WO2023056986A1 - Immunofluorescence assay kit and method for using same, and fluorescence immunoassay device - Google Patents

Abstract

An immunofluorescence assay kit, comprising a kit body. The kit body comprises: a storage chamber and a transfer cavity. An external force is applied to the transfer cavity, so that a negative pressure is created in the transfer cavity by means of increasing the volume of air in the transfer cavity, and then a tiny amount of reagent in the storage chamber flows into the transfer cavity; and/or an external force is applied to the transfer cavity, so that a positive pressure is created in the transfer cavity by means of reducing the volume of air in the transfer cavity, and then a tiny amount of reagent in the transfer cavity is sucked back into the storage chamber. The present invention further relates to a method of transferring a liquid by changing the volume of a transfer cavity, a method for using a kit, and a fluorescence immunoassay device.

Description

Reagent kit and method for immunofluorescence detection, fluorescent immunoassay device 【Technical field】

The invention relates to the technical field of fluorescence detection, in particular to a reagent kit for immunofluorescence detection, a use method, and a fluorescence immunodetection device.

【Background technique】

Traditional nucleic acid detection includes sample pretreatment, nucleic acid extraction, amplification, nucleic acid detection, etc. Each test link requires specific instruments or manual steps to operate. The entire process needs to be performed in a professional P2 laboratory by professional operators. Personnel completed. From sample receipt and registration, laboratory environment preparation, personal protective equipment, and waste disposal, the entire testing process takes several hours. If the time of waiting in line, sample collection, and sample delivery is included in the calculation, it may take several days. In order to get the test result report, the test cost will also increase.

In the face of the sudden outbreak of the new crown epidemic, many medical institutions do not have professional laboratories, nor are they equipped with professional testing instruments and operators, which leads to a delay in the detection speed, thus affecting the control of the epidemic. For this reason, it is urgent to develop a portable POCT nucleic acid real-time detection device, which can complete the nucleic acid detection steps in a short time without professional laboratories and operators, which can not only meet the detection needs in special periods of the epidemic, but also be popularized. Grassroots use.

Therefore, it is necessary to improve the prior art to overcome the defects in the prior art.

【Content of invention】

Aiming at the deficiencies of the prior art, the object of the present invention is to provide a kit for immunofluorescence detection, comprising a box body, the box body comprising:

A storage room, the storage room is used to store trace reagents, the trace reagents include various solvents and samples to be tested;

a transport chamber, which is used to transport the trace reagent between the transport chamber and the storage chamber;

An external force is applied to the transfer chamber to form a negative pressure in the transfer chamber by increasing the volume of air in the transfer chamber, so that the trace reagent in the storage chamber flows into the transfer chamber;

and / or

An external force is applied to the transfer chamber to form a positive pressure in the transfer chamber by reducing the volume of air in the transfer chamber, so that the trace reagent in the transfer chamber is sucked back to the storage chamber.

Preferably, the wall of the transfer chamber includes at least one movable piston end face.

Preferably, at least one electromagnetic switch is further provided on the box body for controlling the flow of the magnetic beads at the position where the electromagnetic switch is located.

Preferably, the storage chamber further includes at least one of a lysate storage area, a magnetic bead solution storage area, a washing liquid storage area, and an eluent storage area.

Preferably, an external force is applied to the transport chamber such that the transport chamber can move between several storage chambers.

The present invention also provides a kit for immunofluorescence detection, including a box body for providing a flow place for trace reagents, and the box body includes:

Several storage chambers are used to store trace reagents; wherein, the storage chambers at least include a transfer chamber and a fluorescence detection area;

a transport chamber, which is used to conditionally communicate with the storage chamber;

Driven by external force, the transport chamber can move among several storage chambers, so as to realize the transfer of trace reagents between the transport chamber and the fluorescent detection area.

Preferably, the wall of the transfer chamber includes at least one movable piston end face.

Preferably, at least one electromagnetic switch is further provided on the box body for controlling the flow of the magnetic beads at the position where the electromagnetic switch is located.

Preferably, the storage chamber further includes at least one of a lysate storage area, a magnetic bead solution storage area, a washing liquid storage area, and an eluent storage area.

The present invention also relates to a method for using the kit as described above, comprising the following steps:

When the transfer chamber is located at a position of the storage chamber, an external driving device is configured to move to increase the volume of the transfer chamber, so that a negative pressure is formed in the transfer chamber, so as to inject the trace reagent in the storage chamber into the transfer chamber;

and / or

When the transfer chamber is located at a position of the storage chamber, an external driving device is configured to move to reduce the volume of the transfer chamber and form a positive pressure in the transfer chamber to inject a small amount of reagent in the transfer chamber into the storage chamber.

Preferably, the following steps are included:

The volume of the transport cavity driven by external force and/or the volume of the transport cavity decreased by external force can be repeatedly performed, so as to realize repeated movement of trace reagents between the transport cavity and a storage chamber.

