In-vitro diagnosis detection method, storage medium and system

Abstract

The invention discloses an in-vitro diagnosis detection method using a micro-fluidic chip, a storage medium and a system.The in-vitro diagnosis detection method comprises the steps that the micro-fluidic chip is provided and comprises a chip bottom shell, a first covering film and a second covering film, a sample adding cavity, an extraction cavity, a detection reagent storage module, a waste liquid cavity and a plurality of amplification cavities of the chip bottom shell are all arranged on a substrate, and the chip bottom shell is an integrally formed part; injecting a to-be-detected sample liquid into the extraction cavity through the sample adding cavity; the extraction reagent, the washing liquid reagent and the elution liquid reagent in the detection reagent storage module are injected into the extraction cavity in batches so as to act with the to-be-detected sample, waste liquid obtained after action is discharged into the waste liquid cavity, and obtained elution liquid containing target detection substances is injected into the multiple amplification cavities; and starting the detection module to detect the mixed liquid in the plurality of amplification cavities. The in-vitro diagnosis detection method, the storage medium and the system are simple to operate and high in detection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic testing technology, and in particular to an in vitro diagnostic testing method, storage medium and system. Background Art

[0002] Microfluidic chips are called "laboratories on a chip". They have the characteristics of small size, low composition, high efficiency, automation, and integration. They are integrated detection chips that can integrate nucleic acid extraction, purification, elution, and detection on a small chip with a complex fluid operation system and functional unit modules. However, if nucleic acid extraction and amplification are to be achieved on microfluidic chips, most of the microfluidic chips contain complex liquid circuits and control valve designs. For example, some microfluidic chips are equipped with a rotary diversion valve, a drive module, and multiple modules such as a sample chamber, a lysate storage chamber, a cleaning liquid storage chamber, an eluate storage chamber, and a waste liquid chamber. When in use, the rotary diversion valve needs to be frequently switched; some microfluidic chips set the mixing chamber and the waste liquid chamber as elastic membrane pressing chambers, and drive the movement of liquids between chambers by multiple pressing. However, the structures of these microfluidic chips are relatively complex and the operation and control are relatively complex, and the detection efficiency is not high enough. Summary of the invention

[0003] The object of the present invention is to overcome the above-mentioned technical problems and provide an in vitro diagnostic detection method, a storage medium and a system, which are simple to operate and have high detection efficiency.

[0004] In order to achieve the above object, the present invention provides an in vitro diagnostic detection method using a microfluidic chip, the in vitro diagnostic detection method comprising:

[0005] S1. Provide a microfluidic chip, which includes a chip bottom shell and a first covering film and a second covering film welded on both sides of the chip bottom shell. The chip bottom shell includes a substrate, a sample loading chamber, an extraction chamber, a detection reagent storage module, a waste liquid chamber and a plurality of amplification chambers. The sample loading chamber, the extraction chamber, the detection reagent storage module, the waste liquid chamber and the plurality of amplification chambers are all arranged on the substrate. The sample loading chamber, the detection reagent storage module, the waste liquid chamber and the plurality of amplification chambers are respectively connected with the extraction chamber through microchannels on the substrate. The chip bottom shell is an integrally formed part. The first covering film and the second covering film are used to cover the microchannel and part of the cavity on the substrate. The detection reagent storage module is provided with an extraction reagent, a washing liquid reagent and an elution liquid reagent, and the amplification chamber is provided with a freeze-dried detection reagent ball;

[0006] S2, injecting the sample liquid to be tested into the extraction chamber through the sample injection chamber;

[0007] S3, injecting the extraction reagent, washing liquid reagent and elution liquid reagent in the detection reagent storage module into the extraction chamber in batches to react with the sample to be tested, discharging the waste liquid after the reaction into the waste liquid chamber respectively, and injecting the obtained eluate containing the target detection substance into multiple amplification chambers;

[0008] S4. Start the fluorescent PCR module to detect the mixed solutions in the multiple amplification chambers.

[0009] Optionally, along the longitudinal direction of the plate surface of the substrate, the sample loading chamber may be located above the extraction chamber in the longitudinal direction, the extraction chamber is connected with an extraction chamber exhaust channel capable of exhausting to the outside, the extraction chamber exhaust channel comprises an extraction chamber external exhaust hole, an extraction chamber exhaust channel and an extraction chamber internal exhaust hole, the extraction chamber exhaust channel is concavely formed on the second plate surface, the extraction chamber external exhaust hole and the extraction chamber internal exhaust hole are both arranged on the substrate along the thickness direction of the substrate, along the longitudinal direction of the plate surface of the substrate, the extraction chamber external exhaust hole is located above the extraction chamber in the longitudinal direction, the extraction chamber internal exhaust hole is arranged on the cavity bottom wall at the longitudinal upper part of the extraction chamber along the thickness direction of the substrate, the extraction chamber, the extraction chamber internal exhaust hole, the extraction chamber exhaust channel and the extraction chamber external exhaust hole are connected in sequence, the sample loading chamber is provided with a sample loading port, and the microfluidic chip further comprises a sample loading sealing plug, the sample loading sealing plug is used to cover the sample loading port and can make piston movement in the sample loading chamber;

[0010] Wherein, the S2 includes:

[0011] S21, lay the microfluidic chip flat so that the sample injection port faces upward, and add the sample to be tested from the sample injection port;

[0012] S22, insert the sample adding sealing plug into the sample adding port and place the microfluidic chip vertically along the longitudinal direction of the substrate at the detection position, so that the sample liquid to be tested in the sample adding cavity flows downward into the extraction cavity.

[0013] Further, the detection reagent storage module may include a plurality of columnar storage cavities that can cooperate with the piston cover and communicate with the extraction chamber, the axes of the plurality of columnar storage cavities are parallel to the plate surface of the substrate, the piston cover cooperation openings of the plurality of columnar storage cavities are arranged on the substrate thickness surface of the substrate, the substrate thickness surface is perpendicular to the plate surface of the substrate, and the microfluidic chip also includes a plurality of piston covers, and the plurality of piston covers are sealed in the plurality of columnar storage cavities in a one-to-one manner;

[0014] Wherein, the S3 includes:

[0015] S31, starting the piston drive motor to push the piston of the columnar storage chamber storing the extraction reagent to move, so as to inject the extraction reagent into the extraction chamber;

[0016] S32, starting the ultrasonic instrument to perform ultrasonic lysis on the sample liquid to be tested in the extraction chamber;

[0017] S33, turning off the ultrasonic instrument, matching the magnetic suction instrument with the extraction chamber, and discharging the waste liquid in the extraction chamber into the waste liquid chamber;

[0018] S34, starting the piston drive motor to push the piston of the columnar storage chamber storing the washing liquid reagent to move, and injecting the washing liquid reagent into the extraction chamber;

[0019] S35, removing the magnetic suction instrument and starting the ultrasonic instrument to wash the substance in the extraction chamber;

[0020] S36, turning off the ultrasonic instrument, matching the magnetic suction instrument with the extraction chamber, and discharging the waste liquid in the extraction chamber into the waste liquid chamber;

[0021] S37, starting the piston drive motor to push the piston of the columnar storage chamber storing the elution liquid reagent to move, and injecting the elution liquid reagent into the extraction chamber;

[0022] S38, removing the magnetic suction instrument and starting the ultrasonic instrument to elute the substance in the extraction chamber;

[0023] S39, turn off the ultrasonic instrument, match the magnetic suction instrument with the extraction chamber, and discharge the eluate containing the target detection substance in the extraction chamber into the amplification chamber.

