are lysed, and the released DNA is combined with the silicon dioxide adsorption film; S3, supernatant removal: the program control opens the heating plate at the bottom of the first paraffin valve and the second paraffin valve, so that the first paraffin valve and the second paraffin valve are opened; the gas source enters through the first gas inlet, and the supernatant in the reaction chamber is pushed to the waste liquid chamber through the second microfluidic channel and the waste channel; at this time, the reaction chamber only leaves the silicon dioxide adsorption film containing bacterial DNA; S4, ethanol cleaning: the program control opens the heating plate at the bottom of the third paraffin valve, so that the third paraffin valve is opened; the gas source enters through the second gas inlet, and the ethanol cleaning liquid in the first liquid storage chamber is pushed into the reaction chamber through the third microfluidic channel, and is kept for 3 min, so as to remove the residual impurities; after cleaning, the heating plate at the bottom of the reaction chamber is heated to 60 DEG C, and at the same time, the air is slowly introduced through the first gas inlet, so as to accelerate the volatilization of the residual ethanol on the silicon dioxide adsorption film; S5, DNA elution: the program control opens the heating plate at the bottom of the fourth paraffin valve, so that the fourth paraffin valve is opened; the gas source enters through the third gas inlet, and the eluent in the second liquid storage chamber is pushed into the reaction chamber through the fourth microfluidic channel; at the same time, the heating plate at the bottom of the reaction chamber is heated to 56 DEG C and kept for 5 min, so as to desorb the DNA from the silicon dioxide adsorption film; S6, eluent collection: the program control opens the heating plate at the bottom of the fifth paraffin valve, so that the paraffin in the fifth paraffin valve melts and flows into the waste channel from the branch, so as to close the waste channel; the program control opens the heating plate at the bottom of the sixth paraffin valve, so that the sixth paraffin valve is opened; the gas source enters through the first gas inlet, and the DNA-containing eluent is pushed to the liquid outlet through the liquid outlet channel, and the DNA-containing eluent is collected by the external collector.

[0013] The bacterial DNA sampling chip and the sampling method have the advantages that all the sample loading, lysis and combination, supernatant removal, ethanol cleaning, blow-drying and DNA elution steps are automatically completed in the same closed microfluidic flow path, without the need for traditional equipment such as centrifuge and pipette and complex manual operation, so that the process is greatly simplified, the detection time is shortened to 10-20 minutes, the low sample and low reagent consumption are realized through the preset heating plate temperature control program and paraffin valve isolation control, the high efficiency extraction with high reproducibility is realized, the cross contamination is effectively avoided by relying on the closed gas source, the chip structure is small and the interface is standardized, and the chip is suitable for various on-site rapid detection scenes. BRIEF DESCRIPTION OF DRAWINGS

[0014] The application will be further described below in combination with the drawings and examples.

[0015] Fig. 1is an exploded structural schematic diagram of an embodiment of a bacterial DNA sampling chip of the present application.

[0016] Fig. 2 is a front view schematic diagram of a bottom plate of an embodiment of a bacterial DNA sampling chip of the present application.

[0017] In the figure: 1, bottom plate, 2, middle plate, 3, upper plate, 4, silica adsorption film, 5, first paraffin valve, 6, second paraffin valve, 7, third paraffin valve, 8, fourth paraffin valve, 9, fifth paraffin valve, 10, sixth paraffin valve, 101, reaction cavity, 102, first microfluidic channel, 103, second microfluidic channel, 104, first gas inlet, 105, gas inlet channel, 106, first liquid storage cavity, 107, second gas inlet, 108, third microfluidic channel, 109, second liquid storage cavity, 1010, liquid outlet, 1011, third gas inlet, 1012, fourth microfluidic channel, 1013, waste liquid cavity, 1014, waste discharge channel, 1015, liquid outlet channel. DETAILED DESCRIPTION

[0018] The embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference signs represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0019] In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential" and the like are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation of the present application.

[0020] In addition, the terms "first", "second" and the like are used only for descriptive purposes and should not be construed as indicating or implying relative importance. In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "connected", "connected" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances. In addition, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0021] As shown in Figs. 1-2 The present application provides an embodiment of a bacterial DNA sampling chip, which comprises a bottom plate 1, a middle plate 2 and an upper plate 3 integrally bonded by hot pressing, the hot pressing simplifies the assembly process and ensures the sealing of the flow path, comprising: A reaction group, the reaction group comprises a reaction cavity 101, a first microfluidic channel 102 and a second microfluidic channel 103 opened in the bottom plate 1, a silicon dioxide adsorption film 4 is arranged in the reaction cavity 101, the silicon dioxide adsorption film 4 utilizes its adsorption characteristics to DNA to realize efficient capture of DNA after lysis, and a sample addition port is opened in the upper plate 3 above the reaction cavity 101. The first microfluidic channel 102 and the second microfluidic channel 103 are respectively connected on both sides of the reaction cavity 101, a first paraffin valve 5 is arranged in the first microfluidic channel 102, and a second paraffin valve 6 is arranged in the second microfluidic channel 103.