Preferably, when the magnetic bead solution is stored in the transport cavity, the following steps are also included:

Control and trigger the electromagnetic switch to fix the magnetic beads in the transfer chamber;

The volume of the transport chamber is driven to shrink, and a positive pressure is formed in the transport chamber so that a small amount of reagent in the transport chamber is injected into the storage chamber.

The present invention also provides a method for using the kit as described above, comprising the following steps:

The transport chamber is driven to move among several storage chambers until the transport chamber moves to the position of the fluorescence detection area.

Preferably, the transfer chamber is reciprocable between several storage chambers.

Preferably, it also includes the steps of:

Obtain the position of the transport chamber located in the box body;

When the transfer chamber is located in a storage chamber that can be used to store waste liquid, the control triggers the electromagnetic switch to fix the magnetic beads in the transfer chamber;

Controlling the injection of trace reagents into the storage chamber for storing waste liquid;

Control the transfer chamber to move to the next storage chamber and mix with the trace reagent in the storage chamber, and then control the transfer chamber to return to the storage chamber for storing waste liquid;

The electromagnetic switch is controlled to be turned off so that the magnetic beads are mixed with the liquid in the transfer chamber; the transfer chamber is then configured to transfer the mixed liquid to the next target storage chamber.

Preferably, it also includes the steps of:

Obtain the position of the transport chamber located in the box body;

When the transfer chamber is located in a storage chamber that can be used to store waste liquid, the control triggers the electromagnetic switch to fix the magnetic beads in the transfer chamber;

Controlling the injection of trace reagents into the storage chamber for storing waste liquid;

Control and turn off the electromagnetic switch to configure the transfer chamber to drive the magnetic beads in it to move to the next storage chamber and mix with the trace reagent in the storage chamber; then configure the transfer chamber to transfer the mixed solution to the next target storage chamber.

The present invention also provides a fluorescent immunoassay device, which includes a housing, and the housing includes a detection device, a display device, and an installation position, and the installation position is used to perform fluorescence detection on the reagent box as described above.

Compared with the prior art, the beneficial effects of the present invention are:

The invention relates to a kit for immunofluorescence detection, which comprises a box body, and the box body includes: a storage chamber and a transfer chamber; an external force is applied to the transfer chamber to increase the volume of air in the transfer chamber to form a Negative pressure makes the trace reagent in the storage chamber flow into the transfer chamber; and/or an external force is applied to the transfer chamber to form a positive pressure in the transfer chamber by reducing the volume of air in the transfer chamber, so that the trace reagent in the transfer chamber is sucked back to storage room. The invention also relates to a method for transporting liquid by changing the volume of the transport chamber, a method for using the kit, and a fluorescent immunoassay device. By manipulating the power device to transfer liquid in a closed environment, the required sample volume is small, the operation is simple, and the transfer can be completed in a short time without professional laboratories and operators.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention and accompanying drawings are described in detail below. The specific embodiment of the present invention is given in detail by the following examples and accompanying drawings.

【Description of drawings】

The accompanying drawings described here are used to provide a further understanding of the present invention and constitute a part of the application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention. In the attached picture:

Fig. 1 is the schematic structural view of the micro reagent transfer device including the power device for immunofluorescence detection of the present invention;

Fig. 2 is the structure schematic diagram of the micro reagent transfer device that does not include the power device for immunofluorescence detection of the present invention;

Fig. 3 is the schematic structural view of the fluorescent immunoassay device of the present invention;

Fig. 4 is a flow chart of the use method of the kit when a magnetic bead solution is included in the present invention;

Fig. 5 is a flow chart of the use method of a kit for immunofluorescence detection in the elution process of the present invention;

Fig. 6 is a flow chart of another method of using the immunofluorescence detection kit for the elution process of the present invention.

Explanation of reference signs:

100. Fluorescence immunoassay device;

110. Shell; 120. Installation position; 130. Display device;

200. Box body;

201, transfer cavity; 202, lysate storage area; 203, magnetic bead solution storage area; 204, washing liquid storage area; 205, eluent storage area; 206, PCR amplification and fluorescence detection area; 207, piston; 208 , micro-flow valve; 209, electromagnetic switch.

【Detailed ways】

The present invention will be further described in detail below in conjunction with the accompanying drawings, and the aforementioned and other objects, features, aspects and advantages of the present invention will become more apparent, so that those skilled in the art can implement them with reference to the description. In the drawings, the shapes and dimensions may be exaggerated for clarity, and the same reference numerals will be used throughout to designate the same or like parts. In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, etc. are based on the orientation or positional relationship shown in the drawings. In particular, "height" corresponds to the dimension from top to bottom, "width" corresponds to the dimension from left to right, and "depth" corresponds to the dimension from front to back. These relative terms are for convenience of description and are generally not intended to require a specific orientation. Terms referring to attachment, coupling, etc. (e.g., "connected" and "attached") refer to a fixed or attached relationship, as well as a movable or rigid attachment or relationship, of structures to one another, directly or indirectly through intermediate structures, unless otherwise stated in The other way is clearly stated.