[0024] Furthermore, the microfluidic chip may further include an upper cover, which is arranged on the bottom shell of the chip and is provided with a sample addition hole and an ultrasonic hole, the sample addition hole is arranged in alignment with the sample addition cavity, and the ultrasonic hole is arranged in alignment with the extraction cavity; wherein the starting ultrasonic instrument in S3 includes:

[0025] After the ultrasonic instrument is matched with the extraction cavity through the ultrasonic hole, the ultrasonic instrument is turned on.

[0026] In addition, the waste liquid chamber is connected with a waste liquid chamber exhaust channel capable of exhausting air to the outside, and the multiple amplification chambers are connected with an amplification chamber exhaust channel capable of exhausting air to the outside. The extraction chamber can be connected with the waste liquid chamber through a waste liquid chamber microchannel, and a waste liquid chamber valve capable of controlling the conduction or closure of the channel is provided on the waste liquid chamber microchannel. The extraction chamber is connected with the multiple amplification chambers through an amplification chamber microchannel, and the amplification chamber microchannel is provided with an amplification chamber valve capable of controlling the conduction or closure of the channel.

[0027] Wherein, in S3, matching the magnetic suction instrument with the extraction chamber and discharging the waste liquid in the extraction chamber into the waste liquid chamber comprises:

[0028] Match the magnetic suction instrument with the extraction chamber;

[0029] The amplification chamber valve is controlled to remain closed, and the waste liquid chamber valve is controlled to open, so that the waste liquid in the extraction chamber is discharged into the waste liquid chamber.

[0030] In addition, the step of cooperating the magnetic suction instrument with the extraction chamber and discharging the eluate containing the target detection substance in the extraction chamber into the amplification chamber in S3 may include:

[0031] Match the magnetic suction instrument with the extraction chamber;

[0032] The waste liquid chamber valve is controlled to remain closed, and the amplification chamber valve is controlled to open, so as to discharge the eluent containing the target detection substance in the extraction chamber into the amplification chamber.

[0033] In some embodiments, the waste liquid chamber microfluidic channel and the amplification chamber microfluidic channel may be both concavely formed on the second plate surface, a waste liquid chamber microfluidic channel cut-off portion for cutting off the flow channel is integrally provided on the waste liquid chamber microfluidic channel, the waste liquid chamber flow channel cut-off portion and the second covering film form the waste liquid chamber valve, an amplification chamber microfluidic channel cut-off portion for cutting off the flow channel is integrally provided on the amplification chamber microfluidic channel, the amplification chamber microfluidic channel cut-off portion and the second covering film form the amplification chamber valve, and the waste liquid chamber valve and the amplification chamber valve are formed as normally open film valves;

[0034] Among them, at the detection position in S22, the waste liquid chamber valve and the amplification chamber valve can be closed by pressing the second film on the waste liquid chamber flow channel cutting part and the amplification chamber microchannel cutting part through the cutting part driving motor.

[0035] In other embodiments, a plurality of amplification chambers may be connected to the extraction chamber via an amplification chamber microchannel, the amplification chamber microchannel includes an amplification chamber main channel and a plurality of amplification chamber branch channels, the first end of the amplification chamber main channel is connected to the extraction chamber, the first ends of the plurality of amplification chamber branch channels are connected to the plurality of amplification chambers one by one, the second ends of the plurality of amplification chamber branch channels are connected to the second end of the amplification chamber main channel, the amplification chamber exhaust channel includes a plurality of amplification chamber exhaust microchannels and a plurality of amplification chamber exhaust holes provided on the plurality of amplification chamber exhaust microchannels, the plurality of amplification chamber exhaust microchannels are connected to the plurality of amplification chambers one by one, the amplification chamber exhaust holes are provided on the substrate along the thickness direction of the substrate, the chip bottom shell also includes a plurality of wax valve chambers provided on the substrate, each amplification chamber branch channel and each amplification chamber exhaust microchannel are connected to a wax valve chamber, paraffin is installed in the wax valve chamber, and the extraction chamber, the plurality of wax valve chambers, the plurality of amplification chamber exhaust holes and the plurality of amplification chambers are arranged in sequence from top to bottom along the longitudinal direction of the board surface of the substrate;

[0036] Wherein, after S9 and before S4, S3 may further include:

[0037] The heating instrument is started to heat the multiple wax valve chambers, so that the paraffin in the multiple wax valve chambers melts and flows downward into the multiple amplification chamber branch channels and the multiple amplification chamber exhaust microchannels accordingly, so as to close the multiple amplification chambers.

[0038] The present invention also provides a computer-readable storage medium, which includes a program or an instruction. When the program or the instruction is executed, the above method is implemented.

[0039] The present invention also provides an in vitro diagnostic detection system using a microfluidic chip, and the in vitro diagnostic detection system includes the above-mentioned computer-readable storage medium.

[0040] The invention discloses an in vitro diagnostic detection method, storage medium and system using a microfluidic chip. The in vitro diagnostic detection method, storage medium and system include: providing a microfluidic chip, the microfluidic chip including a chip bottom shell and a first coating and a second coating welded on both sides of the chip bottom shell, the sample loading chamber, extraction chamber, detection reagent storage module, waste liquid chamber and multiple amplification chambers of the chip bottom shell are all arranged on a substrate, and the chip bottom shell is an integrally formed part. The first coating and the second coating are used to cover the microchannel and part of the cavity on the substrate, the detection reagent storage module is equipped with an extraction reagent, a washing liquid reagent and an elution liquid reagent, and the amplification chamber is equipped with a freeze-dried detection reagent ball; injecting the sample liquid to be tested into the extraction chamber through the sample loading chamber; injecting the extraction reagent, the washing liquid reagent and the elution liquid reagent in the detection reagent storage module into the extraction chamber in batches to act on the sample to be tested, respectively discharging the waste liquid after the action into the waste liquid chamber, and injecting the obtained elution containing the target detection substance into multiple amplification chambers; starting the fluorescent PCR module to detect the mixed liquid in the multiple amplification chambers. Among them, the sample adding chamber, the detection reagent storage module, the waste liquid chamber and the multiple amplification chambers are respectively connected to the extraction chamber through the microfluidic channels on the substrate, and the extraction chamber is directly connected to other chambers through the microfluidic channels. The flow channel layout is simple, and the chip bottom shell is an integrated molded part. The microfluidic chip does not need to be provided with a control valve with a complex structure, and the structure is simple and reasonable. In this way, the in vitro diagnostic detection method, storage medium and system using the microfluidic chip are simple to operate and have high detection efficiency. In addition, the in vitro diagnostic detection method, storage medium and system of the present invention can be used with a microfluidic chip in conjunction with an instrument to achieve a detection with a personnel operation time of less than one minute, sample input and result output, and high detection efficiency.

[0041] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0043] Figure 1 A method flow chart of an in vitro diagnostic detection method according to a specific embodiment of the present invention is shown;

[0044] Figure 2 for Figure 1 Specific method flow chart of step S2 in FIG.

[0045] Figure 3 for Figure 1Specific method flow chart of step S3 in FIG.

[0046] Figure 4 An exploded view of the installation of a microfluidic chip according to a specific embodiment of the present invention is shown;

[0047] Figure 5 for Figure 4 A stereoscopic view of the sample injection sealing plug in FIG.

[0048] Figure 6 for Figure 4 A three-dimensional diagram of the upper cover in FIG.

[0049] Figure 7 for Figure 4 A three-dimensional diagram of a chip bottom shell in FIG.