[0022] An air inlet group, the air inlet group comprises a first air inlet 104 and an air inlet channel 105 opened in the bottom plate 1, and the air inlet channel 105 is connected between the first air inlet 104 and the first microfluidic channel 102; the gas source enters through the first air inlet 104 to push the fluid to move in each step.

[0023] An ethanol cleaning group, the ethanol cleaning group comprises a first liquid storage cavity 106, a second air inlet 107 and a third microfluidic channel 108 opened in the bottom plate 1, the second air inlet 107 and the third microfluidic channel 108 are respectively connected on both sides of the first liquid storage cavity 106, the third microfluidic channel 108 is connected with the first microfluidic channel 102, and a third paraffin valve 7 is arranged in the third microfluidic channel 108. By heating to open the third paraffin valve 7, the ethanol washing liquid is injected into the reaction cavity 101 to remove impurities.

[0024] The elution group comprises a second liquid storage cavity 109, a third gas inlet 1011 and a fourth microfluidic channel 1012 formed in the bottom plate 1. The third gas inlet 1011 and the fourth microfluidic channel 1012 are connected to the two sides of the second liquid storage cavity 109 respectively. The fourth microfluidic channel 1012 is connected to the first microfluidic channel 102. The fourth microfluidic channel 1012 is provided with a fourth paraffin valve 8. The fourth paraffin valve 8 is heated at a set temperature to release the eluent, so as to realize the desorption of DNA from the adsorption film.

[0025] The first paraffin valve 5, the second paraffin valve 6, the third paraffin valve 7, the fourth paraffin valve 8 and the bottom of the reaction cavity 101 are provided with heating sheets. The heating sheets are used to drive the opening / closing of the paraffin valves, and also used to provide the temperature required for lysis, washing and elution.

[0026] The waste liquid group comprises a waste liquid cavity 1013 and a waste liquid discharge channel 1014 formed in the bottom plate 1. The waste liquid discharge channel 1014 is connected between the second microfluidic channel 103 and the waste liquid cavity 1013. A fifth paraffin valve 9 is arranged in a branch of one side of the waste liquid discharge channel 1014. The bottom of the fifth paraffin valve 9 is provided with a heating sheet.

[0027] The bottom plate 1 is also provided with an eluent outlet channel 1015 and an eluent outlet 1010. The eluent outlet channel 1015 is connected between the second microfluidic channel 103 and the eluent outlet 1010. The eluent outlet channel 1015 is provided with a sixth paraffin valve 10. The bottom of the sixth paraffin valve 10 is provided with a heating sheet.

[0028] In the embodiment, after the bottom plate 1, the middle plate 2 and the upper plate 3 are bonded into one body by hot pressing, the bacterial suspension is first injected through the sample injection port of the upper plate 3, so that the bacterial suspension is in contact with the pre-arranged silica adsorption film 4 at the bottom of the reaction cavity 101; then the program is started, the heating sheet at the bottom of the reaction cavity 101 is heated to 40℃ and kept for 10 min, so as to lyse the cells and make the released DNA combine with the adsorption film; then the first paraffin valve 5 and the second paraffin valve 6 are heated to open the flow path, the supernatant is pushed by the gas source through the second microfluidic channel 103 and the waste liquid discharge channel 1014 to the waste liquid cavity 1013 through the first gas inlet 104; then the third paraffin valve 7 is heated, the ethanol washing liquid is injected into the reaction cavity 101 by the gas source through the second gas inlet 107, the bottom of the reaction cavity 101 is heated to 60℃ and slowly aerated to volatilize the residual ethanol after keeping for 3 min; then the fourth paraffin valve 8 and the heating sheet at the bottom of the reaction cavity 101 are heated, the eluent is kept at 56℃ for 5 min to realize the desorption of DNA; finally, the fifth paraffin valve 9 is heated to close the waste liquid discharge channel 1014 and the sixth paraffin valve 10 is heated to open the eluent outlet channel 1015, the gas source pushes the eluent containing DNA to flow out through the eluent outlet channel 1015 and is collected. The whole closed automatic process is completed within 10-20 min, and the bacterial genomic DNA obtained meets the requirements of downstream detection in terms of purity and concentration.