Next, the present invention will be further described in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, on the premise of not conflicting, the various embodiments or technical features described below can be combined arbitrarily to form new implementations. example. It should be understood that terms such as "having", "comprising" and "including" as used herein do not entail the presence or addition of one or more other elements or combinations thereof.

Embodiment one

As shown in Figures 1-2, a kit for immunofluorescence detection includes a box body 200, and the box body 200 includes:

The storage room is used to store trace reagents, which include various solvents and samples to be tested;

A transfer chamber 210, which is used to transfer trace reagents between the transfer chamber 210 and the storage chamber;

An external force is applied to the transfer chamber 210 to create a negative pressure in the transfer chamber 210 by increasing the volume of air in the transfer chamber 210, so that the trace reagent in the storage chamber flows into the transfer chamber 210;

and / or

External force is applied to the transfer chamber 210 to form a positive pressure in the transfer chamber 210 by reducing the volume of the air in the transfer chamber 210, so that the trace reagent in the transfer chamber 210 is sucked back to the storage chamber. The external force can be manpower, and the external force can also be provided by a power device, and the power device can be a device that can provide power commonly used in the art. That is to say, the storage chamber of the kit of this solution can release trace reagents, and can also be used for reabsorbing trace reagents to enrich the use of the storage chamber in the kit; it should be understood that since the kit of this program can be used for reabsorbing trace reagents, The kit can be used to suck back the waste liquid to prevent the waste liquid from affecting the final test results.

Furthermore, more than two storage chambers can be set in the box body 200 for storing or adding different trace reagents to enrich the usage of the kit. When used for immunofluorescence detection, the storage chamber at least includes a transfer chamber 201, a PCR amplification and a fluorescence detection area 206. At this time, it may also include a lysate storage area 202, a magnetic bead solution storage area 203, a washing liquid storage area 204, At least one of the eluent storage areas 205.

Further, among them, the transfer chamber 201 is manually added to the sample and then the entrance is sealed; the lysate storage area 202, the magnetic bead solution storage area 203, the washing liquid storage area 204, and the eluent storage area 205 are pre-packaged with liquid reagents in advance; PCR The amplification and fluorescence detection area 206 is prepackaged with primers/solid reagents in advance.

In some embodiments, the power device is a piston 207, at this time, the cavity wall of the transfer cavity 210 includes at least one movable end surface of the piston. Further, the power device also includes two oppositely arranged pistons 207, the two pistons 207 can move relatively, and a transfer chamber 210 is formed between the opposing surfaces of the two pistons 207, when the two pistons 207 move relatively, the air volume in the transfer chamber 210 is compressed, In order to form a positive pressure in the transfer chamber 210, it is beneficial for the trace reagent in the transfer chamber 210 to be sucked back to the storage chamber; Pressing is used to press the trace reagent in the storage chamber into the transfer chamber 210. The piston is selected because it is simple to operate, low in cost and good in controllability compared with other power devices. It should be understood that other common air pressure changing devices in the field can also be selected as the power device.

Further, the relative distance between the two pistons 207 is smaller than the distance between two adjacent storage chambers; by limiting the relative distance between the two pistons 207, it is avoided that the transfer chamber 210 simultaneously Trace reagents in the two reservoirs cause compression.

Further, at least one electromagnetic switch 209 is provided on the box body 200 for controlling the flow of magnetic beads passing through the position where the electromagnetic switch 209 is located. It should be understood that, according to actual elution requirements, two or more electromagnetic switches may be provided on the cartridge body 200 to fix the magnetic beads during the elution operation.

Further, an external force is applied to the transfer chamber 210 so that the transfer chamber 210 can move between several storage chambers; that is, the kit of this solution can be used to transfer trace reagents in addition to injecting and sucking back trace reagents. In some embodiments, an external force is applied to the piston 207 to push the transport chamber 210 to move between different storage chambers until reaching the fluorescence detection area.

Embodiment two

As shown in Figures 1-2, a kit for immunofluorescence detection includes a box body 200 for providing a flow place for trace reagents, and the box body 200 includes:

Several storage chambers are used to store trace reagents; wherein, the storage chambers at least include a transfer chamber 201 and a detection area (PCR amplification and fluorescence detection area 206); to detect the samples to be tested in the transfer chamber 201;

The transfer chamber 210 is used to conditionally communicate with the storage chamber;

Driven by external force, the transfer chamber 210 can move between several storage chambers, so as to realize the transfer of trace reagents between the transfer chamber 201 and the fluorescence detection area (PCR amplification and fluorescence detection area 206).