[0050] Figure 8 Shows Figure 7 a first board surface of the chip bottom shell;

[0051] Fig. 9 Shows Figure 7 The second board surface of the chip bottom shell.

[0052] Description of Reference Numerals

[0053] 100 chip bottom shell 101 substrate

[0054] 1011 first board surface 1012 second board surface

[0055] 1013 substrate thickness surface 102 sample loading chamber

[0056] 1021 sample chamber outlet hole 1022 sample chamber outlet microchannel

[0057] 103 extraction chamber 1031 extraction chamber outer exhaust hole

[0058] 1032 Extraction chamber exhaust channel 1033 Extraction chamber exhaust hole

[0059] 1034 Extraction chamber drainage hole 1035 Eluent discharge port

[0060] 1036 Extraction chamber liquid inlet 1037 First reagent liquid inlet

[0061] 1038 Second reagent inlet hole 1071 Wax outlet hole

[0062] 104 detection reagent storage module 1041 piston cover matching opening

[0063] 1042 first storage cavity 1043 second storage cavity

[0064] 1044 first flow channel 1045 second flow channel

[0065] 1046 First branch channel cut-off portion 1047 Second branch channel cut-off portion

[0066] 105 waste liquid chamber 1051 exhaust hole outside waste liquid chamber

[0067] 1052 exhaust channel of waste liquid chamber 1053 exhaust hole in waste liquid chamber

[0068] 1054 waste liquid chamber inlet hole 1055 waste liquid chamber microchannel

[0069] 1056 Waste liquid chamber microchannel cut-off part 106 Amplification chamber

[0070] 1061 Amplification chamber microchannel 10611 Amplification chamber main channel

[0071] 10612 Amplification chamber branch channel 1062 Amplification chamber microchannel cut-off part

[0072] 1063 Amplification chamber exhaust microchannel 1064 Amplification chamber exhaust hole

[0073] 1065 amplification chamber branch channel cut-off part 107 wax valve chamber

[0074] 108 Positioning column 200 Sample filling sealing plug

[0075] 300 cover 301 sample hole

[0076] 302 Ultrasonic hole 400 Piston cover

[0077] 303 positioning pin DETAILED DESCRIPTION

[0078] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0079] In the present invention, unless otherwise specified, directional words such as "up, down, top, bottom" are usually used with reference to the directions shown in the drawings or are used to describe the relative positional relationships of components in the vertical, perpendicular or gravity direction.

[0080] The present application provides an in vitro diagnostic detection method using a microfluidic chip, which is simple to operate and has high detection efficiency. In addition, the in vitro diagnostic detection method and system using a microfluidic chip of the present invention can use a microfluidic chip in conjunction with an instrument to achieve multiple fluorescent PCR detection with less than one minute of human operation time, sample input and result output, and high detection efficiency.

[0081] Specifically, Figure 1 As shown, the in vitro diagnostic test method comprises:

[0082] Step S1: Provide a microfluidic chip 1000, the fluidic chip 1000 includes a chip bottom shell 100 and a first cover film and a second cover film welded on both sides of the chip bottom shell 100, the chip bottom shell 100 includes a substrate 101, a sample loading chamber 102, an extraction chamber 103, a detection reagent storage module 104, a waste liquid chamber 105 and a plurality of amplification chambers 106, the sample loading chamber 102, the extraction chamber 103, the detection reagent storage module 104, the waste liquid chamber 105 and the plurality of amplification chambers 106 are all arranged on the substrate On the board 101, the sample loading chamber 102, the detection reagent storage module 104, the waste liquid chamber 105 and the plurality of amplification chambers 106 are respectively connected to the extraction chamber 103 through the microchannels on the substrate 101, the chip bottom shell 100 is an integrally formed part, the first coating and the second coating are used to cover the microchannels and part of the chamber on the substrate 101, the detection reagent storage module 104 is equipped with extraction reagents, washing liquid reagents and elution liquid reagents, and the amplification chamber 106 is equipped with amplification freeze-dried detection reagent balls;

[0083] Step S2: injecting the sample liquid to be tested into the extraction chamber 103 through the sample injection chamber 102;

[0084] Step S3: Inject the extraction reagent, washing liquid reagent and elution liquid reagent in the detection reagent storage module 104 into the extraction chamber 103 in batches to react with the sample to be tested, discharge the waste liquid after the reaction into the waste liquid chamber 105 respectively, and inject the obtained eluate containing the target detection substance into the multiple amplification chambers 106;

[0085] Step S4: Start the detection module to detect the mixed solution in the multiple amplification chambers 106.

[0086] That is Figure 4 , Figures 7 to 9 As shown, the sample adding chamber 102, the detection reagent storage module 104, the waste liquid chamber 105 and the multiple amplification chambers 106 are respectively connected to the extraction chamber 103 through the microchannels on the substrate 101, and the extraction chamber 103 is directly connected to other chambers through the microchannels. The flow channel layout is simple, and the chip bottom shell 100 is an integrally formed part, and there is no need to set a control valve with a complex structure. In this way, the in vitro diagnostic detection method, storage medium and system using the microfluidic chip are simple to operate and have high detection efficiency. In addition, the in vitro diagnostic detection method, storage medium and system of the present invention can be used with a microfluidic chip in conjunction with an instrument to achieve a detection with a personnel operation time of less than one minute, sample input and result output, and high detection efficiency. And the chip bottom shell 100 is an integrally formed part, with a simple and reasonable structure, low requirements for design and material use, and low manufacturing cost.

[0087] Among them, the in vitro diagnostic detection method can be used for nucleic acid extraction fluorescent PCR detection, and can also be used for protein quantitative detection, etc. When the in vitro diagnostic detection method is used for nucleic acid extraction fluorescent PCR detection, the target detection substance is nucleic acid, the detection module is a fluorescent PCR module, and the amplification freeze-dried detection reagent ball is a nucleic acid amplification freeze-dried detection reagent ball. The following will specifically describe the in vitro diagnostic method for nucleic acid extraction fluorescent PCR detection.

[0088] The chip bottom shell 100 and the microfluidic chip 1000 of the present invention are provided with a plurality of amplification chambers 106, such as Figure 4 , Figures 7 to 9 As shown, the chip bottom shell 100 and the microfluidic chip 1000 of the present invention have 8 amplification chambers 106. Of course, the present invention is not limited thereto. Based on the reasonable layout of the flow channel and the chamber of the present invention, the number of amplification chambers 106 can also be set to more, such as 10, 12, etc. The chip bottom shell 100 and the microfluidic chip 1000 of the present invention can be pre-buried with liquid reagents for magnetic bead nucleic acid extraction and freeze-dried reagents for nucleic acid amplification. One consumable is used, and the sample is added once. The operation time of the personnel is less than one minute. The nucleic acid extraction and amplification process of sample input and result output can be completed with the instrument, and the fluorescent PCR detection of up to 32 targets can be realized, and the detection efficiency is high.

[0089] Alternatively, if Figures 7 to 9 As shown, the substrate 101 may include a first plate surface 1011 and a second plate surface 1012 that are arranged opposite to each other along the thickness direction of the substrate, the sample loading chamber 102, the extraction chamber 103, the detection reagent storage module 104, and the waste liquid chamber 105 are all arranged on the first plate surface 1011, and the microchannel and the plurality of amplification chambers 106 are arranged on the second plate surface 1012. In this way, the chamber arrangement and the channel arrangement on the chip bottom shell 100 are more reasonable, which is more conducive to the further miniaturization of the chip bottom shell 100.