[0029] In this embodiment, each module works closely and cooperatively: the bottom plate 1, the middle plate 2 and the upper plate 3 form a closed flow path through thermal compression bonding, providing a stable microenvironment for the reaction cavity 101, the cleaning cavity and the elution cavity; the heating sheet at the bottom of the reaction cavity 101 cooperates with the silicon adsorption membrane 4 to achieve cell disruption and DNA capture through the 40℃ cracking and adsorption stage; subsequently, the first and second paraffin valves 6 are linked to the first gas inlet 104 under program control and sequentially open the flow path to efficiently remove the supernatant; the third paraffin valve 7 cooperates with the second gas inlet 107 to quantitatively introduce ethanol washing solution, which is volatilized with the help of heating sheet warming and aeration; the fourth paraffin valve is linked to the third gas inlet to accurately push the eluent into the reaction cavity 101, and the heating sheet provides a 56℃ constant temperature environment to promote DNA desorption; finally, the fifth and sixth paraffin valves act in sequence with the waste discharge channel and the liquid outlet channel to ensure that the eluent does not backflow and is quickly discharged. The organic cooperation of this series of valve control, heating and pneumatic units enables the entire closed process to be automatically completed within 10-20 min, reducing reagent and sample consumption and avoiding cross contamination, and the purity and concentration of the obtained DNA fully meet the downstream molecular biology detection requirements.

[0030] The application also provides an embodiment of a sampling method of a bacterial DNA sampling chip, which comprises the following steps: S1, sample loading and sealing: inject the bacterial suspension into the sample loading port, mix with the pre-placed silicon adsorption membrane in the reaction cavity, and then seal the sample loading port with a sealing film to ensure no leakage when the gas source is pressurized and avoid external contamination; S2, cell lysis and DNA binding: control the program to start, heat the heating sheet at the bottom of the reaction cavity to 40℃ and keep for 10 min to lyse the bacterial cells, release the DNA and bind it to the silicon adsorption membrane, achieving synchronous and efficient completion of lysis and adsorption; S3, supernatant removal: program control opens the heating sheet at the bottom of the first paraffin valve and the second paraffin valve, so that the first paraffin valve and the second paraffin valve are opened; the gas source enters through the first gas inlet to push the supernatant in the reaction cavity to the waste liquid cavity through the second microfluidic channel and the waste discharge channel, efficiently separating and removing proteins and cell debris; at this time, only the silicon adsorption membrane containing bacterial DNA remains in the reaction cavity; S4, ethanol cleaning: program control opens the heating sheet at the bottom of the third paraffin valve, so that the third paraffin valve is opened; the gas source enters through the second gas inlet to push the ethanol cleaning solution in the first liquid storage cavity into the reaction cavity through the third microfluidic channel, keeping for 3 min to remove residual impurities; after cleaning, the heating sheet at the bottom of the reaction cavity is heated to 60℃, and at the same time, the gas is slowly aerated through the first gas inlet to accelerate the volatilization of residual ethanol on the silicon adsorption membrane, ensuring the subsequent elution efficiency; S5, DNA elution: the program control opens the heating sheet at the bottom of the fourth paraffin valve, so that the fourth paraffin valve is opened; the gas source enters through the third gas inlet, and the elution solution in the second liquid storage cavity is pushed into the reaction cavity through the fourth microfluidic channel; at the same time, the heating sheet at the bottom of the reaction cavity is heated to 56℃ and kept for 5 min, so that the DNA is desorbed from the silica adsorption membrane, and the DNA is fully desorbed under constant temperature conditions, so that a high-concentration and high-purity DNA solution is obtained; S6, elution solution collection: the program control opens the heating sheet at the bottom of the fifth paraffin valve, so that the paraffin in the fifth paraffin valve melts and flows into the waste discharge channel from the branch, so as to close the waste discharge channel; the program control opens the heating sheet at the bottom of the sixth paraffin valve, so that the sixth paraffin valve is opened; the gas source enters through the first gas inlet, and the elution solution containing DNA is pushed into the liquid outlet channel through the liquid outlet channel and then into the liquid outlet, so as to collect the elution solution containing bacterial DNA by an external collector. The whole closed and automatic process is completed within 10-20 min, and the obtained DNA can be directly used for downstream analysis.

[0031] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0032] Based on the above ideal embodiments according to the present application, through the above description, relevant personnel can make various changes and modifications without deviating from the technical idea of the present application. The technical scope of the present application is not limited to the content in the specification, and must be determined according to the scope of claims.