The external force can be manpower, and the external force can also be provided by a power device, and the power device can be a device that can provide power commonly used in the art. Several storage chambers are arranged one by one along the moving direction of the power device, and the external force drives the transfer chamber 210 to move relative to the transfer chamber 201; after the sample in the transfer chamber 201 enters the transfer chamber 210, the external force drives the sample in the transfer chamber 210 to move until the sample Add PCR amplification and detection reagents in the fluorescence detection area 206. That is, the sample is moved to the target position (PCR amplification and fluorescence detection area 206 ) through the transfer chamber, so that the sample is at least mixed with detection reagents to perform relevant detection tests. The reagent kit involved in this solution is a closed transfer device, with little pollution, small volume of equipment, small amount of reagents and samples required, simple operation, and the transfer of trace reagents can be completed in a short time.

Further, the transfer cavity 210, which is used to conditionally communicate with the storage chamber, means that the transfer cavity 210 is not connected under normal conditions, but when there is a need for communication, the storage chamber and the transfer cavity 210 can be communicated by external force. In some embodiments, an external force is applied to the transfer chamber 210 to form a negative pressure in the transfer chamber 210 by increasing the volume of air in the transfer chamber 210, so that the trace reagent in the storage chamber flows into the transfer chamber 210;

and / or

External force is applied to the transfer chamber 210 to form a positive pressure in the transfer chamber 210 by reducing the volume of the air in the transfer chamber 210, so that the trace reagent in the transfer chamber 210 is sucked back to the storage chamber. It should be understood that the communication between the transfer chamber 210 and the storage chamber may also be realized in other ways, such as using a switch device such as a wax valve.

Further, the storage room also includes at least one of a lysate storage area, a magnetic bead solution storage area, a washing liquid storage area, and an eluent storage area, so that the sample to be tested can be mixed with the detection agent after being processed to improve detection accuracy. sex. Specifically, after the sample enters the transfer chamber 201, the sample can also flow to the location of the next storage chamber and the trace reagents in it according to the direction of its movement, so that the sample can be chemically processed by different types of trace reagents, Finally, it is mixed with the PCR amplification and fluorescent detection area 206 to improve the final detection result. Specifically, in some embodiments, as shown in Figures 1 and 2, the storage chamber includes a transfer chamber 201, a lysate storage area 202, a magnetic bead solution storage area 203, a washing solution storage area 204, an eluent storage area 205, PCR amplification and fluorescence detection area 206; among them, the transfer cavity 201 is closed by manually adding samples; the lysate storage area 202, the magnetic bead solution storage area 203, the washing liquid storage area 204, and the eluent storage area 205 are carried out in advance Liquid reagents are prepackaged; PCR amplification and fluorescence detection area 206 is prepackaged with primers/solid reagents in advance. The power device first forces the sample to be tested in the transfer chamber 201 to be transferred to the transfer chamber 210 of the power device along its moving direction, and applies pressure to the power device so that the transfer chamber 210 drives the sample to be tested to move to the lysate storage area 202, releasing the reagent in the lysate storage area 202 so that the lysate is mixed with the sample to be tested; the power device continues to push the mixed liquid to move and store it with the magnetic bead solution storage area 203, the washing liquid storage area 204, and the eluent in sequence. Reagents in zone 205, PCR amplification and fluorescence detection zone 206 are mixed. It should be understood that the release order of the trace reagents in the storage chamber is not necessarily in accordance with the transport chamber 201, the lysate storage area 202, the magnetic bead solution storage area 203, the washing liquid storage area 204, the eluent storage area 205, the PCR amplification and the fluorescence storage area. The sequential release of the detection zone 206 can be selected according to the actual mixing requirements. For example, by changing the direction of the transport chamber 210 , the trace reagent in the transport chamber 210 can move in two directions, for example, it can move away from the transport chamber 201 , and it can also move in a direction close to the transport chamber 201 .

In some embodiments, the power device is a piston 207, at this time, the cavity wall of the transfer cavity 210 includes at least one movable end surface of the piston. Further, the power device further includes two oppositely disposed pistons 207 , the two pistons 207 can move relative to each other, and a transfer chamber 210 is formed between the opposite surfaces of the two pistons 207 . The piston is chosen for its ease of operation and good controllability compared to other power units. It should be understood that other common air pressure changing devices in the field can also be selected as the power device.

Further, when the two pistons 207 move toward the transfer chamber 201, the relative distance between the two pistons 207 is smaller than the distance between the two adjacent storage chambers. On the one hand, the displacement of the transfer chamber 210 can be stably operated, and on the other hand, it can be avoided. The relative distance between the two pistons 207 is too large, causing the transfer chamber 210 to contact trace reagents in other storage chambers in advance, resulting in contamination of the mixed liquid in the transfer chamber 210 .