[0090] Furthermore, if Figures 7 to 9 As shown, the sample loading chamber 102, the extraction chamber 103 and the waste liquid chamber 105 can all be open at the top, and the bottom walls of the sample loading chamber 102, the extraction chamber 103 and the waste liquid chamber 105 are arranged on the first plate surface 1011, and the surrounding walls of the sample loading chamber 102, the extraction chamber 103 and the waste liquid chamber 105 are all convexly formed on the first plate surface 1011, and the plurality of amplification chambers 106 are open and concavely formed on the second plate surface 1012. In this way, the chamber arrangement and the flow channel arrangement on the chip bottom shell 100 are more reasonable, the manufacturing materials and weight of the chip bottom shell 100 can be reduced, the manufacturing cost is low, and it is also more conducive to the further miniaturization and lightweight setting of the chip bottom shell 100, and the space utilization rate is high when stacked, which is convenient for transportation and storage.

[0091] Optionally, along the longitudinal direction of the plate surface of the substrate 101, the sample loading chamber 102 is located above the extraction chamber 103 in the longitudinal direction, and the extraction chamber 103 is connected to an extraction chamber exhaust channel capable of exhausting air to the outside, and the extraction chamber exhaust channel includes an extraction chamber external exhaust hole 1031, an extraction chamber exhaust channel 1032, and an extraction chamber internal exhaust hole 1033. The extraction chamber exhaust channel 1032 is concavely formed on the second plate surface 1012, and the extraction chamber external exhaust hole 1031 and the extraction chamber internal exhaust hole 1033 are both arranged on the substrate 101 through the thickness direction of the substrate. Along the longitudinal direction of the plate surface of the substrate, the extraction chamber external exhaust hole 1031 is located above the extraction chamber 103 in the longitudinal direction. The extraction chamber internal exhaust hole 1033 is arranged on the bottom wall of the chamber body in the longitudinal upper part of the extraction chamber 103 through the thickness direction of the substrate, and the extraction chamber 103, the extraction chamber internal exhaust hole 1033, the extraction chamber exhaust channel 1032, and the extraction chamber external exhaust hole 1031 are connected in sequence, and the sample loading chamber 102 is provided with a sample loading port. Figure 4 and Figure 5 As shown, the microfluidic chip 1000 also includes a sample loading sealing plug 200, which is used to cover the sample loading port and can perform piston movement in the sample loading chamber 102. Figure 2 As shown, step S2 may specifically include:

[0092] Place the microfluidic chip 1000 flatly so that the sample injection port faces upward, and add the sample to be tested from the sample injection port;

[0093] Insert the sample adding sealing plug 200 into the sample adding port and place the microfluidic chip 1000 vertically in the longitudinal direction of the substrate at the detection position. That is, the amplification chamber 106 is placed vertically into the detection instrument in a downward direction, so that the sample liquid to be tested in the sample adding chamber 102 flows downward into the extraction chamber 103. That is, the user uses a quantitative sample adding tool to add the sample liquid into the sample adding chamber 102, plugs the sample adding sealing plug 200, and vertically places the microfluidic chip 1000. The liquid flows vertically into the extraction chamber 103 along the sample adding chamber outlet microchannel 1022 under the action of gravity and extrusion.

[0094] In this way, since the extraction chamber 103 of the present application is provided with an exhaust channel, after the sample liquid is added to the sample loading chamber 102, the sample liquid can be injected into the extraction chamber 103 by plugging the sample loading sealing plug 200, utilizing the positive extrusion of the liquid under gravity and the pressurized sample, without the need for other chambers to provide negative pressure. The microfluidic chip of the present application has a simple structure and low manufacturing cost, can use one consumable, and load samples at one time. The operation time of personnel is less than one minute, the operation is convenient, and the user experience is good. Among them, when the microfluidic chip 1000 is laid flat, the first plate surface faces upward, the second top surface faces downward, and the sample loading port faces upward; when the microfluidic chip 1000 is placed vertically, the sample loading chamber 12 is located at the top, and the amplification chamber 16 is located at the bottom.

[0095] Further, the detection reagent storage module 104 includes a plurality of columnar storage chambers that can cooperate with the piston cover 400 and communicate with the extraction chamber 103, the axes of the plurality of columnar storage chambers are parallel to the board surface of the substrate 101, the piston covers of the plurality of columnar storage chambers cooperate with the opening 1041 arranged on the substrate thickness surface of the substrate 101, the substrate thickness surface 1013 is perpendicular to the board surface of the substrate 101, and the microfluidic chip 1000 also includes a plurality of piston covers, and the plurality of piston covers 400 are sealed in the plurality of columnar storage chambers one by one. The axes of the plurality of columnar storage chambers of the present application are arranged laterally along the board surface of the substrate 101, the structure is reasonable and simple, and it is easy to manufacture, and the space utilization rate is high when stacked, and it is easy to transport and store. The columnar storage chamber is used to pre-set the reagent vesicles. Since the reagent is pre-set in the columnar storage chamber through the vesicles, the liquid is squeezed out by the piston cover only when it is used, and the reagent has good sealing performance, which effectively avoids the contamination of the reagent, and the liquid is squeezed out by the piston, and the reagent supply is more accurate and quantitative, and the detection result is also more accurate and reliable. In addition, the reagents are pre-set in the columnar storage cavity in the form of vesicles and can be replaced in a timely manner according to actual detection needs, with better versatility and scalability.

[0096] Furthermore, the plurality of columnar storage chambers include a plurality of first storage chambers 1042 and a second storage chamber 1043. The plurality of first storage chambers 1042 are connected to the extraction chamber 103 through the first flow channel 1044 and the first reagent inlet hole 1037. The first flow channel includes a first main flow channel and a plurality of first branch flow channels. The first ends of the plurality of first branch flow channels are connected to the plurality of first storage chambers 1042 one by one, and the first main flow channel is connected between the extraction chamber 103 and the second ends of the plurality of first branch flow channels. The second storage chamber 1043 is connected to the extraction chamber 103 through the second flow channel 1045 and the second reagent inlet hole 1038. The plurality of first branch flow channels are provided with a first branch flow channel cutting portion 1046 for cutting off the flow channel, and the second flow channel 1045 is provided with a second flow channel cutting portion 1047 for cutting off the flow channel. Optionally, one end of the plunger cover 400 is a column pressure end, and the other end is provided with a piston rod connecting portion that can be connected to the piston rod.

[0097] In some embodiments, the first storage chamber 1042 can be provided with a certain number of reserved spaces according to the actual number required to meet different types of testing requirements. Figures 7 to 9As shown, the detection reagent storage module 104 includes five first storage chambers 1042 and one second storage chamber 1043. Specifically, the reagents required for nucleic acid extraction can be pre-packed into the first storage chamber 1042 in the form of vesicles, and the eluent is placed in the second storage chamber 1043, and then the 400 plunger is inserted into the piston cover of the multiple columnar storage chambers to cooperate with the opening 1041, and a tearable dustproof paper film is affixed to the surface to complete the packaging. After packaging, the liquid reagent is sealed by the flow channel membrane valve formed by the piston cover 400 and the first branch flow channel cutting part 1046 and the second branch flow channel cutting part 1047. During the experiment, the test instrument pushes a specific plunger through a screw motor to squeeze the vesicle to break it, and the liquid reagent breaks through the membrane valve and flows into the extraction chamber 103.