Claims

1. A DNA sampling chip of bacteria comprising a bottom plate, a middle plate and an upper plate which are integrated by heat-press bonding, characterized in that, The device comprises: a reaction group comprising a reaction cavity, a first microfluidic channel and a second microfluidic channel opened on the bottom plate, the first microfluidic channel and the second microfluidic channel being connected on both sides of the reaction cavity respectively, the first microfluidic channel being provided with a first paraffin valve, and the second microfluidic channel being provided with a second paraffin valve; an air inlet group comprising a first air inlet and an air inlet channel opened on the bottom plate, the air inlet channel being connected between the first air inlet and the first microfluidic channel; an ethanol cleaning group comprising a first liquid storage cavity, a second air inlet and a third microfluidic channel opened on the bottom plate, the second air inlet and the third microfluidic channel being connected on both sides of the first liquid storage cavity respectively, the third microfluidic channel being connected with the first microfluidic channel, and the third microfluidic channel being provided with a third paraffin valve; an elution group comprising a second liquid storage cavity, a third air inlet and a fourth microfluidic channel opened on the bottom plate, the third air inlet and the fourth microfluidic channel being connected on both sides of the second liquid storage cavity respectively, the fourth microfluidic channel being connected with the first microfluidic channel, and the fourth microfluidic channel being provided with a fourth paraffin valve; the first paraffin valve, the second paraffin valve, the third paraffin valve, the fourth paraffin valve and the bottom of the reaction cavity are all provided with heating pieces.

2. The bacterial DNA sampling chip according to claim 1, wherein, Further comprising: a waste liquid group comprising a waste liquid cavity and a waste liquid discharge channel opened on the bottom plate, the waste liquid discharge channel being connected between the second microfluidic channel and the waste liquid cavity, a fifth paraffin valve being arranged in a branch on one side of the waste liquid discharge channel, and the bottom of the fifth paraffin valve being provided with a heating piece.

3. The bacterial DNA sampling chip according to claim 2, characterized in that: an outlet channel and an outlet opening are further opened on the bottom plate, the outlet channel being connected between the second microfluidic channel and the outlet opening, a sixth paraffin valve being arranged in the outlet channel, and the bottom of the sixth paraffin valve being provided with a heating piece.

4. The bacterial DNA sampling chip according to claim 3, characterized in that: a sample inlet is opened on the upper plate above the reaction cavity.

5. The bacterial DNA sampling chip according to claim 4, characterized in that: a silicon dioxide adsorption film is arranged in the reaction cavity.

6. A sampling method of a DNA sampling chip based on the bacterium according to claim 5, characterized by, The device comprises the following steps: S1, sample loading and sealing: injecting a bacterial suspension into the sample inlet, mixing with the silicon dioxide adsorption film pre-placed in the reaction cavity, and then sealing the sample inlet with a sealing film; S2, cell lysis and DNA binding: controlling the program to start, heating the heating piece at the bottom of the reaction cavity to 40 ℃ and keeping for 10 min, so that the bacterial cells are lysed and the released DNA is combined with the silicon dioxide adsorption film; S3, supernatant removal: the program controls the opening of the first paraffin valve and the heating piece at the bottom of the second paraffin valve, so that the first paraffin valve and the second paraffin valve are opened; the air source enters through the first air inlet, and the supernatant in the reaction cavity is pushed to the waste liquid cavity through the second microfluidic channel and the waste liquid discharge channel; At this time, the reaction cavity only remains the silicon dioxide adsorption film containing bacterial DNA; S4, ethanol cleaning: the program control opens the heating piece at the bottom of the third paraffin valve, so that the third paraffin valve is opened; the gas source enters through the second gas inlet, and the ethanol cleaning solution in the first liquid storage cavity is pushed into the reaction cavity through the third microfluidic channel for 3 min to remove residual impurities; after cleaning, the heating piece at the bottom of the reaction cavity is heated to 60 ℃, and at the same time, the first gas inlet is slowly aerated to accelerate the volatilization of residual ethanol on the silica adsorption membrane; S5, DNA elution: the program control opens the heating piece at the bottom of the fourth paraffin valve, so that the fourth paraffin valve is opened; the gas source enters through the third gas inlet, and the eluent in the second liquid storage cavity is pushed into the reaction cavity through the fourth microfluidic channel; at the same time, the heating piece at the bottom of the reaction cavity is heated to 56 ℃ and kept for 5 min to desorb the DNA from the silica adsorption membrane; S6, eluent collection: the program control opens the heating piece at the bottom of the fifth paraffin valve, so that the paraffin in the fifth paraffin valve melts and flows into the waste discharge channel from the branch, thereby closing the waste discharge channel; The program control opens the heating piece at the bottom of the sixth paraffin valve, so that the sixth paraffin valve is opened; the gas source enters through the first gas inlet, and the eluent containing DNA is pushed into the liquid outlet through the liquid outlet channel to the liquid outlet, and the eluent containing bacterial DNA is collected by the external collector.

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