Further, at least one electromagnetic switch 209 is provided on the box body 200 for controlling the flow of magnetic beads passing through the position where the electromagnetic switch 209 is located. It should be understood that, according to actual elution requirements, two or more electromagnetic switches may be provided on the cartridge body 200 to fix the magnetic beads during the elution operation.

Further, the box body 200 includes at least two storage chambers for storing samples to be tested; this solution is suitable for samples to be tested that are not suitable for one-time mixing, and the final micro reagent mixing efficiency is improved by adding samples in batches, so as to Ultimately improve the detection results.

Embodiment Three

As shown in Figures 1-3, a fluorescence immunoassay device 100 includes a housing 110, the housing 110 forms a chamber for fluorescence immunoassay, the housing 110 includes a detection device, a display device 130 and an installation position 120, the The mounting position 120 is used to perform fluorescence detection on the kits in the above embodiments (the first embodiment and the second embodiment).

Furthermore, the installation positions 120 are respectively arranged on two sides of the display device 130 to maximize the use of the limited space of the housing 110 and improve the overall visual effect of the housing 110 . The fluorescent immunoassay device 100 is a portable detection device, which can automatically complete nucleic acid detection in a short time, including nucleic acid extraction, sequence amplification, nucleic acid amplification, fluorescent PCR detection, etc.; Simple, without the need for professional laboratories and operators, the nucleic acid detection steps can be completed in a short time, and multiple samples or multiple detection items can be carried out at the same time.

Embodiment four

A method for using the kit for immunofluorescence detection in embodiment one, comprising the following steps:

Step 110: When the transfer chamber 210 is located at a storage chamber position, configure an external driving device to move to increase the volume of the transfer chamber 210, so that a negative pressure is formed in the transfer chamber 210, so as to inject the trace reagent in the storage chamber into the transfer chamber 210;

and / or

When the transfer chamber 210 is located in a storage chamber (it can be the same storage chamber or a different storage chamber), configure the external driving device to move to reduce the volume of the transfer chamber 210, so that a positive pressure is formed in the transfer chamber 210, so that the transfer chamber The trace reagent in 210 is injected into the storage chamber.

Further, external force can be used to increase the volume of the transport chamber and/or external force can reduce the volume of the transport chamber, so as to realize the repeated movement of trace reagents between the transfer chamber 210 and a storage chamber; The chamber and the transport cavity 210 are freely transported.

Further, when the magnetic bead solution is stored in the transport cavity 210, the following steps are also included, as shown in Figure 4:

S111: controlling and triggering the electromagnetic switch to fix the magnetic beads in the transfer chamber 210;

S112: Drive to reduce the volume of the transfer chamber 210, and form a positive pressure in the transfer chamber 210 so that the trace reagent in the transfer chamber 210 is injected into the storage chamber. This scenario can be applied when the magnetic beads are washed with the eluent and/or washing solution, and the waste liquid after washing the magnetic beads can be injected into the storage chamber that can be used to store the waste liquid by performing S111-S112 to avoid the influence of the waste liquid Final test results.

Embodiment five

A method for using the kit for immunofluorescence detection in Example 2, comprising the following steps:

Step 120: Driving the transport chamber 210 to move among several storage chambers until the transport chamber 210 moves to the position of the fluorescence detection area. That is, the driving power device and the transfer chamber 201 move relative to each other, so as to drive the sample to be tested in the transfer chamber 210 away from the location of the transfer chamber 201 until it flows to the location of the detection area (PCR amplification and fluorescence detection area 206). Specifically, the transfer chamber is driven to move along the arrangement path of the storage chambers, so as to drive the samples to be tested in the transfer chamber 210 to move directly below different storage chambers. At this time, the storage chamber above the transfer chamber 210 can be damaged by manpower or mechanical force. so that the sample to be tested is mixed with the trace reagent in the storage chamber; until the transfer chamber 210 moves to the PCR amplification and fluorescence detection area 206 directly below, human or mechanical force damages the PCR amplification and fluorescence detection area 206 above the transfer chamber 210, and The detection reagent is at least mixed with the sample to be tested, so that the fluorescence immunoassay device can perform fluorescence detection on the mixed trace reagent in the kit.

It should be understood that the transfer chamber 210 can move away from the transfer chamber 201 or move towards the transfer chamber 201 . In addition, the transfer chamber 210 can reciprocate between several storage chambers.

Further, steps are also included, as shown in Figure 5:

S121: Obtain the position of the transfer chamber 210 in the box body 200; the position of the transfer chamber 210 can be obtained through a position sensor or other sensors; the position of the transfer chamber 210 can also be judged by human vision.