[0098] Among them, Figure 3 As shown, step S3 may specifically include:

[0099] S31: starting the piston drive motor to push the piston of the columnar storage chamber storing the extraction reagent to move, so as to inject the extraction reagent into the extraction chamber 103;

[0100] S32: Start the ultrasonic instrument to perform ultrasonic lysis on the sample liquid to be tested in the extraction chamber 103;

[0101] S33: Turn off the ultrasonic instrument, match the magnetic suction instrument with the extraction chamber 103, and discharge the waste liquid in the extraction chamber 103 into the waste liquid chamber 105;

[0102] S34: starting the piston drive motor to push the piston cover 400 of the columnar storage chamber storing the washing liquid reagent to move, and injecting the washing liquid reagent into the extraction chamber;

[0103] S35: remove the magnetic suction instrument and start the ultrasonic instrument to wash the substance in the extraction chamber 103;

[0104] S36: Turn off the ultrasonic instrument, match the magnetic suction instrument with the extraction chamber 103, and discharge the waste liquid in the extraction chamber 103 into the waste liquid chamber 105;

[0105] S37: Start the piston drive motor to push the piston cover 400 of the columnar storage chamber storing the elution liquid reagent to move, and inject the elution liquid reagent into the extraction chamber 103;

[0106] S38: remove the magnetic suction instrument and start the ultrasonic instrument to elute the substance in the extraction chamber 103;

[0107] S39: Turn off the ultrasonic instrument, match the magnetic suction instrument with the extraction chamber 103, and discharge the elution liquid containing nucleic acid in the extraction chamber 103 into the amplification chamber 106.

[0108] Specifically, the extraction chamber 103 is a chamber for nucleic acid extraction, which is connected to the sample chamber 102 through the sample chamber outlet microchannel 1022. Due to the existence of the extraction chamber exhaust channel, after the user adds the sample and closes the cover, the sample solution is squeezed by the sample sealing plug 200 and flows downward into the extraction chamber 103; through the motor control on the detection instrument, the extraction reagent enters the extraction chamber 103 through the first flow channel 1044, and cooperates with the ultrasonic, magnetic, and heating modules on the instrument to achieve ultrasonic, mixing, heating and other nucleic acid extraction steps. During the extraction process, the extraction chamber 103 leads to the waste liquid chamber 105, the flow channel membrane valve of the extraction chamber 103, the waste liquid chamber valve, and the amplification chamber valve are pressed by the instrument motor to ensure that the liquid does not flow down. When discharging waste liquid, the motor of the waste liquid chamber valve is released. Due to the existence of the waste liquid chamber exhaust channel, the waste liquid flows downward into the waste liquid chamber 105. After the nucleic acid extraction is completed, the eluent enters the extraction chamber 103 through the second flow channel 1045. After the elution process is completed, the motor of the amplification chamber valve is released, and the eluent containing nucleic acid breaks through the membrane valve formed by the amplification chamber branch channel cutting part 1065 and flows into the amplification chamber 106.

[0109] Optionally, the extraction chamber 103 is arranged adjacent to and shares part of the chamber wall with the detection reagent storage module 104. In this way, the chamber arrangement and flow channel arrangement on the chip bottom shell 100 are more reasonable, the manufacturing material and weight of the chip bottom shell 100 can be reduced, the manufacturing cost is low, and it is also more conducive to further miniaturization and lightweight setting of the chip bottom shell 100.

[0110] like Figure 4 and Figure 6 As shown, the microfluidic chip 1000 further includes an upper cover 300, which is covered on the chip bottom shell 100 and is provided with a sample addition hole 301 and an ultrasonic hole 302, the sample addition hole 301 is aligned with the sample addition cavity 302, and the ultrasonic hole 301 is aligned with the extraction cavity 302. Among them, starting the ultrasonic instrument in S3 may include: after the ultrasonic instrument is matched with the extraction cavity 103 through the ultrasonic hole 302, the ultrasonic instrument is turned on.

[0111] In addition, along the longitudinal direction of the board surface of the substrate 101, the sample loading chamber 102, the extraction chamber 103 and the waste liquid chamber 105 are arranged in sequence from top to bottom, and a plurality of amplification chambers 106 are located below the longitudinal direction of the extraction chamber 103. The sample loading chamber 102 and the extraction chamber 103 are arranged side by side with the detection reagent storage module 104 along the board surface of the substrate 101. In this way, the structural arrangement of the microfluidic chip of the present application can be made simpler and more reasonable, and the transfer of liquid between chambers can be assisted by gravity, which is convenient to operate and has good performance. The sample loading sealing plug 200 and the piston cover 400 can be made of elastic rubber or silicone material, and the upper cover 300 is a plastic shell, and the upper surface can be attached or printed with information such as chip identification mark. The chip bottom shell 100 is a colorless transparent plastic chip bottom shell, and both sides are covered with transparent pp film by ultrasonic welding. The upper cover 300 and the chip bottom shell 100 can be connected and fixed by positioning columns 108 and positioning pins 303.

[0112] Optionally, the waste liquid chamber 105 is connected with a waste liquid chamber exhaust channel capable of exhausting gas to the outside, and the waste liquid chamber exhaust channel includes an exhaust hole 1051 outside the waste liquid chamber, an exhaust channel 1052 inside the waste liquid chamber, and an exhaust hole 1053 inside the waste liquid chamber. Along the longitudinal direction of the plate surface of the substrate 101, the exhaust hole 1051 outside the waste liquid chamber is arranged on the substrate 101 along the thickness direction of the substrate and is located above the longitudinal direction of the waste liquid chamber 105. The exhaust hole 1053 inside the waste liquid chamber is arranged on the bottom wall of the cavity of the substrate 101 and the waste liquid chamber 105 along the thickness direction of the substrate, and the exhaust channel 1052 of the waste liquid chamber is formed concavely on the second plate surface 1012, and the waste liquid chamber 105, the exhaust hole 1053 inside the waste liquid chamber, the exhaust channel 1052 of the waste liquid chamber, and the exhaust hole 1051 outside the waste liquid chamber are connected in sequence. In this way, the microfluidic chip of the present application does not require additional power, and the liquid can be transferred between the extraction chamber 103 and the waste liquid chamber 105 only by gravity, which is convenient for users to use. The exhaust channel is integrally formed on the chip bottom shell 100, and the structure is simple and reasonable.

[0113] Furthermore, the extraction chamber 103 is connected to the waste liquid chamber 105 through the waste liquid chamber microchannel 1055, the second plate surface 1012 can be recessed to form the waste liquid chamber microchannel 1055, the extraction chamber 103 is provided with an extraction chamber drainage hole 1034 that penetrates the substrate 101 and the bottom wall of the extraction chamber 103 along the thickness direction of the substrate, the waste liquid chamber 105 is provided with a waste liquid chamber inlet hole 1054 that penetrates the substrate 101 and the bottom wall of the waste liquid chamber 105 along the thickness direction of the substrate, and the extraction chamber 103 is connected to the waste liquid chamber 105 through the extraction chamber drainage hole 1034, the waste liquid chamber microchannel 1055 and the waste liquid chamber inlet hole 1054 in sequence.

[0114] In some embodiments, the extraction chamber 103 can be connected to the waste liquid chamber 105 through a waste liquid chamber microchannel 1055, and a waste liquid chamber valve capable of controlling the conduction or closure of the channel is provided on the waste liquid chamber microchannel 1055. The waste liquid chamber microchannel 1055 is formed concave on the second plate surface 1012, and a waste liquid chamber microchannel cut-off portion 1056 for cutting off the channel is integrally provided on the waste liquid chamber microchannel 1055, and the waste liquid chamber channel cut-off portion 1056 and the second covering film form a waste liquid chamber valve.