S122: When the transfer chamber 210 is located in a storage room that can be used to store waste liquid, control and trigger the electromagnetic switch to fix the magnetic beads in the transfer chamber 210;

S123: controlling the injection of trace reagents into the storage chamber for storing the waste liquid; controlling the injection of the mixed liquid in the transfer chamber in step S122 into the storage chamber for storing the waste liquid, the specific method can refer to S110.

S124: Control the transfer chamber 210 to move to the next storage chamber and mix with the trace reagent in the storage chamber, and then control the transfer chamber 210 to return to the storage chamber for storing waste liquid;

S125: Control to turn off the electromagnetic switch so that the magnetic beads are mixed with the liquid in the transfer chamber 210, and then configure the transfer chamber 210 to transfer the mixed liquid to the next target storage chamber. By executing S121-S125, the elution waste liquid can be discharged in time after the magnetic beads are eluted, so as to avoid affecting the final detection result. The trace reagents of the magnetic beads move to the next target storage chamber. This scheme ensures that the magnetic beads have better fluidity during the movement and avoids the loss of biological information on the magnetic beads.

Further, steps are also included, as shown in Figure 6:

S126: Obtain the position of the transfer chamber 210 in the box body 200; the position of the transfer chamber 210 can be obtained through a position sensor or other sensors; the position of the transfer chamber 210 can also be judged by human vision.

S127: When the transfer chamber 210 is located in a storage room that can be used to store waste liquid, control and trigger the electromagnetic switch to fix the magnetic beads in the transfer chamber 210;

S128: Control the injection of trace reagents into the storage chamber for storing waste liquid; control the injection of the mixed liquid in the transfer chamber 210 in step S127 into the storage chamber for storing waste liquid. For specific methods, refer to S110.

S129: Control and turn off the electromagnetic switch to configure the transfer chamber 210 to drive the magnetic beads in it to the next storage chamber and mix with the trace reagent in the storage chamber, and then configure the transfer chamber to transfer the mixed solution to the next target storage chamber. By executing S126-S129. During the process of magnetic bead elution and waste liquid removal, the eluted magnetic beads can continue to be mixed with other trace reagents to complete the final reaction without manipulating the reciprocating movement of the transport chamber.

Human Papillomavirus Nucleic Acid Detection Kit (PCR Fluorescent Probe Method)

Take a cervical cell sample (48 years old, female sample) and transfer it to 1 mL/cell preservation solution, centrifuge at 13,000 RPM for 10 minutes, discard the supernatant, absorb the cell pellet at the bottom of the tube, and manually transfer 50 μL of the cell pellet solution Added to the box body 200. Prepackage 150 μL of lysate in lysate storage area 202; prepackage 50 μL of magnetic bead solution in magnetic bead solution storage area 203; prepackage 200 μL of washing solution in washing solution storage area 204; prepackage 50 μL of eluent In the eluent storage area 205; 25 μL of primer freeze-dried beads are pre-packaged in the PCR amplification and fluorescence detection area 206. Through the use of the microfluidic valve 208 (width 200 microns, depth 200 microns), the prepackaged liquid does not enter the piston area without the pressure of the piston driving air.

Manually add 50 μL of sample to the transfer chamber 201 for sealing, place the box body 200 in the detection device, and the following steps can be automatically completed:

Step 1: Move the piston 207 to the position of the transfer chamber 201, expand and move to both sides (the total stretching distance is 3.5 mm), and transfer 50 μL of sample from the transfer chamber 201 to the area of the piston 207 by the power of air filling.

Step 2: Move the piston 207 to the lysate storage area 202, expand and move to both sides (the total stretching distance is 12.4 mm), and transfer 150 μL of lysate from the lysate storage area 202 to the piston by the power of air filling In area 207, sufficient mixing (cell lysis) is achieved by continuous stretching and compression (the stretching distance is 12.4 mm to 18.2 mm, the total number of stretching times is 20 times, and the reaction time is 10 minutes).

Step 3: Move the piston 207 to the magnetic bead solution storage area 203, expand and move to both sides (the total stretching distance is 15.2 mm), and use the power of air filling to transfer 50 μL of magnetic bead solution from the magnetic bead solution storage area 203 Transfer to the piston 207 area, by continuous stretching and compression (the stretching distance is 15.2mm to 20.4mm, the total number of stretching times is 20 times, and the reaction time is 20 minutes) to achieve sufficient mixing.

Step 4: After the reaction is completed, turn on the electromagnetic switch 209 below the magnetic bead solution storage area 203 to adsorb the magnetic beads to the bottom of the piston. Then squeeze the piston toward the middle (the total stretching distance is 2.3mm), and the waste liquid can be sent to the magnetic bead solution storage area 203 for storage.