[0115] Optionally, multiple amplification chambers 106 may be arranged side by side along the board surface of the substrate 101 in a horizontal direction, and the multiple amplification chambers 106 are connected to an amplification chamber exhaust channel capable of exhausting air to the outside, and the amplification chamber exhaust channel includes multiple amplification chamber exhaust microchannels 1063 and multiple amplification chamber exhaust holes 1064 provided on the multiple amplification chamber exhaust microchannels 1063. Along the longitudinal direction of the board surface of the substrate, multiple amplification chamber exhaust holes 1064 are arranged on the substrate 101 along the thickness direction of the substrate and are located longitudinally above the multiple amplification chambers 106, and the multiple amplification chambers 106, the multiple amplification chamber exhaust microchannels 1063, and the multiple amplification chamber exhaust holes 1064 are connected in sequence one by one. The microfluidic chip 1000 may also include a waterproof and breathable membrane, which covers the amplification chamber exhaust holes 1064 located on the first board surface 1011.

[0116] In some embodiments, the extraction chamber 103 is connected to a plurality of amplification chambers 106 through an amplification chamber microchannel 1061, which is formed concavely on the second plate surface 1012, and is provided with an amplification chamber valve capable of controlling the conduction or closure of the channel. The amplification chamber microchannels 1061 are all formed concavely on the second plate surface 1012, and an amplification chamber microchannel cut-off portion 1062 for cutting off the channel is integrally provided on the amplification chamber microchannel 1061, and the amplification chamber microchannel cut-off portion 1062 and the second covering film form an amplification chamber valve.

[0117] Further, the plurality of amplification chambers 106 are connected to the extraction chamber 103 through the amplification chamber microchannel 1061. The amplification chamber microchannel 1061 may include an amplification chamber main channel 10611 and a plurality of amplification chamber branch channels 10612. The first end of the amplification chamber main channel 10611 is connected to the extraction chamber 103. The first ends of the plurality of amplification chamber branch channels 10612 are connected to the plurality of amplification chambers 106 one by one respectively. The second ends of the plurality of amplification chamber branch channels 10612 are connected to the second end of the amplification chamber main channel 10611. The amplification chamber microchannel cut-off portion 1062 is provided on the amplification chamber main channel 10611. An amplification chamber branch channel cut-off portion 1065 for cutting off the channel is integrally provided on each amplification chamber branch channel 10612. Each amplification chamber branch channel cut-off portion 1065 forms an amplification chamber branch channel valve with the second covering film.

[0118] Furthermore, the extraction chamber 103 may be provided with an eluent discharge hole 1035 that penetrates the bottom wall of the chamber and the substrate 101 along the thickness direction of the substrate. The eluent discharge hole 1035 is located in the longitudinal lower part of the extraction chamber 103, and the amplification chamber main channel 10611 is connected to the extraction chamber 103 through the eluent discharge hole 1035.

[0119] Among them, the waste liquid chamber valve and the amplification chamber valve are formed as normally open membrane valves. At the detection position in S22, the microfluidic chip 1000 is placed at the detection position, and the waste liquid chamber valve and the amplification chamber valve can be closed by pressing the second film on the waste liquid chamber flow channel cutting part 1056 and the amplification chamber microchannel cutting part 1062 through the cutting part driving motor.

[0120] Optionally, the step of matching the magnetic suction instrument with the extraction chamber 103 and discharging the waste liquid in the extraction chamber 103 into the waste liquid chamber 105 in S3 may include:

[0121] Matching the magnetic suction instrument with the extraction chamber 103;

[0122] The amplification chamber valve is controlled to remain closed, and the waste liquid chamber valve is controlled to open, so as to discharge the waste liquid in the extraction chamber 103 into the waste liquid chamber 105 .

[0123] In addition, the step of cooperating the magnetic suction instrument with the extraction chamber 103 and discharging the eluate containing the nucleic acid in the extraction chamber 103 into the amplification chamber 106 in S3 may include:

[0124] Matching the magnetic suction instrument with the extraction chamber 103;

[0125] The waste liquid chamber valve is controlled to remain closed, and the amplification chamber valve is controlled to open, so as to discharge the elution liquid containing nucleic acid in the extraction chamber 103 into the amplification chamber 106 .

[0126] Specifically, the instrument motor pushes the piston cover 400, so that the pre-packaged vesicle extraction reagent (lysis solution, proteinase K and magnetic bead mixed solution) in the first storage chamber 1042 is squeezed along the first flow channel 1044 into the extraction chamber 103, and cooperates with the ultrasonic instrument to perform the ultrasonic lysis process. Then, in cooperation with the instrument magnetic suction module, the magnetic beads adsorb nucleic acids and stay on the wall of the extraction chamber 103, and the motor pressing the waste liquid chamber microchannel cut-off part 1056 is moved away, and the waste liquid enters the waste liquid chamber 105 along the waste liquid chamber microchannel 1055, and the membrane valve of the waste liquid chamber microchannel cut-off part 1056 is re-sealed; then the instrument motor pushes the piston cover 400, so that the pre-packaged vesicle washing liquid reagent in the first storage chamber 1042 is squeezed along the first flow channel 1044 into the extraction chamber 103, and the instrument magnetic suction module is moved away, ultrasonic mixing is performed, and the magnetic beads adsorbed with nucleic acids are washed; the instrument magnetic suction module is restarted, pressing the waste liquid chamber microchannel cut-off part 1056 The motor is moved away, and the washing waste liquid enters the waste liquid chamber 105 along the waste liquid chamber microchannel 1055, and the membrane valve of the waste liquid chamber microchannel cut-off part 1056 is re-sealed; the instrument motor pushes the piston cover 400, so that the pre-packaged vesicle elution liquid reagent in the second storage chamber 1043 is squeezed and enters the extraction chamber 103 along the second channel 1045, and is ultrasonically mixed. The instrument magnetic suction module is restarted, and the motor pressing the amplification chamber microchannel cut-off part 1062 is moved away, and the eluent containing nucleic acid enters the amplification chamber main channel 10611, breaks through the membrane valve of the amplification chamber branch channel cut-off part 1065, and is injected into the amplification chamber 106 from the bottom along the amplification chamber branch channel 10612.

[0127] Optionally, the chip bottom shell 100 may further include a plurality of wax valve chambers 107 disposed on the substrate 101, wherein the plurality of wax valve chambers 107 are integrally formed on the substrate 101, the wax valve chambers 107 are open at the top and the bottom wall of the chamber is disposed on the first plate surface 1011. The waste liquid chamber 105 is located between the extraction chamber 103 and the wax valve chamber 107 and extends from one end to the other end of the plate surface of the substrate 101 in the horizontal direction. The plurality of wax valve chambers 107 and the plurality of amplification chambers 106 are arranged side by side in the horizontal direction of the plate surface of the substrate 101.

[0128] The middle of the flow channel of each amplification chamber branch channel 10612 and the end of the flow channel of each amplification chamber exhaust microchannel 1063 are respectively connected to a wax valve chamber 107, and paraffin is installed in the wax valve chamber 107. Along the longitudinal direction of the plate surface of the substrate 101, the extraction chamber 103, multiple wax valve chambers 107, multiple amplification chamber exhaust holes 1064 and multiple amplification chambers 106 are arranged in sequence from top to bottom. The end of the flow channel of the amplification chamber exhaust microchannel 1063 extends upward along the longitudinal direction of the plate surface of the substrate 101, and the amplification chamber exhaust hole 1064 is located on the flow channel between the end of the flow channel of the amplification chamber exhaust microchannel 1063 and the amplification chamber 106.