Step 5: Move the piston 207 to the washing liquid storage area 204, expand and move to both sides (the total stretching distance is 14.4 mm), and transfer 200 μL of washing liquid from the washing liquid storage area 204 to the piston by the power of air filling 207 area. Move the piston 207 to the storage area of the magnetic bead solution, turn off the electromagnetic switch 209, release the magnetic beads into the washing solution, move the piston 207 to the storage area 204 of the washing solution, and stretch and compress it continuously (the stretching distance is 14.4 mm) to 22.2mm, the total number of stretches is 15 times, and the reaction time is 15 minutes), in order to achieve sufficient washing.

Step 6: After the washing is completed, turn on the electromagnetic switch 209 below the washing liquid storage area 204 to adsorb the magnetic beads to the bottom of the piston, and then squeeze the piston to the middle (the total stretching distance is 2.3mm), and the waste The liquid is sent to the washing liquid storage area 204 for storage.

Step 7: Move the piston 207 to the eluent storage area 205, expand and move to both sides (the total stretching distance is 5.5 mm), and use the power of air filling to transfer 50 μL of eluent from the eluent storage area 205 Transfer to piston 207 area. Then the piston 207 is moved to the washing solution storage area 204, the electromagnetic switch 209 is turned off, the magnetic beads are released into the eluent, and the piston 207 is moved to the eluent storage area 205, and by continuous stretching and compression (stretching distance) 5.5mm to 10.1mm, the total number of stretches is 15 times, and the reaction time is 25 minutes), in order to achieve sufficient elution.

Step 8: Open the electromagnetic switch 209 below the eluent storage area 205, the magnetic beads can be adsorbed on the bottom of the piston, and then the piston 207 is moved to the PCR amplification and fluorescence detection area 206, and the piston is squeezed toward the middle (stretched) The total distance is 0mm), and the eluate can be sent to the PCR amplification and fluorescence detection area (206). The primers can be dissolved after standing for 1 minute.

Step 9: Start the PCR step: UNG enzyme reaction at 50°C, 2 minutes/1 cycle; pre-denaturation at 95°C, 10 minutes/1 cycle; denaturation at 95°C, 10 seconds/45 cycles; annealing extension at 55°C, 45 seconds/45 cycles. The reporter fluorescence adopts FAM and VIC, the Ct value is 45, and the report result is negative for high-risk HPV (the detected genotypes are HPV26, 39, 51, 59, 68, 82).

Verify the operational accuracy of the piston transferring liquid

Experimental parameters: moving volume 10 microliters, 20 microliters, 50 microliters, 100 microliters, 200 microliters. Experimental analysis: drive the piston 207 to move to the reagent pre-encapsulation area, expand and move to both sides, transfer the liquid in the reagent pre-encapsulation area to the piston 207 area, and then move the piston 207 to another reagent pre-encapsulation area, take out the liquid and carry out Weigh it to infer the volume of the transferred liquid. If the volume is within V±5%, the transfer is considered successful, and the test stretching distance is recorded. Experimental results: the original volume is 10 microliters, the standard is 9.5 microliters-10.5 microliters, the experimental results show that the transfer volume is 9.8 microliters, which meets the experimental standard, and the stretching distance is 3.2mm; the original volume is 20 microliters, the standard is 19 microliters-21 microliters, the experimental results show that the transfer volume is 19.3 microliters, which meets the experimental standards, and the stretching distance is 6.3mm; the original volume is 50 microliters, the standard is 47.5 microliters-52.5 microliters, the experimental results show that the transfer The volume is 49.8 microliters, which meets the experimental standard, and the stretching distance is 14.8mm; the experimental results show that the transfer volume is 19.3 microliters, which meets the experimental standard, and the stretching distance is 6.3mm; the original volume is 100 microliters, and the standard is 95 microliters -105 microliters, the experimental results show that the transfer volume is 102 microliters, which meets the experimental standards, and the stretching distance is 30.5mm; the original volume is 200 microliters, the standard is 190 microliters-210 microliters, the experimental results show that the transfer volume is 201 Microliter, in line with the experimental standard, the stretching distance is 60.4mm. The above results show that the present invention can precisely control liquid transfer.

Each embodiment in this specification is described in a progressive manner, the same and similar parts of each embodiment can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and for relevant parts, refer to part of the description of the method embodiment.