[0129] Optionally, the waste liquid chamber 105 is located longitudinally above the wax valve chamber 107 , and the waste liquid chamber 105 and the wax valve chamber 107 are arranged adjacent to each other and share part of the chamber wall.

[0130] After the obtained eluate containing nucleic acid is injected into the multiple amplification chambers 106 and before the fluorescent PCR module is started, the nucleic acid extraction fluorescent PCR detection method further includes:

[0131] The heating instrument is started to heat the multiple wax valve chambers 107 , so that the paraffin in the multiple wax valve chambers 107 melts and flows downward into the multiple amplification chamber branch channels 10611 and the multiple amplification chamber exhaust microchannels 1063 accordingly, so as to seal the multiple amplification chambers 106 .

[0132] Specifically, nucleic acid amplification freeze-dried detection reagent balls containing different primers are pre-embedded in the amplification chamber 106. Since the front side of the amplification chamber exhaust hole 1064 is covered with a waterproof and breathable membrane, when the wax valve chamber 107 is not heated, the solid paraffin in the wax valve chamber 107 can respectively block the hole at the end of the channel of the amplification chamber exhaust microchannel 1063 connecting with the wax valve chamber 107 and the wax outlet hole 1071 in the middle of the channel of the amplification chamber branch channel 10612 connecting with the wax valve chamber 107, and the freeze-dried balls inside the amplification chamber 106 are relatively closed to the outside world. After the eluent containing nucleic acid is injected from the bottom of the amplification chamber 106 to fill the amplification chamber 106, the heating module on the instrument melts the paraffin inside the wax valve chamber 107, and the melted paraffin flows into the flow channel along the amplification chamber exhaust microchannel 1063 and the amplification chamber branch channel 10612 to seal the amplification chamber exhaust hole 1064, the amplification chamber exhaust microchannel 1063 and the amplification chamber branch channel 10612, thereby completely sealing the amplification chamber 106.

[0133] Optionally, step S4 includes: starting the fluorescence PCR module of the instrument, and completing the nucleic acid amplification detection PCR process through thermal cycling, lighting, and detection.

[0134] The present application also provides a computer-readable storage medium, which includes a program or instruction. When the program or instruction is executed, the in vitro diagnostic detection method as described above is implemented.

[0135] The present application also provides an in vitro diagnostic detection system using a microfluidic chip, which includes the above-mentioned computer-readable storage medium. The microfluidic chip 1000 is used to cooperate with a detection instrument having an ultrasonic module, a heating module, a magnetic suction module, and a four-color fluorescent PCR module, and uses a magnetic bead method to extract nucleic acids and perform PCR detection of up to 32 target genes.

[0136] The above description is a chip bottom shell 100, a microfluidic chip 1000, an in vitro diagnostic detection method, an in vitro diagnostic detection system, and a computer-readable storage medium. The microfluidic chip 1000 and the chip bottom shell 100 of the in vitro diagnostic detection system have a simple flow channel layout, and there is no need to set a complex control valve. The structure is simple and reasonable, and the design and material use requirements are not high. The chip bottom shell is an integrally formed part with low manufacturing cost. In addition, the chip bottom shell of the present application has multiple amplification chambers, which can be pre-buried with liquid reagents for magnetic bead nucleic acid extraction and freeze-dried reagents for nucleic acid amplification. One consumable is used, and the sample is added once. The operator operation time is less than one minute. The nucleic acid extraction and amplification process of sample input and result output is completed in cooperation with the instrument, and fluorescent PCR detection of multiple targets can be realized with high detection efficiency.

[0137] The preferred embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0138] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0139] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An in vitro diagnostic detection method using a microfluidic chip, characterized in that: The in vitro diagnostic test method comprises: S1. A microfluidic chip (1000) is provided, wherein the microfluidic chip (1000) comprises a chip bottom shell (100) and a first coating and a second coating welded on both sides of the chip bottom shell (100), wherein the chip bottom shell (100) comprises a substrate (101), a sample loading chamber (102), an extraction chamber (103), a detection reagent storage module (104), a waste liquid chamber (105), and a plurality of amplification chambers (106), wherein the sample loading chamber (102), the extraction chamber (103), the detection reagent storage module (104), the waste liquid chamber (105), and the plurality of amplification chambers (106) are all arranged in the chip bottom shell (100). On the substrate (101), the sample loading chamber (102), the detection reagent storage module (104), the waste liquid chamber (105) and the plurality of amplification chambers (106) are respectively connected to the extraction chamber (103) through microchannels on the substrate (101); the chip bottom shell (100) is an integrally formed part; the first coating and the second coating are used to cover the microchannels and part of the chamber on the substrate (101); the detection reagent storage module (104) is provided with an extraction reagent, a washing liquid reagent and an elution liquid reagent; and the amplification chamber (106) is provided with a freeze-dried detection reagent ball; S2, injecting the sample liquid to be tested into the extraction chamber (103) through the sample injection chamber (102); S3, injecting the extraction reagent, washing liquid reagent and elution liquid reagent in the detection reagent storage module (104) into the extraction chamber (103) in batches to react with the sample to be tested, discharging the waste liquid after the reaction into the waste liquid chamber (105), and injecting the obtained eluate containing the target detection substance into the plurality of amplification chambers (106); S4, starting the detection module to detect the mixed solution in the plurality of amplification chambers (106).

2. The in vitro diagnostic test method according to claim 1, characterized in that: Along the longitudinal direction of the plate surface of the substrate (101), the sample loading chamber (102) is located above the extraction chamber (103) in the longitudinal direction. The extraction chamber (103) is connected to an extraction chamber exhaust channel capable of exhausting gas to the outside. The extraction chamber exhaust channel includes an extraction chamber external exhaust hole (1031), an extraction chamber exhaust flow channel (1032) and an extraction chamber internal exhaust hole (1033). The extraction chamber exhaust flow channel (1032) is concavely formed on the second plate surface (1012). The extraction chamber external exhaust hole (1031) and the extraction chamber internal exhaust hole (1033) are both arranged on the substrate (101) in the thickness direction of the substrate. Along the longitudinal direction of the plate surface of the substrate, the extraction chamber (103) is connected to an extraction chamber exhaust channel capable of exhausting gas to the outside. The outer exhaust hole (1031) of the extraction chamber is located at the upper longitudinal part of the extraction chamber (103); the exhaust hole (1033) in the extraction chamber is arranged on the bottom wall of the chamber body at the upper longitudinal part of the extraction chamber (103) along the thickness direction of the substrate; the extraction chamber (103), the exhaust hole (1033) in the extraction chamber, the exhaust channel (1032) of the extraction chamber, and the exhaust hole (1031) outside the extraction chamber are connected in sequence; the sample loading chamber (102) is provided with a sample loading port; and the microfluidic chip (1000) further comprises a sample loading sealing plug (200); the sample loading sealing plug (200) is used to cover the sample loading port and can perform piston movement in the sample loading chamber (102); Wherein, the S2 includes: S21, placing the microfluidic chip (1000) flat so that the sample adding port faces upward, and adding the sample to be tested from the sample adding port; S22, inserting the sample injection sealing plug (200) into the sample injection port and placing the microfluidic chip (1000) vertically along the longitudinal direction of the substrate at a detection position, so that the sample liquid to be tested in the sample injection chamber (102) flows downward into the extraction chamber (103).

3. The in vitro diagnostic test method according to claim 2, characterized in that: The detection reagent storage module (104) comprises a plurality of columnar storage cavities capable of cooperating with the piston cover (400) and communicating with the extraction chamber (103), the axes of the plurality of columnar storage cavities being parallel to the plate surface of the substrate (101), the piston cover cooperating openings (1041) of the plurality of columnar storage cavities being arranged on the substrate thickness surface of the substrate (101), the substrate thickness surface (1013) being perpendicular to the plate surface of the substrate (101), and the microfluidic chip (1000) further comprising a plurality of piston covers, the plurality of piston covers (400) being cooperating one by one and sealing in the plurality of columnar storage cavities; Wherein, the S3 includes: S31, starting the piston drive motor to push the piston cover (400) of the columnar storage chamber storing the extraction reagent to move, so as to inject the extraction reagent into the extraction chamber (103); S32, starting an ultrasonic instrument to perform ultrasonic lysis on the sample liquid to be tested in the extraction chamber (103); S33, turning off the ultrasonic instrument, matching the magnetic suction instrument with the extraction chamber (103), and discharging the waste liquid in the extraction chamber (103) into the waste liquid chamber (105); S34, starting the piston drive motor to push the piston cover (400) of the columnar storage chamber storing the washing liquid reagent to move, so as to inject the washing liquid reagent into the extraction chamber (103); S35, removing the magnetic suction instrument and starting the ultrasonic instrument to wash the substance in the extraction chamber (103); S36, turning off the ultrasonic instrument, matching the magnetic suction instrument with the extraction chamber (103), and discharging the waste liquid in the extraction chamber (103) into the waste liquid chamber (105); S37, starting the piston cover driving motor to push the piston cover (400) of the columnar storage chamber storing the elution liquid reagent to move, and injecting the elution liquid reagent into the extraction chamber (103); S38, removing the magnetic suction instrument and starting the ultrasonic instrument to elute the substance in the extraction chamber (103); S39, turn off the ultrasonic instrument, match the magnetic suction instrument with the extraction chamber (103), and discharge the eluate containing the target detection substance in the extraction chamber (103) into the amplification chamber (106).

4. The in vitro diagnostic test method according to claim 3, characterized in that: The microfluidic chip (1000) further comprises an upper cover (300), the upper cover (300) being arranged on the chip bottom shell (100) and being provided with a sample addition hole (301) and an ultrasonic hole (302), the sample addition hole (301) being arranged in alignment with the sample addition cavity (302), and the ultrasonic hole (301) being arranged in alignment with the extraction cavity (302); Wherein, starting the ultrasonic instrument in S3 includes: After the ultrasonic instrument is matched with the extraction chamber (103) through the ultrasonic hole (302), the ultrasonic instrument is turned on.

5. The in vitro diagnostic test method according to claim 3, characterized in that: The waste liquid chamber (105) is connected to a waste liquid chamber exhaust channel capable of exhausting gas to the outside, and the plurality of amplification chambers (106) are connected to an amplification chamber exhaust channel capable of exhausting gas to the outside. The extraction chamber (103) is connected to the waste liquid chamber (105) via a waste liquid chamber microchannel (1055), and the waste liquid chamber microchannel (1055) is provided with a waste liquid chamber valve capable of controlling the conduction or closure of the channel. The extraction chamber (103) is connected to the plurality of amplification chambers (106) via an amplification chamber microchannel (1061), and the amplification chamber microchannel (1061) is provided with an amplification chamber valve capable of controlling the conduction or closure of the channel. Wherein, the step of cooperating the magnetic suction instrument with the extraction chamber (103) and discharging the waste liquid in the extraction chamber (103) into the waste liquid chamber (105) in S3 includes: Matching a magnetic suction instrument with the extraction chamber (103); The amplification chamber valve is controlled to remain closed, and the waste liquid chamber valve is controlled to open, so as to discharge the waste liquid in the extraction chamber (103) into the waste liquid chamber (105).

6. The in vitro diagnostic test method according to claim 5, characterized in that: The step of matching the magnetic suction instrument with the extraction chamber (103) and discharging the eluate containing the target detection substance in the extraction chamber (103) into the amplification chamber (106) in S3 includes: Matching a magnetic suction instrument with the extraction chamber (103); The waste liquid chamber valve is controlled to remain closed, and the amplification chamber valve is controlled to open, so as to discharge the eluate containing the target detection substance in the extraction chamber (103) into the amplification chamber (106).

7. The in vitro diagnostic test method according to claim 6, characterized in that: The waste liquid chamber microfluidic channel (1055) and the amplification chamber microfluidic channel (1061) are both formed concavely on the second plate surface (1012); a waste liquid chamber microfluidic channel cut-off portion (1056) for cutting off the flow channel is integrally provided on the waste liquid chamber microfluidic channel (1055); the waste liquid chamber flow channel cut-off portion (1056) and the second covering film form the waste liquid chamber valve; an amplification chamber microfluidic channel cut-off portion (1062) for cutting off the flow channel is integrally provided on the amplification chamber microfluidic channel (1061); the amplification chamber microfluidic channel cut-off portion (1062) and the second covering film form the amplification chamber valve; the waste liquid chamber valve and the amplification chamber valve are formed as normally open film valves; Among them, at the detection position in S22, the waste liquid chamber valve and the amplification chamber valve can be closed by pressing the second coating on the waste liquid chamber flow channel cutting part (1056) and the amplification chamber microchannel cutting part (1062) through the cutting part driving motor.

8. The in vitro diagnostic test method according to claim 6, characterized in that: The plurality of amplification chambers (106) are connected to the extraction chamber (103) through an amplification chamber microchannel (1061), the amplification chamber microchannel (1061) comprising an amplification chamber main channel (10611) and a plurality of amplification chamber branch channels (10612), the first end of the amplification chamber main channel (10611) being connected to the extraction chamber (103), the first ends of the plurality of amplification chamber branch channels (10612) being connected to the plurality of amplification chambers (106) one by one, the second ends of the plurality of amplification chamber branch channels (10612) being connected to the second end of the amplification chamber main channel (10611), the amplification chamber exhaust channel comprising a plurality of amplification chamber exhaust microchannels (1063) and a plurality of amplification chamber exhaust holes (10612) provided on the plurality of amplification chamber exhaust microchannels (1063) 064), a plurality of amplification chamber exhaust microchannels (1063) are respectively connected to the plurality of amplification chambers (106), the amplification chamber exhaust holes (1064) are arranged on the substrate (101) along the thickness direction of the substrate, the chip bottom shell (100) further comprises a plurality of wax valve chambers (107) arranged on the substrate (101), each of the amplification chamber branch channels (10612) and each of the amplification chamber exhaust microchannels (1063) are respectively connected to a wax valve chamber (107), and paraffin is installed in the wax valve chamber (107), and along the longitudinal direction of the plate surface of the substrate (101), the extraction chamber (103), the plurality of wax valve chambers (107), the plurality of amplification chamber exhaust holes (1064) and the plurality of amplification chambers (106) are arranged in sequence from top to bottom; Wherein, after S39 and before S4, S3 further includes: The heating instrument is started to heat the multiple wax valve chambers (107), so that the paraffin in the multiple wax valve chambers (107) melts and flows downward into the multiple amplification chamber branch channels (10611) and the multiple amplification chamber exhaust microchannels (1063) to seal the multiple amplification chambers (106).

9. A computer-readable storage medium, characterized in that: The invention comprises a program or an instruction, and when the program or the instruction is executed, the in vitro diagnostic detection method according to any one of claims 1 to 8 is implemented.

10. An in vitro diagnostic detection system using a microfluidic chip, characterized in that: The in vitro diagnostic testing system comprises the computer readable storage medium of claim 9.

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