Claims (17)

1. A kit for immunofluorescence detection, comprising a box body, characterized in that the box body includes:

   A storage room, the storage room is used to store trace reagents, the trace reagents include various solvents and samples to be tested;

   a transport chamber, which is used to transport the trace reagent between the transport chamber and the storage chamber;

   An external force is applied to the transfer chamber to form a negative pressure in the transfer chamber by increasing the volume of air in the transfer chamber, so that the trace reagent in the storage chamber flows into the transfer chamber;

   and / or

   An external force is applied to the transfer chamber to form a positive pressure in the transfer chamber by reducing the volume of air in the transfer chamber, so that the trace reagent in the transfer chamber is sucked back to the storage chamber.
2. The kit for immunofluorescence detection according to claim 1, wherein the wall of the transport chamber comprises at least one movable piston end face.
3. The reagent kit for immunofluorescence detection according to claim 1 or 2, wherein at least one electromagnetic switch is provided on the box body to control the flow of magnetic beads flowing through the position where the electromagnetic switch is located .
4. The kit for immunofluorescence detection according to claim 1 or 2, wherein the storage chamber also includes a lysate storage area, a magnetic bead solution storage area, a washing liquid storage area, and an eluent storage area. at least one of .
5. The kit for immunofluorescence detection according to claim 1 or 2, characterized in that an external force is applied to the transport chamber so that the transport chamber can move between several storage chambers.
6. A kit for immunofluorescence detection, comprising a box body for providing a flow place for trace reagents, characterized in that the box body includes:

   Several storage chambers are used to store trace reagents; wherein, the storage chambers at least include a transfer chamber and a fluorescence detection area;

   a transport chamber, which is used to conditionally communicate with the storage chamber;

   Driven by external force, the transport chamber can move among several storage chambers, so as to realize the transfer of trace reagents between the transport chamber and the fluorescent detection area.
7. The kit for immunofluorescence detection according to claim 6, wherein the wall of the transport chamber comprises at least one movable piston end face.
8. The reagent kit for immunofluorescence detection according to claim 6 or 7, wherein at least one electromagnetic switch is also provided on the box body to control the flow of magnetic beads flowing through the position where the electromagnetic switch is located .
9. The reagent kit for immunofluorescence detection according to claim 6 or 7, wherein the storage chamber also includes a lysate storage area, a magnetic bead solution storage area, a washing liquid storage area, and an eluent storage area. at least one of .
10. A method for using the test kit according to claim 1, comprising the following steps:

    When the transfer chamber is located at a position of the storage chamber, an external driving device is configured to move to increase the volume of the transfer chamber, so that a negative pressure is formed in the transfer chamber, so as to inject the trace reagent in the storage chamber into the transfer chamber;

    and / or

    When the transfer chamber is located at a position of the storage chamber, an external driving device is configured to move to reduce the volume of the transfer chamber and form a positive pressure in the transfer chamber to inject a small amount of reagent in the transfer chamber into the storage chamber.
11. The use method of the kit for immunofluorescence detection as claimed in claim 10, is characterized in that, comprises the following steps:

    The volume of the transport cavity driven by external force and/or the volume of the transport cavity decreased by external force can be repeatedly performed, so as to realize repeated movement of trace reagents between the transport cavity and a storage chamber.
12. The use method of the kit for immunofluorescence detection as claimed in claim 10, is characterized in that, when the magnetic bead solution is stored in the transport cavity, further comprising the following steps:

    Control and trigger the electromagnetic switch to fix the magnetic beads in the transfer chamber;

    The volume of the transport chamber is driven to shrink, and a positive pressure is formed in the transport chamber so that a small amount of reagent in the transport chamber is injected into the storage chamber.
13. A method for using the test kit according to claim 6, comprising the following steps:

    The transport chamber is driven to move between several storage chambers until the transport chamber moves to the position of the fluorescence detection area.
14. The method of using the kit according to claim 13, characterized in that the transport chamber can reciprocate between several storage chambers.
15. The method for using the test kit according to claim 13, further comprising the steps of:

    Obtain the position of the transport chamber located in the box body;

    When the transfer chamber is located in a storage chamber that can be used to store waste liquid, the control triggers the electromagnetic switch to fix the magnetic beads in the transfer chamber;

    Controlling the injection of trace reagents into the storage chamber for storing waste liquid;

    Control the transfer chamber to move to the next storage chamber and mix with the trace reagent in the storage chamber, and then control the transfer chamber to return to the storage chamber for storing waste liquid;

    The electromagnetic switch is controlled to be turned off so that the magnetic beads are mixed with the liquid in the transfer chamber; the transfer chamber is then configured to transfer the mixed liquid to the next target storage chamber.
16. The method for using the test kit according to claim 13, further comprising the steps of:

    Obtain the position of the transport chamber located in the box body;

    When the transfer chamber is located in a storage chamber that can be used to store waste liquid, the control triggers the electromagnetic switch to fix the magnetic beads in the transfer chamber;

    Controlling the injection of trace reagents into the storage chamber for storing waste liquid;

    Control and turn off the electromagnetic switch to configure the transfer chamber to drive the magnetic beads in it to move to the next storage chamber and mix with the trace reagent in the storage chamber; then configure the transfer chamber to transfer the mixed solution to the next target storage chamber.
17. A fluorescent immunoassay device, comprising a housing, characterized in that the housing includes a detection device, a display device, and an installation position, and the installation position is used to execute the test kit according to claim 1 or claim 6. Fluorescent detection.

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